U.S. patent application number 13/294911 was filed with the patent office on 2012-06-07 for minicell based delivery of biologically active compounds.
This patent application is currently assigned to Vaxiion Therapeutics, Inc.. Invention is credited to Neil Berkley, Robert Klepper, Roger A. Sabbadini, Mark W. Surber.
Application Number | 20120142079 13/294911 |
Document ID | / |
Family ID | 27808541 |
Filed Date | 2012-06-07 |
United States Patent
Application |
20120142079 |
Kind Code |
A1 |
Sabbadini; Roger A. ; et
al. |
June 7, 2012 |
MINICELL BASED DELIVERY OF BIOLOGICALLY ACTIVE COMPOUNDS
Abstract
Minicells are used to deliver biologically active compounds
including radioisotopes, polypeptides, nucleic acids, small
molecules, drug molecules, and chemotherapeutic agents. In some
cases, the minicell displays ligands or binding moieties that
target the minicell to a desired host cell.
Inventors: |
Sabbadini; Roger A.;
(Lakeside, CA) ; Berkley; Neil; (San Diego,
CA) ; Surber; Mark W.; (San Diego, CA) ;
Klepper; Robert; (San Diego, CA) |
Assignee: |
Vaxiion Therapeutics, Inc.
San Diego
CA
|
Family ID: |
27808541 |
Appl. No.: |
13/294911 |
Filed: |
November 11, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11725196 |
Mar 16, 2007 |
8101396 |
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13294911 |
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11580095 |
Oct 11, 2006 |
7871815 |
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11725196 |
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10156902 |
May 28, 2002 |
7183105 |
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11580095 |
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10154951 |
May 24, 2002 |
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10156902 |
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60359843 |
Feb 25, 2002 |
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60293566 |
May 24, 2001 |
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Current U.S.
Class: |
435/252.1 |
Current CPC
Class: |
Y02A 90/10 20180101;
Y02A 90/26 20180101; C12N 15/70 20130101; G01N 33/543 20130101;
G01N 33/60 20130101; C12N 15/74 20130101; G01N 33/5432 20130101;
C12P 21/02 20130101; C40B 40/02 20130101; C07K 16/00 20130101; C12N
15/1037 20130101; G01N 33/5005 20130101; C12N 15/00 20130101 |
Class at
Publication: |
435/252.1 |
International
Class: |
C12N 1/20 20060101
C12N001/20 |
Claims
1. A fully intact eubacterial minicell derived from a eubacterial
parent cell, wherein the minicell comprises a biologically active
compound and displays an antibody or antibody derivative, wherein
the biologically active compound is selected from the group
consisting of a polypeptide, a small molecule, and a nucleic acid,
and wherein the biologically active compound and the antibody or
antibody derivative are exogenous to the parent cell and distinct
from each other.
2. The minicell of claim 1, wherein the biologically active
compound is a polypeptide.
3. The minicell of claim 2, wherein the polypeptide is a fusion
protein.
4. The minicell of claim 2, wherein the polypeptide is a soluble
polypeptide.
5. The minicell of claim 2, wherein the polypeptide is a protein
toxin.
6. The minicell of claim 1, wherein the biologically active
compound is a small molecule.
7. The minicell of claim 6, wherein the small molecule is a drug
molecule.
8. The minicell of claim 1, wherein the biologically active
compound is a nucleic acid.
9. The minicell of claim 8, wherein the nucleic acid comprises an
antisense nucleic acid.
10. The minicell of claim 8, wherein the nucleic acid comprises a
ribozyme.
11. The minicell of claim 8, wherein the nucleic acid comprises an
aptamer.
12. The minicell of claim 1, wherein the minicell displays an
antibody.
13. The minicell of claim 1, wherein the minicell displays an
antibody derivative.
14. A fully intact eubacterial minicell derived from a eubacterial
parent cell, wherein the minicell comprises a biologically active
compound and displays an antibody or antibody derivative, wherein
the biologically active compound is a therapeutic polypeptide, and
wherein the biologically active compound and the antibody or
antibody derivative are exogenous to the parent cell and distinct
from each other.
15. The minicell of claim 14, wherein the therapeutic polypeptide
is a soluble polypeptide.
16. The minicell of claim 14, wherein the therapeutic polypeptide
is a protein toxin.
17. A fully intact eubacterial minicell derived from a eubacterial
parent cell, wherein the minicell comprises a biologically active
compound and displays an antibody or antibody derivative, wherein
the biologically active compound is a therapeutic nucleic acid, and
wherein the biologically active compound and the antibody or
antibody derivative are exogenous to the parent cell and distinct
from each other.
18. The minicell of claim 17, wherein the therapeutic nucleic acid
comprises an antisense nucleic acid.
19. The minicell of claim 17, wherein the therapeutic nucleic acid
comprises a ribozyme.
20. The minicell of claim 17, wherein the therapeutic nucleic acid
comprises an aptamer.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 11/725,196, filed Mar. 16, 2007, which is a divisional of U.S.
application Ser. No. 11/580,095, filed Oct. 11, 2006, now U.S. Pat.
No. 7,871,815, which is a divisional of U.S. application Ser. No.
10/156,902, filed May 28, 2002, now U.S. Pat. No. 7,183,105, which
is a divisional of U.S. application Ser. No. 10/154,951, filed May
24, 2002, which claims the benefit of U.S. Provisional Application
Nos. 60/359,843, filed Feb. 25, 2002, and 60/293,566, filed May 24,
2001, all of which are incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The invention is drawn to compositions and methods for the
production of achromosomal archeabacterial, eubacterial and
anucleate eukaryotic cells that are used as, e.g., therapeutics
and/or diagnostics, reagents in drug discovery and functional
proteomics, research tools, and in other applications as well.
BACKGROUND OF THE INVENTION
[0003] The following description of the background of the invention
is provided to aid in understanding the invention, but is not
admitted to describe or constitute prior art to the invention. The
contents of the articles, patents, and patent applications, and all
other documents and electronically available information mentioned
or cited in this application, are hereby incorporated by reference
in their entirety to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference. Applicants reserve the right to
physically incorporate into this application any and all materials
and information from any such articles, patents, patent
applications, or other documents.
[0004] Minicells are achromosomal cells that are products of
aberrant cell division, and contain RNA and protein, but little or
no chromosomal DNA. Clark-Curtiss and Curtiss III, Analysis of
Recombinant DNA Using Escherichia coli Minicells, 101 Methods in
Enzymology 347 (1983); Reeve and Mendelson, Minicells of Bacillus
subtilis. A new system for transport studies in absence of
macromolecular biosynthesis, 352 Biochim. Biophys. Acta 298-305
(1974). Minicells are capable of plasmid-directed synthesis of
discrete polypeptides in the absence of synthesis directed by mRNA
from the bacterial chromosome. Meagher et al., Protein Expression
in E. coli Minicells by Recombinant Plasmids, 10 Cell 521, 523
(1977); Roozen et al., Synthesis of Ribonucleic Acid and Protein in
Plasmid-Containing Minicells of Escherichia coli K-12, 107(1) J. of
Bacteriology 21 (1971); and Curtiss III, Research on bacterial
conjugation with minicells and minicell-producing E. coli strains,
In: Microbial Drug Resistance, Editors Susumu Mitsuhashi &
Hajime Hashimoto, p. 169 (Baltimore: University Park Press 1976).
Early descriptions of minicells include those of Adler et al.,
Genetic control of cell division in bacteria, 154 Science 417
(1966), and Adler et al. (Miniature Escherichia coli cells
deficient in DNA, 57 Proc. Nat. Acad. Sci. (Wash.) 321 (1967)).
However, discovery of the production of minicells can arguably be
traced to the 1930's (Frazer and Curtiss III, Production,
Properties and Utility of Bacterial Minicells, 69 Curr. Top.
Microbiol. Immunol. 1-3 (1975)).
[0005] Prokaryotic (a.k.a. eubacterial) minicells have been used to
produce various eubacterial proteins. See, e.g., Michael Gadel, et
al., The kdpF Subunit Is Part of the K.sup.+-translocating Kdp
Complex of Escherichia coli and Is Responsible for Stabilization of
the Complex in vitro, 274(53) Jn. of Biological Chemistry 37901
(1999); Harlow, et al., Cloning and Characterization of the gsk
Gene Encoding Guanosine Kinase of Escherichia coli, 177(8) J. of
Bacteriology 2236 (1995); Carol L. Pickett, et al., Cloning,
Sequencing, and Expression of the Escherichia coli Cytolethal
Distinding Toxin Genes, 62(3) Infection & Immunity 1046 (1994);
Raimund Eck & Jorn Belter, Cloning and characterization of a
gene coding for the catechol 1,2 dioxygenase of Arthrobacter sp.
mA3, 123 Gene 87 (1993); Andreas Schlossser, et al, Subcloning,
Nucleotide Sequence, and Expression of trkG, a Gene That Encodes an
Integral Membrane Protein Involved in Potassium Uptake via the Trk
System of Escherichia coli, 173(10) J. of Bacteriology 3170 (1991);
Mehrdad Jannatipour, et al., Translocation of Vibrio harveyi
N,N'-Diacetylchitobiase to the Outer Membrane of Escherichia coli
169(8) J. of Bacteriology 3785 (1987); and Jacobs et al.,
Expression of Mycobacterium leprae genes from a Streptococcus
mutans promoter in Escherichia coli K-12, 83(6) Proc. Natl. Acad.
Sci. USA 1926 (1986);
[0006] Various bacteria have been used, or proposed to be used, as
gene delivery vectors to mammalian cells. For reviews, see
Grillot-Courvalin et al., Bacteria as gene delivery vectors for
mammalian cells, 10 Current Opinion in Biotechnology 477 (1999);
Johnsen et al., Transfer of DNA from Genetically Modified Organisms
(GMOs), Biotechnological Institute, 1-70 (2000); Sizemore et al.,
Attenuated Shigella as a DNA delivery vehicle for DNA-mediated
immunization, 270(5234) Science 299 (1995); Patrice Courvalin, et
al., Gene transfer from bacteria to mammalian cells, 318 C. R.
Acad. Sci. 1207 (1995); Sizemore, et al. Attenuated bacteria as a
DNA delivery vehicle for DNA-mediated immunization, 15(8) Vaccine
804 (1997).
[0007] U.S. Pat. No. 4,190,495, which issued Feb. 26, 1980, to
Curtiss is drawn to minicell producing strains of E. coli that are
stated to be useful for the recombinant expression of proteins.
[0008] U.S. Pat. No. 4,311,797, which issued Jan. 19, 1982 to
Khachatourians is stated to be drawn to a minicell based vaccine.
The vaccine is stated to induce the production of antibodies
against enteropathogenic E. coli cells in cattle and is stated to
be effective against coliform enteritis.
[0009] Eubacterial minicells expressing immunogens from other
prokaryotes have been described. Purcell et al., Molecular cloning
and characterization of the 15-kilodalton major immunogen of
Treponema pallidum, Infect. Immun. 57:3708, 1989.
[0010] In "Biotechnology: Promise . . . and Peril" (IDRC Reports
9:4-7, 1980) authors Fleury and Shirkie aver that George
Khachatourians at the University of Saskatchewan, Canada, "is
working on a vaccine against cholera using `minicells.`" The
minicells are said to contain "genes from the pathogenic agent,"
and the "pathogen antigens are carried on the surface of the
minicells" (p. 5, paragraph bridging the central and right
columns).
[0011] Lundstrom et al., Secretion of Semliki Forest virus membrane
glycoprotein E1 from Bacillus subtilis, Virus Res. 2:69-83, 1985,
describe the expression of the E1 protein of the eukaryotic virus,
Semliki Forest virus (SFV), in Bacillus minicells. The SFV E1
protein used in these studies is not the native E1 protein. Rather,
it is a fusion protein in which the N-terminal signal sequence and
C-terminal transmembrane domain have been removed and replaced with
signal sequences from a gene from Bacillus amyloliquefaciens. The
authors aver that "E1 is properly translocated through the cell
membrane and secreted" (p. 81, 1.1. 19-20), and note that "it has
been difficult to express viral membrane proteins in prokaryotes"
(p. 81, 1. 27).
[0012] U.S. Pat. No. 4,237,224, which issued Dec. 2, 1980, to Cohen
and Boyer, describes the expression of X. Laevis DNA in E. coli
minicells.
[0013] U.S. patent application Ser. No. 60/293,566 (attorney docket
Nos. 078853-0401 and 089608-0201), is entitled "Minicell
Compositions and Methods," and was filed May 24, 2001, by
Sabbadini, Roger A., Berkley, Neil L., and Klepper, Robert E., and
is hereby incorporated in its entirety by reference.
[0014] Jespersen et al. describes the use of "proteoliposomes" to
generate antibodies to the AMPA receptor. Jespersen L K, Kuusinen
A, Orellana A, Keinanen K, Engberg J. Use of proteoliposomes to
generate phage antibodies against native AMPA receptor. Eur J.
Biochem. 2000 March; 267(5): 1382-9
SUMMARY OF THE INVENTION
[0015] The invention is drawn to compositions and methods for the
production and use of minicells, including but not limited to
eubacterial minicells, in applications such as diagnostics,
therapeutics, research, compound screening and drug discovery, as
well as agents for the delivery of nucleic acids and other
bioactive compounds to cells.
[0016] Minicells are derivatives of cells that lack chromosomal DNA
and which are sometimes referred to as anucleate cells. Because
eubacterial and archeabacterial cells, unlike eukaryotic cells, do
not have a nucleus (a distinct organelle that contains
chromosomes), these non-eukaryotic minicells are more accurately
described as being "without chromosomes" or "achromosomal," as
opposed to "anucleate." Nonetheless, those skilled in the art often
use the term "anucleate" when referring to bacterial minicells in
addition to other minicells. Accordingly, in the present
disclosure, the term "minicells" encompasses derivatives of
eubacterial cells that lack a chromosome; derivatives of
archeabacterial cells that lack their chromosome(s), and anucleate
derivatives of eukaryotic cells. It is understood, however, that
some of the relevant art may use the terms "anucleate minicells" or
anucleate cells" loosely to refer to any of the preceeding types of
minicells.
[0017] In one aspect, the invention is drawn to a eubacterial
minicell comprising a membrane protein that is not naturally found
in a prokaryote, i.e., a membrane protein from a eukaryote or an
archeabacterium. Such minicells may, but need not, comprise an
expression element that encodes and expresses the membrane protein
that it comprises. The membrane protein may be one found in any
non-eubacterial membrane, including, by way of non-limiting
example, a cellular membrane, a nuclear membrane, a nucleolar
membrane, a membrane of the endoplasmic reticulum (ER), a membrane
of a Golgi body, a membrane of a lysosome a membrane of a
peroxisome, a caveolar membrane, an outer membrane of a
mitochondrion or a chloroplast, and an inner membrane of a
mitochondrion or a chloroplast. By way of non-limiting example, a
membrane protein may be a receptor, such as a G-protein coupled
receptor; an enzyme, such as ATPase or adenylate cyclase, a
cytochrome; a channel; a transporter; or a membrane-bound nucleic
acid binding factor, such as a transcription and/or translation
factor; signaling components; components of the electron transport
chain (ETC); or cellular antigens. A membrane fusion protein, which
is generated in vitro using molecular cloning techniques, does not
occur in nature and is thus a membrane protein that is not
naturally found in a prokaryote, even if the fusion protein is
prepared using amino acid sequences derived from eubacterial
proteins.
[0018] Minicells that have segregated from parent cells lack
chromosomal and/or nuclear components, but retain the cytoplasm and
its contents, including the cellular machinery required for protein
expression. Although chromosomes do not segregate into minicells,
extrachromosomal and/or episomal genetic expression elements will
segregate, or may be introduced into minicells after segregation
from parent cells. Thus, in one aspect, the invention is drawn to
minicells comprising an expression element, which may be an
inducible expression element, that comprises expression sequences
operably linked to an open reading frame (ORF) that encodes the
non-eubacterial membrane protein. In a related aspect, the
invention is drawn to minicell-producing host cells having an
expression element, which may be an inducible expression element,
that comprises expression sequences operably linked to an ORF that
encodes a non-eubacterial membrane protein. In a related aspect,
the invention is drawn to a method of making a eubacterial minicell
comprising a membrane protein that is not naturally found in a
prokaryote, the method comprising growing minicell-producing host
cells, the host cells having an expression element, which may be an
inducible expression element, that comprises expression sequences
operably linked to an ORF that encodes a non-eubacterial membrane
protein; and preparing minicells from the host cells. Optionally,
at any point in the method, an inducing agent is provided in order
to induce expression of an ORF that encodes a non-eubacterial
membrane protein.
[0019] In one aspect, the invention is drawn to display produced
membrane-associated protein(s) on the surface of the minicell. For
purposes of this document, the term "display" is defined as
exposure of the structure of interest on the outer surface of the
minicell. By way of non-limiting example, this structure may be an
internally expressed membrane protein or chimeric construct to be
inserted in or associated with the minicell membrane such that the
extracellular domain or domain of interest is exposed on the outer
surface of the minicell (expressed and displayed on the surface of
the minicell or expressed in the parental cell to be displayed on
the surface of the segregated minicell). In any scenario, the
"displayed" protein or protein domain is available for interaction
with extracellular components. A membrane-associated protein may
have more than one extracellular domain, and a minicell of the
invention may display more than one membrane-associated
protein.
[0020] A membrane protein displayed by eubacterial minicells may be
a receptor. Receptors include, by way of non-limiting example,
G-coupled protein receptors, hormone receptors, and growth factor
receptors. Minicells displaying a receptor may, but need not, bind
ligands of the receptor. In therapeutic applications of this aspect
of the invention, the ligand is an undesirable compound that is
bound to its receptor and, in some aspects, is internalized or
inactivated by the minicells. In drug discovery applications of
this aspect of the invention, the ligand for the receptor may be
detectably labeled so that its binding to its receptor may be
quantified. In the latter circumstance, the minicells may be used
to identify and isolate, from a pool of compounds, one or more
compounds that inhibit or stimulate the activity of the receptor.
That is, these minicells can be used in screening assays, including
assays such as those used in high throughput screening (HTS)
systems and other drug discovery methods, for the purpose of
preparing compounds that influence the activity of a receptor of
interest.
[0021] The displayed domain of a membrane protein may be an
enzymatic domain such as on having oxidoreductase, transferase,
hydrolase, lyase, isomerase ligase, lipase, kinase, phosphatase,
protease, nuclease and/or synthetase activity. Contacting such
minicells with the appropriate substrate of the enzyme allows the
substrate to be converted to reactant. When either the substrate or
reactant is detectable, the reaction catalyzed by the
membrane-bound enzyme may be quantified. In the latter instance,
the minicells may be used to identify and isolate, from a pool of
compounds, one or more compounds that inhibit or stimulate the
activity of the enzyme represented by the displayed enzymatic
moiety. That is, these minicells can be used in screening assays,
including assays such as those used in high throughput screening
(HTS) systems and other drug discovery methods, for the purpose of
preparing compounds that influence the activity of an enzyme or
enzymatic moiety of interest.
[0022] The membrane protein displayed by minicells may be a fusion
protein, i.e., a protein that comprises a first polypeptide having
a first amino acid sequence and a second polypeptide having a
second amino acid sequence, wherein the first and second amino acid
sequences are not naturally present in the same polypeptide. At
least one polypeptide in a membrane fusion protein is a
"transmembrane domain" or "membrane-anchoring domain". The
transmembrane and membrane-anchoring domains of a membrane fusion
protein may be selected from membrane proteins that naturally occur
in a eucaryote, such as a fungus, a unicellular eucaryote, a plant
and an animal, such as a mammal including a human. Such domains may
be from a viral membrane protein naturally found in a virus such as
a bacteriophage or a eucaryotic virus, e.g., an adenovirus or a
retrovirus. Such domains may be from a membrane protein naturally
found in an archaebacterium such as a thermophile.
[0023] The displayed domain of a membrane fusion protein may be an
enzymatic domain such as one having oxidoreductase, transferase,
hydrolase, lyase, isomerase ligase, lipase, kinase, phosphatase,
protease, nuclease and/or synthetase activity. Contacting such
minicells with the appropriate substrate of the enzyme allows the
substrate to be converted to reactant. When either the substrate or
reactant is detectable, the reaction catalyzed by the
membrane-bound enzyme may be quantified. In the latter instance,
the minicells may be used to identify and isolate, from a pool of
compounds, one or more compounds that inhibit or stimulate the
activity of the enzyme represented by the displayed enzymatic
moiety. That is, these minicells can be used in screening assays,
including assays such as those used in high throughput screening
(HTS) systems and other drug discovery methods, for the purpose of
preparing compounds that influence the activity of an enzyme or
enzymatic moiety of interest.
[0024] The displayed domain of a membrane fusion protein may be a
binding moiety. By way of non-limiting example, binding moieties
used for particular purposes may be a binding moiety directed to a
compound or moiety displayed by a specific cell type or cells found
predominantly in one type of tissue, which may be used to target
minicells and their contents to specific cell types or tissues; or
a binding moiety that is directed to a compound or moiety displayed
by a pathogen, which may be used in diagnostic or therapeutic
methods; a binding moiety that is directed to an undesirable
compound, such as a toxin, which may be used to bind and preferably
internalize and/or neutralize the undesirable compound; a diseased
cell; or the binding moiety may be a domain that allows for the
minicells to be covalently or non-covalently attached to a support
material, which may be used in compositions and methods for
compound screening and drug discovery. By "diseased cell" it is
meant pathogen-infected cells, malfunctioning cells, and
dysfunctional cells, e.g., cancer cells.
[0025] In various aspects, the minicells of the invention comprise
one or more biologically active compounds. The term "biologically
active" (synonymous with "bioactive") indicates that a composition
or compound itself has a biological effect, or that it modifies,
causes, promotes, enhances, blocks, reduces, limits the production
or activity of, or reacts with or binds to an endogenous molecule
that has a biological effect. A "biological effect" may be but is
not limited to one that stimulates or causes an immunoreactive
response; one that impacts a biological process in an animal; one
that impacts a biological process in a pathogen or parasite; one
that generates or causes to be generated a detectable signal; and
the like. Biologically active compositions, complexes or compounds
may be used in therapeutic, prophylactic and diagnostic methods and
compositions. Biologically active compositions, complexes or
compounds act to cause or stimulate a desired effect upon an
animal. Non-limiting examples of desired effects include, for
example, preventing, treating or curing a disease or condition in
an animal suffering therefrom; limiting the growth of or killing a
pathogen in an animal infected thereby; augmenting the phenotype or
genotype of an animal; stimulating a prophylactic immunoreactive
response in an animal; or diagnosing a disease or disorder in an
animal.
[0026] In the context of therapeutic applications of the invention,
the term "biologically active" indicates that the composition,
complex or compound has an activity that impacts an animal
suffering from a disease or disorder in a positive sense and/or
impacts a pathogen or parasite in a negative sense. Thus, a
biologically active composition, complex or compound may cause or
promote a biological or biochemical activity within an animal that
is detrimental to the growth and/or maintenance of a pathogen or
parasite; or of cells, tissues or organs of an animal that have
abnormal growth or biochemical characteristics, such as cancer
cells.
[0027] In the context of diagnostic applications of the invention,
the term "biologically active" indicates that the composition,
complex or compound can be used for in vivo or ex vivo diagnostic
methods and in diagnostic compositions and kits. For diagnostic
purposes, a preferred biologically active composition or compound
is one that can be detected, typically (but not necessarily) by
virtue of comprising a detectable polypeptide. Antibodies to an
epitope found on composition or compound may also be used for its
detection.
[0028] In the context of prophylactic applications of the
invention, the term "biologically active" indicates that the
composition or compound induces or stimulates an immunoreactive
response. In some preferred embodiments, the immunoreactive
response is designed to be prophylactic, i.e., prevents infection
by a pathogen. In other preferred embodiments, the immunoreactive
response is designed to cause the immune system of an animal to
react to the detriment of cells of an animal, such as cancer cells,
that have abnormal growth or biochemical characteristics. In this
application of the invention, compositions, complexes or compounds
comprising antigens are formulated as a vaccine.
[0029] It will be understood by those skilled in the art that a
given composition, complex or compound may be biologically active
in therapeutic, diagnostic and prophylactic applications. A
composition, complex or compound that is described as being
"biologically active in a cell" is one that has biological activity
in vitro (i.e., in a cell culture) or in vivo (i.e., in the cells
of an animal). A "biologically active component" of a composition
or compound is a portion thereof that is biologically active once
it is liberated from the composition or compound. It should be
noted, however, that such a component may also be biologically
active in the context of the composition or compound.
[0030] In one aspect, the minicells of the invention comprise a
therapeutic agent. Such minicells may be used to deliver
therapeutic agents. In a preferred embodiment, a minicell
comprising a therapeutic agent displays a binding moiety that
specifically binds a ligand present on the surface of a cell, so
that the minicells may be "targeted" to the cell. The therapeutic
agent may be any type of compound or moiety, including without
limitation small molecules, polypeptides, antibodies and antibody
derivatives and nucleic acids. The therapeutic agent may be a drug;
a prodrug, i.e., a compound that becomes biologically active in
vivo after being introduced into a subject in need of treatment; or
an immunogen.
[0031] In one aspect, the minicells of the invention comprise a
detectable compound or moiety. As is understood by those of skill
in the art, a compound or moiety that is "detectable" produces a
signal that can detected by spectroscopic, photochemical,
biochemical, immunochemical, electromagnetic, radiochemical, or
chemical means such as fluorescence, chemifluoresence, or
chemiluminescence, electrochemilumenscence, or any other
appropriate means. A detectable compound may be a detectable
polypeptide, and such polypeptides may, but need not, be
incorporated into fusion membrane proteins of the minicell.
Detectable polypeptides or amino acid sequences, includes, by way
of non-limiting example, a green fluorescent protein (GFP), a
luciferase, a beta-galactosidase, a His tag, an epitope, or a
biotin-binding protein such as streptavidin or avidin. The
detectable compound or moiety may be a radiolabeled compound or a
radioisotope. A detectable compound or moiety may be a small
molecule such as, by way of non-limiting example, a fluorescent
dye; a radioactive isotope; or a compound that may be detected by
x-rays or electromagnetic radiation. Image enhancers as those used
for CAT and PET scans (e.g., calcium, gallidium) may be used. In
another non-limiting example, detectable labels may also include
loss of catalytic substrate or gain of catalytic product following
catalysis by a minicell displayed, solule cytoplasmic, or secreted
enzyme.
[0032] In one aspect, the invention is drawn to a minicell
comprising one or more bioactive nucleic acids or templates
thereof. By way of non-limiting example, a bioactive nucleic acid
may be an antisense oligonucleotide, an aptamer, an antisense
transcript, a ribosomal RNA (rRNA), a transfer RNA (tRNA), a
molecular decoy, or an enzymatically active nucleic acid, such as a
ribozyme. Such minicells can, but need not, comprise a displayed
polypeptide or protein on the surface of the minicell. The
displayed polypeptide or protein may be a binding moiety directed
to a compound or moiety displayed by a particular type of cell, or
to a compound or moiety displayed by a pathogen. Such minicells can
further, but need not, comprise an expression element having
eubacterial, archael, eucaryotic, or viral expression sequences
operably linked to a nucleotide sequence that serves as a template
for a bioactive nucleic acid.
[0033] In one aspect, the invention is drawn to immunogenic
minicells, i.e., minicells that display an immunogen, vaccines
comprising immunogenic minicells, antibodies and antibody
derivatives directed to immunogens displayed on immunogenic
minicells, and method of making and using immunogenic minicells and
antibodies and antibody derivatives produced therefrom in
prophylactic, diagnostic, therapeutic and research applications. A
preferred immunogen displayed by a minicell is an immunogenic
polypeptide, which is preferably expressed from an expression
element contained within the minicell in order to maximize the
amount of immunogen displayed by the immunogenic minicells. The
immunogenic polypeptide can be derived from any organism, obligate
intracelluar parasite, organelle or virus with the provisio that,
in prophylactic applications, the immunogenic polypeptide is not
derived from a prokaryote, including a eubacterial virus. The
source organism for the immunogen may be a pathogen. A minicell
displaying an immunogen derived from a pathogen is formulated into
a vaccine and, in a prophylactic application, used to treat or
prevent diseases and disorders caused by or related to the
eukaryotic or archeabacterial pathogen.
[0034] In a separate aspect, the invention is drawn to minicells
that display an immunogen derived from a nonfunctional,
dysfunctional and/or diseased cell. By way of non-limiting example,
the minicells display an immunogenic polypeptide derived from a
hyperproliferative cell, i.e., a cell that is tumorigenic, or part
of a tumor or cancer. As another non-limiting example, a cell that
is infected with a virus or an obligate intracellular parasite
(e.g., Rickettsiae) displays an immunogenic polypeptide that is
encoded by the genome of the infected cell but is aberrantly
expressed in an infected cell. A vaccine comprising a minicell
displaying an immunogen derived from a nonfunctional, dysfunctional
and/or diseased cell is used in methods of treating or preventing
hyperproliferative diseases or disorders, including without
limitation a cell comprising an intracellular pathogen.
[0035] In one aspect, the invention is drawn to methods of using
minicells, and expression systems optimized therefore, to
manufacture, on a large scale, proteins using recombinant DNA
technology. In a related aspect, the invention is drawn to the
production, via recombinant DNA technology, and/or segration of
exogenous proteins in minicells. The minicells are enriched for the
exogenous protein, which is desirable for increased yield and
purity of the protein. In addition to protein purification, the
minicells can be used for crystallography, the study of
intracellular or extracellular protein-protein interactions, the
study of intracellular or extracellular protein-nucleic acid
interactions, the study of intracellular or extracellular
protein-membrane interactions, and the study of other biological,
chemical, or physiological event(s).
[0036] In one aspect, the invention is drawn to minicells having a
membrane protein that has an intracellular domain. By way of
non-limiting example, the intracellular domain is exposed on the
inner surface of the minicell membrane oriented towards the
cytoplasmic compartment. The intracellular protein domain is
available for interaction with intracellular components.
Intracellular components may be naturally present in the minicells
or their parent cells, or may be introduced into minicells after
segregation from parent cells. A membrane-associated protein may
have more than one intracellular domain, and a minicell of the
invention may display more than one membrane-associated
protein.
[0037] In one aspect, the invention is drawn to a minicell
comprising a membrane protein that is linked to a conjugatable
compound (a.k.a. "attachable compound"). The conjugatable compound
may be of any chemical nature and have one or more therapeutic or
detectable moities. By way of non-limiting example, a protein
having a transmembrane or membrane anchoring domain is displayed
and has the capacity to be specifically cross-linked on its
extracellular domain. Through this approach, any conjugatable
compound of interest may be quickly and easily attached to the
outer surface of minicells containing this expressed
membrane-spanning domain. In aspects of the invention wherein
minicells are used for drug delivery in vivo, a preferred
conjugatable compound is polyethylene glycol (PEG), which provides
for "stealth" minicells that are not taken as well and/or as
quickly by the reticuloendothelial system (RES). Other conjugatable
compounds include polysaccharides, polynucleotides,
lipopolysaccharides, lipoproteins, glycosylated proteins, synthetic
chemical compounds, and/or chimeric combinations of these examples
listed.
[0038] In various aspects of the invention, the minicell displays a
polypeptide or other compound or moiety on its surface. By way of
non-limiting example, a non-eubacterial membrane protein displayed
by eubacterial minicells may be a receptor. Minicells displaying a
receptor may, but need not, bind ligands of the receptor. In
therapeutic applications of this aspect of the invention, the
ligand is an undesirable compound that is bound to its receptor
and, in some aspects, is internalized by the minicells. In drug
discovery applications of this aspect of the invention, the ligand
for the receptor may be detectably labeled so that its binding to
its receptor may be quantified. In the latter circumstance, the
minicells may be used to identify and isolate, from a pool of
compounds, one or more compounds that inhibit or stimulate the
activity of the receptor. That is, these minicells can be used in
screening assays, including assays such as those used in high
throughput screening (HTS) systems and other drug discovery
methods, for the purpose of preparing compounds that influence the
activity of a receptor of interest.
[0039] The non-eubacterial membrane protein displayed by minicells
may be a fusion protein, i.e., a protein that comprises a first
polypeptide having a first amino acid sequence and a second
polypeptide having a second amino acid sequence, wherein the first
and second amino acid sequences are not naturally present in the
same polypeptide. At least one polypeptide in a membrane fusion
protein is a "transmembrane domain" or "membrane-anchoring domain".
The transmembrane and membrane-anchoring domains of a membrane
fusion protein may be selected from membrane proteins that
naturally occur in a eukaryote, such as a fungus, a unicellular
eukaryote, a plant and an animal, such as a mammal including a
human. Such domains may be from a viral membrane protein naturally
found in a virus such as a bacteriophage or a eukaryotic virus,
e.g., an adenovirus or a retrovirus. Such domains may be from a
membrane protein naturally found in an archaebacterium such as a
thermophile.
[0040] The displayed domain of a membrane fusion protein may be an
enzymatic domain such as one having the activity of a lipase, a
kinase, a phosphatase, a reductase, a protease, or a nuclease.
Contacting such minicells with the appropriate substrate of the
enzyme allows the substrate to be converted to reactant. When
either the substrate or reactant is detectable, the reaction
catalyzed by the membrane-bound enzyme may be quantified. In the
latter instance, the minicells may be used to identify and isolate,
from a pool of compounds, one or more compounds that inhibit or
stimulate the activity of the enzyme represented by the displayed
enzymatic moiety. That is, these minicells can be used in screening
assays, including assays such as those used in high throughput
screening (HTS) systems and other drug discovery methods, for the
purpose of preparing compounds that influence the activity of an
enzyme or enzymatic moiety of interest.
[0041] The displayed domain of a membrane fusion protein may be a
binding moiety. By way of non-limiting example, binding moieties
used for particular purposes may be a binding moiety directed to a
compound or moiety displayed by a specific cell type or cells found
predominantly in one type of tissue, which may be used to target
minicells and their contents to specific cell types or tissues; or
a binding moiety that is directed to a compound or moiety displayed
by a pathogen, which may be used in diagnostic or therapeutic
methods; a binding moiety that is directed to an undesirable
compound, such as a toxin, which may be used to bind and preferably
internalize and/or neutralize the undesirable compound; a diseased
cell; or the binding moiety may be a domain that allows for the
minicells to be covalently or non-covalently attached to a support
material, which may be used in compositions and methods for
compound screening and drug discovery.
[0042] In one aspect, the invention provides compositions and
methods for preparing a soluble and/or secreted protein where the
protein remains in the cytoplasm of the minicell or is secreted
following native secretory pathways for endogenous secreted
proteins or is secreted using chimeric fusion to secretory
signaling sequences. By way of non-limiting example, secreted or
cytoplasmic soluble proteins may be produced for purification,
targeted therapeutic applications where the protein produced is a
therapeutic agent and is produced at the desired site of, detection
for screening or diagnostic purposes where the protein is produced
in response to a simulous and/or localization event, or to
stimulate targeted minicell-cell fusion or interaction events where
the protein produced stimulates cell-cell fusion upon targeted
stimulation.
[0043] In one aspect, the invention provides compositions and
methods for preparing antibodies and/or antibody derivatives that
recognize an immunogenic epitope present on the native form of a
membrane protein, but which is not immunogenic when the membrane
protein is denatured or when prepared as a synthetic oligopeptide.
Such antibodies and antibody derivatives are said to be
"conformation sensitive." Unlike most antibodies and antibody
derivatives prepared by using a denatured membrane protein or an
oligopeptide derived from the membrane protein, conformation
sensitive antibodies and antibody derivatives specifically bind
membrane proteins in their native state (i.e., in a membrane) with
high affinity. Conformation sensitive antibodies and antibody
derivatives are used to target compounds and compositions,
including a minicell of the invention, to a cell displaying the
membrane protein of choice. Conformation sensitive antibodies and
antibody derivatives are also used to prevent receptors from
binding their natural ligands by specifically binding to the
receptor with a high affinity and thereby limiting access of the
ligand to the receptor. Conformation sensitive antibodies and
antibody derivatives can be prepared that are specific for a
specific isoform or mutant of a membrane protein, which can be
useful in research and medical applications.
[0044] In one aspect, the invention provides biosensors comprising
minicells including, not limited to, the minicells of the
invention. An exemplary biosensor of the invention is a BIAcore
chip, i.e., a chip onto which minicells are attached, where the
minicells undergo some change upon exposure to a preselected
compound, and the change is detected using surface plasmon
resonance. A biosensor comprising minicells can be used in methods
of detecting the presence of an undesirable compound. Undesirable
compounds include but are not limited to, toxins; pollutants;
explosives, such as those in landmines or illegally present;
illegal narcotics; components of biological or chemical weapons. In
a related aspect, the invention provides a device comprising a
microchip operatively associated with a biosensor comprising a
minicell. The device can further comprise an actuator that performs
a responsive function when the sensor detects a preselected level
of a marker.
[0045] In one aspect, the invention provides minicells that may be
used as research tools and/or kits comprising such research tools.
The minicells of the invention may be used as is, or incorporated
into research tools useful for scientific research regarding all
amino acid comprising compounds including, but not limited to
membrane-associated proteins, chimeric membrane fusion proteins,
and soluble proteins. Such scientific research includes, by way of
non-limiting example, basic research, as well as pharmacological,
diagnostic, and pharmacogenetic studies. Such studies may be
carried out in vivo or in vitro.
[0046] In one aspect, the invention is drawn to archaebacterial
minicells. In a related aspect, the invention is drawn to
archaebacterial minicells comprising at least one exogenous
protein, that is, a protein that is not normally found in the
parent cell, including without limitation fusion proteins. The
archaebacterial minicells of the invention optionally comprise an
expression element that directs the production of the exogenous
protein(s).
[0047] In other aspects, the invention is drawn to methods of
preparing the minicells, protoplasts, and Poroplasts.TM. of the
invention for various applications including but not limited to
diagnostic, therapeutic, research and screening applications. In a
related aspect, the invention is drawn to pharmaceutical
compositions, reagents and kits comprising minicells.
[0048] In each aspect and embodiment of the invention, unless
stated otherwise, embodiments wherein the minicell is a eubacterial
minicell, a poroplast, a spheroplast or a protoplast exist.
[0049] In a first aspect, the invention provides a minicell
comprising a membrane protein selected from the group consisting of
a eukaryotic membrane protein, an archeabacterial membrane protein
and an organellar membrane protein. In another embodiment, wherein
the minicell comprises a biologically active compound. By way of
non-limiting example, the biologically active compound is a
radioisotope, a polypeptide, a nucleic acid or a small
molecule.
[0050] In another embodiment, the minicell comprises a expression
construct, wherein the first expression construct comprises
expression sequences operably linked to an ORF that encodes a
protein. In another embodiment, the ORF encodes the membrane
protein. In another embodiment, the expression sequences that are
operably linked to an ORF are inducible and/or repressible.
[0051] In another aspect, the minicell comprises a second
expression construct, wherein the second expression construct
comprises expression sequences operably linked to a gene. In
another embodiment, the expression sequences that are operably
linked to a gene are inducible and/or repressible. In a related
embodiment, the gene product of the gene regulates the expression
of the ORF that encodes the protein. A factor that "regulates" the
expression of a gene or a gene product directly or indirectly
initiates, enhances, quickens, slows, terminates, limits or
completely blocks expression of a gene. In different embodiments,
the gene product of the gene is a nucleic acid or a polypeptide.
The polypeptide can be of any type, including but not limited to a
membrane protein, a soluble protein or a secreted protein. A
membrane protein can be a membrane fusion protein comprising a
first polypeptide, which comprises at least one transmembrane
domain or at least one membrane anchoring domain; and a second
polypeptide.
[0052] In one aspect, the invention provides a minicell comprising
a membrane fusion protein, the fusion protein comprising a first
polypeptide, the first polypeptide comprising at least one
transmembrane domain or at least one membrane anchoring domain; and
a second polypeptide, wherein the second polypeptide is not derived
from a eubacterial protein and is neither a His tag nor a
glutathione-S-transferase polypeptide. In various embodiments, the
minicell is a eubacterial minicell, a poroplast, a spheroplast or a
protoplast. In one embodiment, the minicell comprises a
biologically active compound.
[0053] In one aspect, the invention provides a minicell comprising
a membrane conjugate, wherein the membrane conjugate comprises a
membrane protein chemically linked to a conjugated compound. In one
embodiment, the conjugated compound is selected from the group
consisting of a nucleic acid, a polypeptide, a lipid and a small
molecule.
[0054] In one aspect, the invention provides a method for making
minicells, comprising (a) culturing a minicell-producing parent
cell, wherein the parent cell comprises an expression construct,
wherein the expression construct comprises a gene operably linked
to expression sequences that are inducible and/or repressible, and
wherein induction or repression of the gene causes or enhances the
production of minicells; and (b) separating the minicells from the
parent cell, thereby generating a composition comprising minicells,
wherein an inducer or repressor is present within the parent cells
during one or more steps and/or between two or more steps of the
method. In one embodiment, the method further comprises (c)
purifying the minicells from the composition.
[0055] Relevant gene products are factors involved in or modulating
DNA replication, cellular division, cellular partitioning,
septation, transcription, translation, or protein folding. The
minicells are separated from parent cells by processes such as
centrifugation, ultracentrifugation, density gradation,
immunoaffinity, immunoprecipitation and other techniques described
herein.
[0056] In one embodiment, the minicell is a poroplast, and the
method further comprises (d) treating the minicells with an agent,
or incubating the minicells under a set of conditions, that
degrades the outer membrane of the minicell. The outer membrane is
degraded by treatment with an agent selected from the group
consisting of EDTA, EGTA, lactic acid, citric acid, gluconic acid,
tartaric acid, polyethyleneimine, polycationic peptides, cationic
leukocyte peptides, aminoglycosides, aminoglycosides, protamine,
insect cecropins, reptilian magainins, polymers of basic amino
acids, polymixin B, chloroform, nitrilotriacetic acid and sodium
hexametaphosphate; by exposure to conditions selected from the
group consisting of osmotic shock and insonation; and by other
methods described herein.
[0057] In one embodiment, further comprising removing one or more
contaminants from the composition. Representative contaminants are
LPS and peptidoglycan. In a representative embodiment, LPS is
removed by contacting the composition to an agent that binds or
degrades LPS. At least about 50%, preferably about 65% to about
75%, more preferably 95%, most preferably 99% or >99% of LPS is
removed from an initial preparation of minicells. In a related
embodiment, the minicell-producing parent cell comprises a mutation
in a gene required for lipopolysaccharide synthesis.
[0058] In on embodiment, the minicell is a spheroplast, and the
method further comprises (d) treating the minicells with an agent,
or incubating the minicells under a set of conditions, that
disrupts or degrades the outer membrane; and (e) treating the
minicells with an agent, or incubating the minicells under a set of
conditions, that disrupts or degrades the cell wall. The agent that
disrupts or degrades the cell wall can be. e.g., a lysozyme, and
the set of conditions that disrupts or degrades the cell wall can
be, e.g., incubation in a hypertonic solution.
[0059] In one embodiment, the minicell is a protoplast, and the
method further comprises (d treating the minicells with an agent,
or incubating the minicells under a set of conditions, that disrupt
or degrade the outer membrane; (e) treating the minicells with an
agent, or incubating the minicells under a set of conditions, that
disrupts or degrades the cell wall, in order to generate a
composition that comprises protoplasts; and (f) purifying
protoplasts from the composition. In one embodiment, the method
further comprises preparing a denuded minicell from the minicell.
In one embodiment, the method further comprises covalently or
non-covalently linking one or more components of the minicell to a
conjugated moiety.
[0060] In one aspect, the invention provides a L-form minicell
comprising (a) culturing an L-form eubacterium, wherein the
eubacterium comprises one or more of the following: (i) an
expression element that comprises a gene operably linked to
expression sequences that are inducible and/or repressible, wherein
induction or repression of the gene regulates the copy number of an
episomal expression construct; (ii) a mutation in an endogenous
gene, wherein the mutation regulates the copy number of an episomal
expression construct; (iii) an expression element that comprises a
gene operably linked to expression sequences that are inducible
and/or repressible, wherein induction or repression of the gene
causes or enhances the production of minicells; and (iv) a mutation
in an endogenous gene, wherein the mutation causes or enhances
minicell production; (b) culturing the L-form minicell-producing
parent cell in media under conditions wherein minicells are
produced; and (c) separating the minicells from the parent cell,
thereby generating a composition comprising L-form minicells,
wherein an inducer or repressor is present within the minicells
during one or more steps and/or between two or more steps of the
method. In one embodiment, the method further comprises (d)
purifying the L-form minicells from the composition.
[0061] In one aspect, the invention provides a method of producing
a protein, comprising (a) transforming a minicell-producing parent
cell with an expression element that comprises expression sequences
operably linked to a nucleic acid having an ORF that encodes the
protein; (b) culturing the minicell-producing parent cell under
conditions wherein minicells are produced; and (c) purifying
minicells from the parent cell, (d) purifying the protein from the
minicells, wherein the ORF is expressed during step (b), between
steps (b) and (c), and during step (c).
[0062] In one embodiment, the expression elements segregate into
the minicells, and the ORF is expressed between steps (c) and (d).
In one embodiment, the protein is a soluble protein contained
within the minicells, and the method further comprises (e) lysing
the minicells.
[0063] In one embodiment, the protein is a secreted protein, and
the method further comprises (e) collecting a composition in which
the minicells are suspended or with which the minicells are in
contact.
[0064] In one embodiment, the expression sequences to which the ORF
is operably linked are inducible, wherein the method further
comprises adding an inducing agent between steps (a) and (b);
during step (b); and between steps (b) and (c).
[0065] In one embodiment, the expression sequences to which the ORF
is operably linked are inducible, wherein the expression elements
segregate into the minicells, the method further comprises adding
an inducing agent after step (c).
[0066] In one embodiment, the method further comprises (e)
preparing poroplasts from the minicells, wherein the ORF is
expressed during step (b); between steps (b) and (c); during step
(c); between step (c) and step (d) when the expression elements
segregate into the minicells; and/or after step (d) when the
expression elements segregate into the minicells.
[0067] In one embodiment, the method further comprises (f)
purifying the protein from the poroplasts.
[0068] In one embodiment, the method further comprises (e)
preparing spheroplasts from the minicells, wherein the ORF is
expressed during step (b), between steps (b) and (c), during step
(c), between steps (c) and (d) and/or after step (d).
[0069] In one embodiment, the method further comprises (f)
purifying the protein from the spheroplasts.
[0070] In one embodiment, the method further comprises (e)
preparing protoplasts from the minicells, wherein the ORF is
expressed during step (b), between steps (b) and (c), during step
(c), between steps (c) and (d) and/or after step (d).
[0071] In one embodiment, the method further comprises (f)
purifying the protein from the protoplasts.
[0072] In one embodiment, the method further comprises (e)
preparing membrane preparations from the minicells, wherein the ORF
is expressed during step (b), between steps (b) and (c), during
step (c), between steps (c) and (d) and/or after step (d).
[0073] In one embodiment, the method further comprises (f)
purifying the protein from the membrane preparations.
[0074] In one embodiment, the minicell-producing parent cell is an
L-form bacterium.
[0075] In one aspect, the invention provides a method of producing
a protein, comprising (a) transforming a minicell with an
expression element that comprises expression sequences operably
linked to a nucleic acid having an ORF that encodes the protein;
and (b) incubating the minicells under conditions wherein the ORF
is expressed.
[0076] In one embodiment, the method further comprises (c)
purifying the protein from the minicells.
[0077] In one aspect, the invention provides a method of producing
a protein, comprising (a) transforming a minicell-producing parent
cell with an expression element that comprises expression sequences
operably linked to a nucleic acid having an ORF that encodes a
fusion protein comprising the protein and a polypeptide, wherein a
protease-sensitive amino acid sequence is positioned between the
protein and the polypeptide; (b) culturing the minicell-producing
parent cell under conditions wherein minicells are produced; (c)
purifying minicells from the parent cell, wherein the ORF is
expressed during step (b); between steps (b) and (c); and/or after
step (c) when the expression elements segregate into the minicells;
and (d) treating the minicells with a protease that cleaves the
sensitive amino acid sequence, thereby separating the protein from
the polypeptide.
[0078] In one aspect, the invention provides a poroplast, the
poroplast comprising a vesicle, bonded by a membrane, wherein the
membrane is an eubacterial inner membrane, wherein the vesicle is
surrounded by a eubacterial cell wall, and wherein the eubacterial
inner membrane is accessible to a compound in solution with the
poroplast. In one embodiment, the poroplast is a cellular
poroplast. The compound has a molecular weight of at least 1 kD,
preferably at least about 0.1 to about 1 kD, more preferably from
about 1, 10 or 25 kD to about 50 kD, and most preferably from about
75 or about 100 kD to about 150 or 300 kD.
[0079] In one embodiment, the poroplast comprises an exogenous
nucleic acid, which may be an expression construct. In one
embodiment, the expression construct comprises an ORF that encodes
an exogenous protein, wherein the ORF is operably linked to
expression sequences. In one embodiment, the exogenous protein is a
fusion protein, a soluble protein or a secreted protein. In one
embodiment, the exogenous protein is a membrane protein, and is
preferably accessible to compounds in solution with the poroplast.
In one embodiment, poroplasts are placed in a hypertonic solution,
wherein 90% or more of an equivalent amount of spheroplasts or
protoplasts lyse in the solution under the same conditions.
[0080] In one embodiment, the membrane protein is selected from the
group consisting of a eukaryotic membrane protein, an
archeabacterial membrane protein, and an organellar membrane
protein. In one embodiment, the membrane protein is a fusion
protein, the fusion protein comprising a first polypeptide, the
first polypeptide comprising at least one transmembrane domain or
at least one membrane anchoring domain; and a second polypeptide,
wherein the second polypeptide is displayed by the poroplast. In
one embodiment, the second polypeptide is displayed on the external
side of the eubacterial inner membrane. The second polypeptide can
be an enzyme moiety, a binding moiety, a toxin, a cellular uptake
sequence, an epitope, a detectable polypeptide, and a polypeptide
comprising a conjugatable moiety. An enzyme moiety is a polypeptide
derived from, by way of non-limiting example, a cytochrome P450, an
oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase,
a ligase or a synthetase.
[0081] In one embodiment, the poroplast comprises a membrane
component that is chemically linked to a conjugated compound.
[0082] In one embodiment, the expression construct comprises one or
more DNA fragments from a genome or cDNA. In one embodiment, the
exogenous protein has a primary amino acid sequence predicted from
a nucleic acid sequence.
[0083] In one aspect, the invention provides a solid support
comprising a minicell. In various embodiments, the solid support is
a dipstick, a bead or a microtiter multiwell plate. In one
embodiment, the minicell comprises a detectable compound, which may
be a colorimetric, fluorescent or radioactive compound.
[0084] In one embodiment, the minicell displays a membrane
component selected from the group consisting of (i) a eukaryotic
membrane protein, (ii) an archeabacterial membrane protein, (iii)
an organellar membrane protein, (iv) a fusion protein comprising at
least one transmembrane domain or at least one membrane anchoring
domain, and (v) a membrane conjugate comprising a membrane
component chemically linked to a conjugated compound.
[0085] In one embodiment, the membrane component is a receptor. In
a related embodiment, the solid support further comprises a
co-receptor. In one embodiment, the minicell displays a binding
moiety.
[0086] In one aspect, the invention provides a solid support
comprising a minicell, wherein the minicell displays a fusion
protein, the fusion protein comprising a first polypeptide that
comprises at least one transmembrane domain or at least one
membrane anchoring domain, and a second polypeptide. In various
embodiments, the second polypeptide comprises a binding moiety or
an enzyme moiety.
[0087] In one aspect, the invention provides a solid support
comprising a minicell, wherein the minicell comprises a membrane
conjugate comprising a membrane component chemically linked to a
conjugated compound. In one embodiment, the conjugated compound is
a spacer. In one embodiment, the spacer is covalently linked to the
solid support. In one embodiment, the conjugated compound is
covalently linked to the solid support.
[0088] In one aspect, the invention provides a minicell comprising
a biologically active compound, wherein the minicell displays a
ligand or binding moiety, wherein the ligand or binding moiety is
part of a fusion protein comprising a first polypeptide that
comprises at least one transmembrane domain or at least one
membrane anchoring domain and a second polypeptide that comprises a
binding moiety, and the minicell is a poroplast, spheroplast or
protoplast.
[0089] In one aspect, the invention provides a eubacterial minicell
comprising a biologically active compound, wherein the minicell
displays a binding moiety, wherein the binding moiety is selected
from the group consisting of (a) a eukaryotic membrane protein; (b)
an archeabacterial membrane protein; (c) an organellar membrane
protein; and (d) a fusion protein, the fusion protein comprising a
first polypeptide, the first polypeptide comprising at least one
transmembrane domain or at least one membrane anchoring domain; and
a second polypeptide, wherein the second polypeptide is not derived
from a eubacterial protein and is neither a His tag nor a
glutathione-S-transferase polypeptide, and wherein the polypeptide
comprises a binding moiety.
[0090] In one embodiment, the binding moiety is selected from the
group consisting of an antibody, an antibody derivative, a receptor
and an active site of a non-catalytic derivative of an enzyme. In a
preferred embodiment, the binding moiety is a single-chain
antibody. In one embodiment, one of the ORFs encodes a protein that
comprises the binding moiety.
[0091] In one embodiment, the binding moiety is directed to a
ligand selected from the group consisting of an epitope displayed
on a pathogen, an epitope displayed on an infected cell and an
epitope displayed on a hyperproliferative cell.
[0092] In one embodiment, the invention further comprises a first
and second nucleic acid, wherein the first nucleic acid comprises
eukaryotic expression sequences operably linked to a first ORF, and
a second nucleic acid, wherein the second nucleic acid comprises
eubacterial expression sequences operably linked to a second
ORF.
[0093] In one embodiment, the eubacterial expression sequences are
induced and/or derepressed when the binding moiety is in contact
with a target cell. In a variant embodiment, the eukaryotic
expression sequences are induced and/or derepressed when the
nucleic acid is in the cytoplasm of a eukaryotic cell. In related
embodiments, the protein encoded by the first ORF comprises
eukaryotic secretion sequences and/or the protein encoded by the
second ORF comprises eubacterial secretion sequences.
[0094] In one aspect, the invention provides a method of
associating a radioactive compound with a cell, wherein the cell
displays a ligand specifically recognized by a binding moiety,
comprising contacting the cell with a minicell that comprises the
radioactive compound and displays the binding moiety. In a
diagnostic embodiment, the amount of radiation emitted by the
radioactive isotope is sufficient to be detectable. In a
therapeutic embodiment, the amount of radiation emitted by the
radioactive isotope is sufficient to be cytotoxic. In one
embodiment, the ligand displayed by the cell is selected from the
group consisting of an epitope displayed on a pathogen, an epitope
displayed on an infected cell and an epitope displayed on a
hyperproliferative cell. In one embodiment, the binding moiety is
selected from the group consisting of an antibody, an antibody
derivative, a channel protein and a receptor, and is preferably a
single-chain antibody. In other embodiments, the binding moiety is
an aptamer or a small molecule. In one embodiment, the ligand is
selected from the group consisting of a cytokine, hormone, and a
small molecule.
[0095] In one aspect, the invention provides a method of delivering
a biologically active compound to a cell, wherein the cell displays
a ligand specifically recognized by a binding moiety, comprising
contacting the cell with a minicell that displays the binding
moiety, wherein the minicell comprises the biologically active
compound, and wherein the contents of the minicell are delivered
into the cell from a minicell bound to the cell. In one embodiment,
the biologically active compound is selected from the group
consisting of a nucleic acid, a lipid, a polypeptide, a radioactive
compound, an ion and a small molecule.
[0096] In one embodiment, the membrane of the minicell comprises a
system for transferring a molecule from the interior of a minicell
into the cytoplasm of the cell. A representative system for
transferring a molecule from the interior of a minicell into the
cytoplasm of the cell is a Type III secretion system.
[0097] In one embodiment, the minicell further comprises a first
and second nucleic acid, wherein the first nucleic acid comprises
eukaryotic expression sequences operably linked to a first ORF, and
a second nucleic acid, wherein the second nucleic acid comprises
eubacterial expression sequences operably linked to a second ORF.
In one embodiment, one of the ORFs encodes a protein that comprises
the binding moiety. In one embodiment, the eubacterial expression
sequences are induced and/or derepressed when the binding moiety is
in contact with a target cell. In one embodiment, the eukaryotic
expression sequences are induced and/or derepressed when the
nucleic acid is in the cytoplasm of a eukaryotic cell. In one
embodiment, the protein encoded by the first ORF comprises
eukaryotic secretion sequences and/or the protein encoded by the
second ORF comprises eubacterial secretion sequences. In one
embodiment, the ligand is selected from the group consisting of a
cytokine, hormone, and a small molecule.
[0098] In one aspect, the invention provides a minicell displaying
a synthetic linking moiety, wherein the synthetic linking moiety is
covalently or non-covalently attached to a membrane component of
the minicell.
[0099] In one aspect, the invention provides a sterically
stabilized minicell comprising a displayed moiety that has a longer
half-life in vivo than a wild-type minicell, wherein the displayed
moiety is a hydrophilic polymer that comprises a PEG moiety, a
carboxylic group of a polyalkylene glycol or PEG stearate.
[0100] In one aspect, the invention provides a minicell having a
membrane comprising an exogenous lipid, wherein a minicell
comprising the exogenous lipid has a longer half-life in vivo than
a minicell lacking the exogenous lipid, and wherein the minicell is
selected from the group consisting of a eubacterial minicell, a
poroplast, a spheroplast and a protoplast. In one embodiment, the
exogenous lipid is a derivatized lipid which may, by way of
non-limiting example, be phosphatidylethanolamine derivatized with
PEG, DSPE-PEG, PEG stearate; PEG-derivatized phospholipids, a PEG
ceramide or DSPE-PEG.
[0101] In one embodiment, the exogenous lipid is not present in a
wild-type membrane, or is present in a different proportion than is
found in minicells comprising a wild-type membrane. The exogenous
lipid can be a ganglioside, sphingomyelin, monosialoganglioside
GM1, galactocerebroside sulfate,
1,2-sn-dimyristoylphosphatidylcholine, phosphatidylinositol and
cardiolipin.
[0102] In one embodiment, the linking moiety is non-covalently
attached to the minicell. In one embodiment, one of the linking
moiety and the membrane component comprises biotin, and the other
comprises avidin or streptavidin. In one embodiment, the synthetic
linking moiety is a cross-linker. In one embodiment, the
cross-linker is a bifunctional cross-linker.
[0103] In one aspect, the invention provides a method of
transferring a membrane protein from a minicell membrane to a
biological membrane comprising contacting a minicell to the
biological membrane, wherein the minicell membrane comprises the
membrane protein, and allowing the minicell and the biological
membrane to remain in contact for a period of time sufficient for
the transfer to occur.
[0104] In one embodiment, the biological membrane is a cytoplasmic
membrane or an organellar membrane. In one embodiment, the
biological membrane is a membrane selected from the group
consisting of a membrane of a pathogen, a membrane of an infected
cell and a membrane of a hyperproliferative cell. In one
embodiment, the biological membrane is the cytoplasmic membrane of
a recipient cell, which may be a cultured cell and a cell within an
organism. In one embodiment, the biological membrane is present on
a cell that has been removed from an animal, the contacting occurs
in vitro, after which the cell is returned to the organism.
[0105] In one embodiment, the membrane protein is an enzyme. In
this embodiment, the membrane protein having enzymatic activity is
selected from the group consisting of a cytochrome P450 and a
fusion protein, the fusion protein comprising a first polypeptide,
the first polypeptide comprising at least one polypeptide, wherein
the second polypeptide has enzymatic activity.
[0106] In one embodiment, the membrane protein is a membrane fusion
protein, the membrane fusion protein comprising a first
polypeptide, the first polypeptide comprising at least one
transmembrane domain or at least one membrane anchoring domain; and
a second polypeptide.
[0107] In one embodiment, the second polypeptide is a biologically
active polypeptide. In one embodiment, the minicell displays ligand
or a binding moiety.
[0108] In one aspect, the invention provides a minicell that
comprises an expression construct comprising an ORF encoding a
membrane protein operably linked to expression sequences, wherein
the expression sequences are induced and/or derepressed when the
minicell is in contact with a target cell.
[0109] In one embodiment, the biological membrane is a cytoplasmic
membrane or an organellar membrane. In one embodiment, the
biological membrane is a membrane selected from the group
consisting of a membrane of a pathogen, a membrane of an infected
cell and a membrane of a hyperproliferative cell. In one
embodiment, the minicell displays a ligand or a binding moiety
selected from the group consisting of an antibody, an antibody
derivative, an aptamer and a small molecule. In one embodiment, the
membrane protein is a membrane fusion protein, the membrane fusion
protein comprising a first polypeptide, the first polypeptide
comprising at least one transmembrane domain or at least one
membrane anchoring domain; and a second polypeptide. In one
embodiment, the ligand is selected from the group consisting of a
cytokine, hormone, and a small molecule.
[0110] In one aspect, the invention provides a pharmaceutical
composition comprising a minicell, wherein the minicell displays a
membrane protein, wherein the membrane protein is selected from the
group consisting of a eukaryotic membrane protein, an
archeabacterial membrane protein and an organellar membrane
protein. In one embodiment, the membrane protein is selected from
the group consisting of a receptor, a channel protein, a cellular
adhesion factor and an integrin. In one embodiment, the
pharmaceutical formulation further comprises an adjuvant. In one
embodiment, the membrane protein comprises a polypeptide epitope
displayed by a hyperproliferative cell. In one embodiment, the
membrane protein comprises an epitope displayed by a eukaryotic
pathogen, an archeabacterial pathogen, a virus or an infected
cell.
[0111] In one aspect, the invention provides a pharmaceutical
composition comprising a minicell, wherein the minicell displays a
membrane protein that is a fusion protein, the fusion protein
comprising (i) a first polypeptide, the first polypeptide
comprising at least one transmembrane domain or at least one
membrane anchoring domain; and (ii) a second polypeptide, wherein
the second polypeptide is not derived from a eubacterial protein.
In one embodiment, the pharmaceutical formulation further comprises
an adjuvant. In one embodiment, the second polypeptide comprises a
polypeptide epitope displayed by a hyperproliferative cell. In one
embodiment, the membrane protein comprises an epitope displayed by
a eukaryotic pathogen, an archeabacterial pathogen, a virus or an
infected cell.
[0112] In one aspect, the invention provides a pharmaceutical
composition comprising a minicell, wherein the minicell displays a
membrane conjugate, wherein the membrane conjugate comprises a
membrane component chemically linked to a conjugated compound. In
one embodiment, the membrane protein is selected from the group
consisting of a receptor, a channel protein, a cellular adhesion
factor and an integrin. In one embodiment, the pharmaceutical
further comprises an adjuvant. In one embodiment, the membrane
component is a polypeptide comprising at least one transmembrane
domain or at least one membrane anchoring domain, or a lipid that
is part of a membrane. In one embodiment, the conjugated compound
is a polypeptide, and the chemical linkage between the membrane
compound and the conjugated compound is not a peptide bond. In one
embodiment, the conjugated compound is a nucleic acid. In one
embodiment, the conjugated compound is an organic compound. In one
embodiment, the organic compound is selected from the group
consisting of a narcotic, a toxin, a venom, a sphingolipid and a
soluble protein.
[0113] In one aspect, the invention provides a method of making a
pharmaceutical composition comprising a minicell, wherein the
minicell displays a membrane protein, wherein the membrane protein
is selected from the group consisting of a eukaryotic membrane
protein, an archeabacterial membrane protein and an organellar
membrane protein. In one embodiment, the method further comprises
adding an adjuvant to the pharmaceutical formulation. In one
embodiment, the method further comprises desiccating the
formulation. In one embodiment, the method further comprises adding
a suspension buffer to the formulation. In one embodiment, the
method further comprises making a chemical modification of the
membrane protein. In one embodiment, the chemical modification is
selected from the group consisting of glycosylation,
deglycosylation, phosphorylation, dephosphorylation and
proteolysis. In one aspect, the invention provides a method of
making a pharmaceutical composition comprising a minicell, wherein
the minicell displays a membrane protein that is a fusion protein,
the fusion protein comprising (i) a first polypeptide, the first
polypeptide comprising at least one transmembrane domain or at
least one membrane anchoring domain; and (ii) a second polypeptide,
wherein the second polypeptide is not derived from a eubacterial
protein.
[0114] In one aspect, the invention provides a method of making a
pharmaceutical formulation comprising a minicell, wherein the
minicell displays a membrane conjugate, wherein the membrane
conjugate comprises a membrane component chemically linked to a
conjugated compound. In one embodiment, the method further
comprises adding an adjuvant to the pharmaceutical formulation. In
one embodiment, the membrane component is a polypeptide comprising
at least one transmembrane domain or at least one membrane
anchoring domain, or a lipid that is part of a membrane. In one
embodiment, the conjugated compound is a polypeptide, and the
chemical linkage between the membrane compound and the conjugated
compound is not a peptide bond. In one embodiment, the conjugated
compound is a nucleic acid. In one embodiment, the conjugated
compound is an organic compound. In one embodiment, the organic
compound is selected from the group consisting of a narcotic, a
toxin, a venom, and a sphingolipid.
[0115] In one aspect, the invention provides a method of detecting
an agent that is specifically bound by a binding moiety, comprising
contacting a minicell displaying the binding moiety with a
composition known or suspected to contain the agent, and detecting
a signal that is modulated by the binding of the agent to the
binding moiety. In one embodiment, the agent is associated with a
disease. In one embodiment, the minicell comprises a detectable
compound. In one embodiment, the binding moiety is antibody or
antibody derivative. In one embodiment, the composition is an
environmental sample. In one embodiment, the composition is a
biological sample. In one embodiment, the biological sample is
selected from the group consisting of blood, serum, plasma, urine,
saliva, a biopsy sample, feces and a skin patch.
[0116] In one aspect, the invention provides a method of in situ
imaging of a tissue or organ, comprising administering to an
organism a minicell comprising an imaging agent and a binding
moiety and detecting the imaging agent in the organism.
[0117] In one embodiment, the minicell is a eubacterial minicell, a
poroplast, a spheroplast or a protoplast. In one embodiment, the
binding moiety is an antibody or antibody derivative. In one
embodiment, the binding moiety specifically binds a cell surface
antigen. In one embodiment, the cell surface antigen is an antigen
displayed by a tumorigenic cell, a cancer cell, and an infected
cell. In one embodiment, the cell surface antigen is a
tissue-specific antigen. In one embodiment, the method of imaging
is selected from the group consisting of magnetic resonance
imaging, ultrasound imaging; and computer axaial tomography (CAT).
In one aspect, the invention provides a device comprising a
microchip operatively associated with a biosensor comprising a
minicell, wherein the microchip comprises or contacts the minicell,
and wherein the minicell displays a binding moiety.
[0118] In one embodiment, the invention provides a method of
detecting a substance that is specifically bound by a binding
moiety, comprising contacting the device of claim 16 with a
composition known or suspected to contain the substance, and
detecting a signal from the device, wherein the signal changes as a
function of the amount of the substance present in the composition.
In one embodiment, the composition is a biological sample or an
environmental sample.
[0119] In one aspect, the invention provides a method of
identifying an agent that specifically binds a target compound,
comprising contacting a minicell displaying the target compound
with a library of compounds, and identifying an agent in the
library that binds the target compound. In one embodiment, the
library of compounds is a protein library. In one embodiment, the
protein library is selected from the group consisting of a phage
display library, a phagemid display library, a baculovirus library,
a yeast display library, and a ribosomal display library. In one
embodiment, the library of compounds is selected from the group
consisting of a library of aptamers, a library of synthetic
peptides and a library of small molecules.
[0120] In one embodiment, the target compound is a target
polypeptide. In one embodiment, the minicell comprises an
expression construct comprising expression sequences operably
linked to an ORF encoding the target polypeptide. In one
embodiment, the target polypeptide is a membrane protein. In one
embodiment, the membrane protein is a receptor or a channel
protein. In one embodiment, the membrane protein is an enzyme. In
one embodiment, the target compound is a membrane fusion protein,
the membrane fusion protein comprising a first polypeptide, wherein
the first polypeptide comprises at least one transmembrane domain
or at least one membrane anchoring domain; and a second
polypeptide, wherein the second polypeptide comprises amino acid
sequences derived from a target polypeptide. In one embodiment, the
method further comprises comparing the activity of the target
compound in the presence of the agent to the activity of the target
compound in the absence of the agent.
[0121] In one embodiment, the activity of the target compound is an
enzyme activity. In one embodiment, the activity of the target
compound is a binding activity. In one embodiment, the invention
further comprises comparing the binding of the agent to the target
compound to the binding of a known ligand of the target compound.
In one embodiment, a competition assay is used for the
comparing.
[0122] In one aspect, the invention provides a device comprising
microchips operatively associated with a biosensor comprising a set
of minicells in a prearranged pattern, wherein the each of the
microchips comprise or contact a minicell, wherein each of the
minicell displays a different target compound, and wherein binding
of a ligand to a target compound results in an increased or
decreased signal. In one embodiment, the invention provides a
method of identifying an agent that specifically binds a target
compound, comprising contacting the device with a library of
compounds, and detecting a signal from the device, wherein the
signal changes as a function of the binding of an agent to the
target compound. In one embodiment, the invention provides a method
of identifying an agent that specifically blocks the binding of a
target compound to its ligand, comprising contacting the device
with a library of compounds, and detecting a signal from the
device, wherein the signal changes as a function of the binding of
an agent to the target compound.
[0123] In one aspect, the invention provides a method of making a
antibody that specifically binds a protein domain, wherein the
domain is in its native conformation, wherein the domain is
contained within a protein displayed on a minicell, comprising
contacting the minicell with a cell, wherein the cell is competent
for producing antibodies to an antigen contacted with the cell, in
order to generate an immunogenic response in which the cell
produces the antibody.
[0124] In one embodiment, the protein displayed on a minicell is a
membrane protein. In one embodiment, the membrane protein is a
receptor or a channel protein. In one embodiment, the domain is
found within the second polypeptide of a membrane fusion protein,
wherein the membrane fusion protein comprises a first polypeptide,
wherein the first polypeptide comprises at least one transmembrane
domain or at least one membrane anchoring domain. In one
embodiment, the contacting occurs in vivo. In one embodiment, the
antibody is a polyclonal antibody or a monoclonal antibody. In one
embodiment, the contacting occurs in an animal that comprises an
adjuvant.
[0125] In one aspect, the invention provides the method of making
an antibody derivative that specifically binds a protein domain,
wherein the domain is in its native conformation, wherein the
domain is displayed on a minicell, comprising contacting the
minicell with a protein library, and identifying an antibody
derivative from the protein library that specifically binds the
protein domain. In one embodiment, the protein library is selected
from the group consisting of a phage display library, a phagemid
display library, and a ribosomal display library.
[0126] In one aspect, the invention provides a method of making an
antibody or antibody derivative that specifically binds an epitope,
wherein the epitope is selected from the group consisting of (i) an
epitope composed of amino acids found within a membrane protein,
(ii) an epitope present in an interface between a membrane protein
and a membrane component, (iii) an epitope present in an interface
between a membrane protein and one or more other proteins and (iv)
an epitope in a fusion protein, the fusion protein comprising a
first polypeptide, the first polypeptide comprising at least one
transmembrane domain or at least one membrane anchoring domain, and
a second polypeptide, the second polypeptide comprising the
epitope; comprising contacting a minicell displaying the epitope
with a protein library, or to a cell, wherein the cell is competent
for producing antibodies to an antigen contacted with the cell, in
order to generate an immunogenic response in which the cell
produces the antibody.
[0127] In one embodiment, the cell is contacted in vivo. In various
embodiments, the antibody is a polyclonal antibody or a monoclonal
antibody. In one embodiment, the protein library is contacted in
vitro. In one embodiment, the protein library is selected from the
group consisting of a phage display library, a phagemid display
library, and a ribosomal display library.
[0128] In one aspect, the invention provides a method of
determining the rate of transfer of nucleic acid from a minicell to
a cell, comprising (a) contacting the cell to the minicell, wherein
the minicell comprises the nucleic acid, for a measured period of
time; (b) separating minicells from the cells; (c) measuring the
amount of nucleic acid in the cells, wherein the amount of nucleic
acid in the cells over the set period of time is the rate of
transfer of a nucleic acid from a minicell.
[0129] In one aspect, the invention provides a method of
determining the amount of a nucleic acid transferred to a cell from
a minicell, comprising (a) contacting the cell to the minicell,
wherein the minicell comprises an expression element having
eukaryotic expression sequences operably linked to an ORF encoding
a detectable polypeptide, wherein the minicell displays a binding
moiety, and wherein the binding moiety binds an epitope of the
cell; and (b) detecting a signal from the detectable polypeptide,
wherein a change in the signal corresponds to an increase in the
amount of a nucleic acid transferred to a cell.
[0130] In one embodiment, the cell is a eukaryotic cell. By way of
non-limiting example, a eukaryotic cell can be a plant cell, a
fungal cell, a unicellular eukaryote, an animal cell, a mammalian
cell, a rat cell, a mouse cell, a primate cell or a human cell.
[0131] In one embodiment, the binding moiety is an antibody or
antibody derivative. In one embodiment, the binding moiety is a
single-chain antibody. In one embodiment, the binding moiety is an
aptamer. In one embodiment, the binding moiety is an organic
compound. In one embodiment, the detectable polypeptide is a
fluorescent polypeptide.
[0132] In one aspect, the invention provides a method of detecting
the expression of an expression element in a cell, comprising (a)
contacting the cell to a minicell, wherein the minicell comprises
an expression element having cellular expression sequences operably
linked to an ORF encoding a detectable polypeptide, wherein the
minicell displays a binding moiety, and wherein the binding moiety
binds an epitope of the cell; (b) incubating the cell and the
minicell for a period of time effective for transfer of nucleic
acid from the minicell to the cell; and (c) detecting a signal from
the detectable polypeptide, wherein an increase in the signal
corresponds to an increase in the expression of the expression
element.
[0133] In one embodiment, the cell is a eukaryotic cell and the
expression sequences are eukaryotic expression sequences. In one
embodiment, the eukaryotic cell is a mammalian cell. In one
embodiment, the binding moiety is an antibody or antibody
derivative. In one embodiment, the binding moiety is a single-chain
antibody. In one embodiment, the binding moiety is an aptamer. In
one embodiment, the binding moiety is an organic compound.
[0134] In a related aspect, the invention provides methods of
detecting the transfer of a fusion protein from the cytosol to an
organelle of a eukaryotic cell, comprising (a) contacting the cell
to a minicell, wherein (i) the minicell comprises an expression
element having eukaryotic expression sequences operably linked to
an ORF encoding a fusion protein, wherein the fusion protein
comprises a first polypeptide that comprises organellar delivery
sequences, and a second polypeptide that comprises a detectable
polypeptide; and (ii) the minicell displays a binding moiety that
binds an epitope of the cell, or an epitope of an organelle; (b)
incubating the cell and the minicell for a period of time effective
for transfer of nucleic acid from the minicell to the cell and
production of the fusion protein; and (c) detecting a signal from
the detectable polypeptide, wherein a change in the signal
corresponds to an increase in the amount of the fusion protein
transferred to the organelle.
[0135] In one aspect, the invention provides a minicell comprising
at least one nucleic acid, wherein the minicell displays a binding
moiety directed to a target compound, wherein the binding moiety is
selected from the group consisting of (i) a eukaryotic membrane
protein; (ii) an archeabacterial membrane protein; (iii) an
organellar membrane protein; and (iv) a fusion protein, the fusion
protein comprising a first polypeptide, the first polypeptide
comprising at least one transmembrane domain or at least one
membrane anchoring domain; and a second polypeptide, wherein the
second polypeptide is not derived from a eubacterial protein and is
neither a His tag nor a glutathione-S-transferase polypeptide, and
wherein the polypeptide comprises a binding moiety.
[0136] In one embodiment, the nucleic acid comprises an expression
construct comprising expression sequences operably linked to an ORF
encoding a protein selected from the group consisting of (i) the
eukaryotic membrane protein, (ii) the archeabacterial membrane
protein, (iii) the organellar membrane protein; and (iv) the fusion
protein.
[0137] In one embodiment, the nucleic acid comprises an expression
construct comprising expression sequences operably linked to an
ORF, wherein the ORF encodes a therapeutic polypeptide. In one
embodiment, the therapeutic polypeptide is a membrane polypeptide.
In one embodiment, the therapeutic polypeptide is a soluble
polypeptide. In one embodiment, the soluble polypeptide comprises a
cellular secretion sequence. In one embodiment, the expression
sequences are inducible and/or repressible.
[0138] In one embodiment, the expression sequences are induced
and/or derepressed when the binding moiety displayed by the
minicell binds to its target compound. In one embodiment, the
nucleic acid comprises an expression construct comprising
expression sequences operably linked to an ORF, wherein the ORF
encodes a polypeptide having an amino acid sequence that
facilitates cellular transfer of a biologically active compound
contained within or displayed by the minicell. In one embodiment,
the membrane of the minicell comprises a system for transferring a
molecule from the interior of a minicell into the cytoplasm of the
cell. In one embodiment, the system for transferring a molecule
from the interior of a minicell into the cytoplasm of the cell is a
Type III secretion system.
[0139] In one aspect, the invention provides a method of
introducing a nucleic acid into a cell, comprising contacting the
cell with a minicell that comprises the nucleic acid, wherein the
minicell displays a binding moiety, wherein the binding moiety is
selected from the group consisting of (i) a eukaryotic membrane
protein; (ii) an archeabacterial membrane protein; (iii) an
organellar membrane protein; and (iv) a fusion protein, the fusion
protein comprising a first polypeptide, the first polypeptide
comprising at least one transmembrane domain or at least one
membrane anchoring domain; and a second polypeptide, wherein the
second polypeptide is not derived from a eubacterial protein and is
neither a His tag nor a glutathione-S-transferase polypeptide, and
wherein the polypeptide comprises a binding moiety; and wherein the
binding moiety binds an epitope of the cell.
[0140] In one embodiment, the nucleic acid comprises an expression
construct comprising expression sequences operably linked to an ORF
encoding a protein selected from the group consisting of (i) the
eukaryotic membrane protein, (ii) the archeabacterial membrane
protein, (iii) the organellar membrane protein; and (iv) a fusion
protein.
[0141] In one embodiment, the nucleic acid comprises an expression
construct comprising expression sequences operably linked to an
ORF, wherein the ORF encodes a therapeutic polypeptide. In one
embodiment, the expression sequences are inducible and/or
derepressible. In one embodiment, the expression sequences are
induced or derepressed when the binding moiety displayed by the
minicell binds its target compound. In one embodiment, the
expression sequences are induced or derepressed by a
transactivation or transrepression event. In one embodiment, the
nucleic acid comprises an expression construct comprising
expression sequences operably linked to an ORF, wherein the ORF
encodes a polypeptide having an amino acid sequence that
facilitates cellular transfer of a biologically active compound
contained within or displayed by the minicell.
[0142] In one aspect, the invention provides a minicell comprising
a nucleic acid, wherein the nucleic acid comprises eukaryotic
expression sequences and eubacterial expression sequences, each of
which is independently operably linked to an ORF.
[0143] In one embodiment, the minicell displays a binding moiety.
In one embodiment, the eubacterial expression sequences are induced
and/or derepressed when the binding moiety is in contact with a
target cell. In one embodiment, the eukaryotic expression sequences
are induced and/or derepressed when the nucleic acid is in the
cytoplasm of a eukaryotic cell. In one embodiment, the protein
encoded by the ORF comprises eubacterial or eukaryotic secretion
sequences.
[0144] In one aspect, the invention provides a minicell comprising
a first and second nucleic acid, wherein the first nucleic acid
comprises eukaryotic expression sequences operably linked to a
first ORF, and a second nucleic acid, wherein the second nucleic
acid comprises eubacterial expression sequences operably linked to
a second ORF.
[0145] In one embodiment, the minicell displays a binding moiety.
In one embodiment, the eubacterial expression sequences are induced
and/or derepressed when the binding moiety is in contact with a
target cell. In one embodiment, the eukaryotic expression sequences
are induced and/or derepressed when the nucleic acid is in the
cytoplasm of a eukaryotic cell. In one embodiment, the protein
encoded by the first ORF comprises eukaryotic secretion sequences
and/or the protein encoded by the second ORF comprises eubacterial
secretion sequences.
[0146] In one aspect, the invention provides a method of
introducing into and expressing a nucleic acid in an organism,
comprising contacting a minicell to a cell of the organism, wherein
the minicell comprises the nucleic acid.
[0147] In one embodiment, the minicell displays a binding moiety.
In one embodiment, the nucleic acid comprises a eukaryotic
expression construct, wherein the eukaryotic expression construct
comprises eukaryotic expression sequences operably linked to an
ORF. In one embodiment, the ORF encodes a protein selected from the
group consisting of a membrane protein, a soluble protein and a
protein comprising eukaryotic secretion signal sequences. In one
embodiment, the nucleic acid comprises a eubacterial expression
construct, wherein the eubacterial expression construct comprises
eubacterial expression sequences operably linked to an ORF. In one
embodiment, the minicell displays a binding moiety, wherein the
eubacterial expression sequences are induced and/or derepressed
when the binding moiety is in contact with a target cell. In one
embodiment, the protein encoded by the ORF comprises eubacterial
secretion sequences. In one aspect, the invention provides a
minicell comprising a crystal of a membrane protein. In one
embodiment, the minicell is a eubacterial minicell, a poroplast, a
spheroplast or a protoplast. In one embodiment, the membrane
protein is a receptor. In one embodiment, the receptor is a
G-protein coupled receptor. In one embodiment, the crystal is
displayed.
[0148] In a related aspect, the invention provides a minicell
membrane preparation comprising a crystal of a membrane
protein.
[0149] In one embodiment, the membrane protein is a fusion protein,
the fusion protein comprising a first polypeptide, the first
polypeptide comprising at least one transmembrane domain or at
least one membrane anchoring domain, and a second polypeptide. In
one embodiment, the crystal is a crystal of the second polypeptide.
In one embodiment, the crystal is displayed.
[0150] In one aspect, the invention provides a method of
determining the three-dimensional structure of a membrane protein,
comprising preparing a crystal of the membrane protein in a
minicell, and determining the three-dimensional structure of the
crystal.
[0151] In one aspect, the invention provides a method for
identifying ligand-interacting atoms in a defined three-dimensional
structure of a target protein, comprising (a) preparing one or more
variant proteins of a target protein having a known or predicted
three-dimensional structure, wherein the target protein binds a
preselected ligand; (b) expressing and displaying a variant protein
in a minicell; and (c) determining if a minicell displaying the
variant protein binds the preselected ligand with increased or
decreased affinity as compared to the binding of the preselected
ligand to the target protein.
[0152] In one embodiment, the ligand is a protein that forms a
multimer with the target protein, and the ligand interacting atoms
are atoms in the defined three-dimensional structure are atoms that
are involved in protein-protein interactions. In one embodiment,
the ligand is a compound that induces a conformational change in
the target protein, and the defined three-dimensional structure is
the site of the conformational change. In one embodiment, the
method for identifying ligands of a target protein, further
comprising identifying the chemical differences in the variant
proteins as compared to the target protein. In one embodiment, the
invention further comprises mapping the chemical differences onto
the defined three-dimensional structure, and correlating the effect
of the chemical differences on the defined three-dimensional
structure. In one embodiment, the target protein is a wild-type
protein. In one aspect, the invention provides a minicell library,
comprising two or more minicells, wherein each minicell comprises a
different exogenous protein. In one embodiment, the minicell is a
eubacterial minicell, a poroplast, a spheroplast or a protoplast.
In one embodiment, the exogenous protein is a displayed protein. In
one embodiment, the exogenous protein is a membrane protein. In one
embodiment, the membrane protein is a receptor. In one embodiment,
the protein is a soluble protein that is contained within or
secreted from the minicell. In one embodiment, minicells within the
library comprise an expression element that comprises expression
sequences operably linked to a nucleic acid having an ORF that
encodes the exogenous protein. In one embodiment, the nucleic acid
has been mutagenized; the mutagenesis can be site-directed or
random. In one embodiment, an active site of the exogenous protein
has a known or predicted three-dimensional structure, and the a
portion of the ORF encoding the active site has been mutagenized.
In one embodiment, each of the minicells comprises an exogenous
protein that is a variant of a protein having a known or predicted
three-dimensional structure.
[0153] In one aspect, the invention provides a minicell library,
comprising two or more minicells, wherein each minicell comprises a
different fusion protein, each of the fusion protein comprising a
first polypeptide that is a constant polypeptide, wherein the
constant polypeptide comprises at least one transmembrane domain or
at least one membrane anchoring domain, and a second polypeptide,
wherein the second polypeptide is a variable amino acid sequence
that is different in each fusion proteins. In one embodiment,
minicells within the library comprise an expression element that
comprises expression sequences operably linked to a nucleic acid
having an ORF that encodes the fusion protein. In one embodiment,
the second polypeptide of the fusion protein is encoded by a
nucleic acid that has been cloned. In one embodiment, each of the
second polypeptide of each of the fusion proteins comprises a
variant of an amino acid sequence from a protein having a known or
predicted three-dimensional structure.
[0154] In one aspect, the invention provides a minicell library,
comprising two or more minicells, wherein each minicell comprises a
constant protein that is present in each minicell and a variable
protein that differs from minicell to minicell. In one embodiment,
one of the constant and variable proteins is a receptor, and the
other of the constant and variable proteins is a co-receptor. In
one embodiment, each of the constant and variable proteins is
different from each other and is a factor in a signal transduction
pathway. In one embodiment, one of the constant and variable
proteins is a G-protein, and the other of the constant and variable
proteins is a G-protein coupled receptor.
[0155] In one embodiment, one of the constant and variable proteins
comprises a first transrepression domain, and the other of the
constant and variable comprises a second transrepression domain,
wherein the transrepression domains limit or block expression of a
reporter gene when the constant and variable proteins associate
with each other.
[0156] In one embodiment, one of the constant and variable proteins
comprises a first transactivation domain, and the other of the
constant and variable comprises a second transactivation domain,
wherein the transactivation domains stimulate expression of a
reporter gene when the constant and variable proteins associate
with each other.
[0157] In one aspect, the invention provides a method of
identifying a nucleic acid that encodes a protein that binds to or
chemically alters a preselected ligand, comprising (a) separately
contacting the ligand with individual members of a minicell
library, wherein minicells in the library comprise expression
elements, wherein the expression elements comprise DNA inserts,
wherein an ORF in the DNA insert is operably linked to expression
sequences, in order to generate a series of reaction mixes, each
reaction mix comprising a different member of the minicell library;
(b) incubating the reaction mixes, thereby allowing a protein that
binds to or chemically alters the preselected ligand to bind or
chemically alter the preselected ligand; (c) detecting a change in
a signal from reaction mixes in which the ligand has been bound or
chemically altered; (d) preparing DNA from reaction mixes in which
the ligand has been bound or chemically altered; wherein the DNA is
a nucleic acid that encodes a protein that binds to or chemically
alters the preselected ligand.
[0158] In one embodiment, the minicell is a eubacterial minicell, a
poroplast, a spheroplast or a protoplast. In one embodiment, the
preselected ligand is a biologically active compound. In one
embodiment, the preselected ligand is a therapeutic drug. In one
embodiment, a protein that binds or chemically alters the
preselected ligand is a target protein for compounds that are
therapeutic for a disease that is treated by administering the drug
to an organism in need thereof. In one embodiment, the preselected
ligand is detectably labeled, the minicell comprises a detectable
compound, and/or a chemically altered derivative of the protein is
detectably labeled.
[0159] In one aspect, the invention provides a method of
determining the amino acid sequence of a protein that binds or
chemically alters a preselected ligand, comprising: (a) contacting
the ligand with a minicell library, wherein minicells in the
library comprise expression elements, wherein the expression
elements comprise DNA inserts, wherein an ORF in the DNA insert is
operably linked to expression sequences; (b) incubating the mixture
of ligand and minicells, under conditions which allow complexes
comprising ligands and minicells to form and/or chemical reactions
to occur; (c) isolating or identifying the complexes from the
ligand and the mixture of ligand and minicells; (d) preparing DNA
from an expression element found in one or more of the complexes,
or in a minicell thereof; (e) determining the nucleotide sequence
of the ORF in the DNA; and (f) generating an amino sequence by in
silico translation, wherein the amino acid sequence is or is
derived from a protein that binds or chemically alters a
preselected ligand.
[0160] In one embodiment, the minicell is a eubacterial minicell, a
poroplast, a spheroplast or a protoplast. In one embodiment, the
DNA is prepared by isolating DNA from the complexes, or in a
minicell thereof. In one embodiment, the DNA is prepared by
amplifying DNA from the complexes, or in a minicell thereof. In one
embodiment, the protein is a fusion protein. In one embodiment, the
protein is a membrane or a soluble protein. In one embodiment, the
protein comprises secretion sequences. In one embodiment, the
preselected ligand is a biologically active compound. In one
embodiment, the preselected ligand is a therapeutic drug. In one
embodiment, the preselected ligand is a therapeutic drug, and the
protein that binds the preselected ligand is a target protein for
compounds that are therapeutic for a disease that is treated by
administering the drug to an organism in need thereof.
[0161] In one aspect, the invention provides a method of
identifying a nucleic acid that encodes a protein that inhibits or
blocks an agent from binding to or chemically altering a
preselected ligand, comprising: (a) separately contacting the
ligand with individual members of a minicell library, wherein
minicells in the library comprise expression elements, wherein the
expression elements comprise DNA inserts, wherein an ORF in the DNA
insert is operably linked to expression sequences, in order to
generate a series of reaction mixes, each reaction mix comprising a
different member of the minicell library; (b) incubating the
reaction mixes, thereby allowing a protein that binds to or
chemically alters the preselected ligand to bind or chemically
alter the preselected ligand; (c) detecting a change in a signal
from reaction mixes in which the ligand has been bound or
chemically altered; (d) preparing DNA from reaction mixes in which
the change in signal ligand has been bound or chemically altered;
wherein the DNA is a nucleic acid that encodes a protein that
inhibits or blocks the agent from binding to or chemically altering
the preselected ligand
[0162] In one embodiment, the minicell is a eubacterial minicell, a
poroplast, a spheroplast or a protoplast. In one embodiment, the
DNA has a nucleotide sequence that encodes the amino acid sequence
of the protein that inhibits or blocks the agent from binding to or
chemically altering the preselected ligand. In one embodiment, a
protein that binds or chemically alters the preselected ligand is a
target protein for compounds that are therapeutic for a disease
that is treated by administering the drug to an organism in need
thereof.
[0163] In one aspect, the invention provides a method of
identifying an agent that effects the activity of a protein,
comprising contacting a library of two or more candidate agents
with a minicell comprising the protein or a polypeptide derived
from the protein, assaying the effect of candidate agents on the
activity of the protein, and identifying agents that effect the
activity of the protein.
[0164] In one embodiment, the protein or the polypeptide derived
from the protein is displayed on the surface of the minicell. In
one embodiment, the protein is a membrane protein. In one
embodiment, the membrane protein is selected from the group
consisting of a receptor, a channel protein and an enzyme. In one
embodiment, the activity of a protein is a binding activity or an
enzymatic activity. In one embodiment, the library of compounds is
a protein library. In one embodiment, the protein library is
selected from the group consisting of a phage display library, a
phagemid display library, and a ribosomal display library. In one
embodiment, the library of compounds is a library of aptamers. In
one embodiment, the library of compounds is a library of small
molecules.
[0165] In one aspect, the invention provides a method of
identifying an agent that effects the activity of a protein domain
containing a library of two or more candidate agents with a
minicell displaying a membrane fusion protein, the fusion protein
comprising a first polypeptide, the first polypeptide comprising at
least one transmembrane domain or at least one membrane anchoring
domain, and a second polypeptide, wherein the second polypeptide
comprises the protein domain.
[0166] In one aspect, the invention provides a method of
identifying undesirable side-effects of a biologically active
compound that occur as a result of binding of the compound to a
protein, wherein binding a compound to the protein is known to
result in undesirable side effects, comprising contacting a
minicell that comprises the protein to the biologically active
compound. In one embodiment, the invention provides comprises
characterizing the binding of the biologically active compound to
the protein. In one embodiment, the invention provides comprises
characterizing the effect of the biologically active compound on
the activity of the protein.
[0167] In one aspect, the invention provides a method for
identifying an agent that effects the interaction of a first
signaling protein with a second signaling protein, comprising (a)
contacting a library of compounds with a minicell, wherein the
minicell comprises: (i) a first protein comprising the first
signaling protein and a first trans-acting regulatory domain; (ii)
a second protein comprising the second signaling protein and a
second trans-acting regulatory domain; and (iii) a reporter gene,
the expression of which is modulated by the interaction between the
first trans-acting regulatory domain and the second trans-acting
regulatory domain; and (b) detecting the gene product of the
reporter gene.
[0168] In one embodiment, the trans-acting regulatory domains are
transactivation domains. In one embodiment, the trans-acting
regulatory domains are transrepression domains.
[0169] In one embodiment, the reporter gene is induced by the
interaction of the first trans-acting regulatory domain and the
second trans-acting regulatory domain. In one embodiment, the agent
that effects the interaction of the first signaling protein with
the second signaling protein is an agent that causes or promotes
the interaction. In one embodiment, the reporter gene is repressed
by the interaction of the first trans-acting regulatory domain and
the second trans-acting regulatory domain. In one embodiment, the
agent that effects the interaction of the first signaling protein
with the second signaling protein is an agent that inhibits or
blocks the interaction.
[0170] In one embodiment, the first signaling protein is a GPCR. In
one embodiment, the GPCR is an Edg receptor or a ScAMPER.
[0171] In one embodiment, the second signalling protein is a
G-protein. In related embodiments, G-protein is selected from the
group consisting of G-alpha-i, G-alpha-s, G-alpha-q, G-alpha-12/13
and Go. In one embodiment, the library of compounds is a protein
library. In one embodiment, the protein library is selected from
the group consisting of a phage display library, a phagemid display
library, and a ribosomal display library. In one embodiment, the
library of compounds is a library of aptamers. In one embodiment,
the library of compounds is a library of small molecules.
[0172] In one aspect, the invention provides a method for
identifying an agent that effects the interaction of a first
signaling protein with a second signaling protein, comprising
contacting a library of two or more candidate agents with a
minicell, wherein the minicell comprises (a) a first fusion protein
comprising the first signaling protein and a first detectable
domain; and (b) a second fusion protein comprising the second
signaling protein and a second detectable domain, wherein a signal
is generated when the first and second signaling proteins are in
close proximity to each other, and detecting the signal.
[0173] In one embodiment, the signal is fluorescence. In one
embodiment, the first detectable domain and the second detectable
domain are fluorescent and the signal is generated by FRET. In one
embodiment, the first and second detectable domains are
independently selected from the group consisting of a green
fluorescent protein, a blue-shifted green fluorescent protein, a
cyan-shifted green fluorescent protein; a red-shifted green
fluorescent protein; a yellow-shifted green fluorescent protein,
and a red fluorescent protein, wherein the first fluorescent domain
and the second fluorescent domain are not identical.
[0174] In one aspect, the invention provides a method of
bioremediation, the method comprising contacting a composition that
comprises an undesirable substance with a minicell, wherein the
minicell alters the chemical structure and/or binds the undesirable
substance.
[0175] In one aspect, the invention provides a method of
bioremediation, the method comprising contacting a composition that
comprises an undesirable substance with a minicell, wherein the
minicell comprises an agent that alters the chemical structure of
the undesirable substance. In one embodiment, the agent that alters
the chemical structure of the undesirable substance is an inorganic
catalyst. In one embodiment, the agent that alters the chemical
structure of the undesirable substance is an enzyme. In one
embodiment, the enzyme is a soluble protein contained within the
minicell. In one embodiment, the enzyme is a secreted protein. In
one embodiment, the enzyme is a membrane protein. In one
embodiment, the membrane enzyme is selected from the group
consisting of a cytochrome P450, an oxidoreductase, a transferase,
a hydrolase, a lyase, an isomerase, a ligase and a synthetase. In
one embodiment, the agent that alters the chemical structure of the
undesirable substance is a fusion protein comprising a first
polypeptide that comprises a transmembrane domain or at least one
membrane-anchoring domain, and a second polypeptide, wherein the
second polypeptide is an enzyme moiety.
[0176] In one aspect, the invention provides a method of
bioremediation, the method comprising contacting a composition that
comprises an undesirable substance with a minicell, wherein the
minicell comprises an agent that binds an undesirable substance. In
one embodiment, the undesirable substance binds to and is
internalized by the minicell or is otherwise inactivated by
selective absorption. In one embodiment, the agent that binds the
undesirable substance is a secreted soluble protein. In one
embodiment, the secreted protein is a transport accessory protein.
In one embodiment, the agent that binds the undesirable substance
is a membrane protein. In one embodiment, the undesirable substance
is selected from the group consisting of a toxin, a pollutant and a
pathogen. In one embodiment, the agent that binds the undesirable
substance is a fusion protein comprising a first polypeptide that
comprises a transmembrane domain or at least one membrane-anchoring
domain, and a second polypeptide, wherein the second polypeptide is
a binding moiety. In one embodiment, wherein the binding moiety is
selected from the group consisting of an antibody, an antibody
derivative, the active site of a non-enzymatically active mutant
enzyme, a single-chain antibody and an aptamer.
[0177] In one aspect, the invention provides a minicell-producing
parent cell, wherein the parent cell comprises one or more of the
following (a) an expression element that comprises a gene operably
linked to expression sequences that are inducible and/or
repressible, wherein induction or repression of the gene regulates
the copy number of an episomal expression construct; (b) a mutation
in an endogenous gene, wherein the mutation regulates the copy
number of an episomal expression construct; (c) an expression
element that comprises a gene operably linked to expression
sequences that are inducible and/or repressible, wherein induction
or repression of the gene causes or enhances the production of
minicells; and (d) a mutation in an endogenous gene, wherein the
mutation causes or enhances minicell production.
[0178] In one embodiment, the invention comprises an episomal
expression construct. In one embodiment, the invention further
comprises a chromosomal expression construct. In one embodiment,
the expression sequences of the expression construct are inducible
and/or repressible. In one embodiment, the minicell-producing
parent cell comprises a biologically active compound. In one
embodiment, the gene that causes or enhances the production of
minicells has a gene product that is involved in or regulates DNA
replication, cellular division, cellular partitioning, septation,
transcription, translation, or protein folding.
[0179] In one aspect, the invention provides a minicell-producing
parent cell, wherein the parent cell comprises an expression
construct, wherein the expression construct comprises expression
sequences operably linked to an ORF that encodes a protein, and a
regulatory expression element, wherein the regulatory expression
element comprises expression sequences operably linked to a
regulatory gene that encodes a factor that regulates the expression
of the ORF. In one embodiment, the expression sequences of the
expression construct are inducible and/or repressible. In one
embodiment, the expression sequences of the regulatory expression
construct are inducible and/or repressible. In one embodiment, one
or more of the expression element or the regulatory expression
element is located on a chromosome of the parent cell. In one
embodiment, one or more of the expression element or the regulatory
expression element is located on an episomal expression construct.
In one embodiment, both of the expression element and the
regulatory expression element are located on an episomal expression
construct, and one or both of the expression element and the
regulatory expression element segregates into minicells produced
from the parent cell. In one embodiment, the minicell-producing
parent cell comprises a biologically active compound. In one
embodiment, the biologically active compound segregates into
minicells produced from the parent cell. In one embodiment, the ORF
encodes a membrane protein or a soluble protein. In one embodiment,
the protein comprises secretion sequences. In one embodiment, the
gene product of the gene regulates the expression of the ORF. In
one embodiment, the gene product is a transcription factor. In one
embodiment, the gene product is a RNA polymerase. In one
embodiment, the parent cell is MC-T7.
[0180] In one aspect, the invention provides a minicell comprising
a biologically active compound, wherein the minicell displays a
binding moiety, wherein the minicell selectively absorbs and/or
internalizes an undesirable compound, and the minicell is a
poroplast, spheroplast or protoplast. In one embodiment, the
binding moiety is selected from the group consisting of an
antibody, an antibody derivative, a receptor and an active site of
a non-catalytic derivative of an enzyme. In one embodiment, the
binding moiety is a single-chain antibody. In one embodiment, the
binding moiety is directed to a ligand selected from the group
consisting of an epitope displayed on a pathogen, an epitope
displayed on an infected cell and an epitope displayed on a
hyperproliferative cell. In one embodiment, the biologically active
compound is selected from the group consisting of a radioisotope, a
polypeptide, a nucleic acid and a small molecule. In one
embodiment, a ligand binds to and is internalized by the minicell
or is otherwise inactivated by selective absorption. In one
embodiment, the invention provides a pharmaceutical composition
comprising the minicell. In one aspect, the invention provides a
method of reducing the free concentration of a substance in a
composition, wherein the substance displays a ligand specifically
recognized by a binding moiety, comprising contacting the
composition with a minicell that displays the binding moiety,
wherein the binding moiety binds the substance, thereby reducing
the free concentration of the substance in the composition. In one
embodiment, the substance is selected from the group consisting of
a nucleic acid, a lipid, a polypeptide, a radioactive compound, an
ion and a small molecule. In one embodiment, the binding moiety is
selected from the group consisting of an antibody, an antibody
derivative, a channel protein and a receptor.
[0181] In one embodiment, the composition is present in an
environment including but not limited to water, air or soil. In one
embodiment, the composition is a biological sample from an
organism, including but not limited to blood, serum, plasma, urine,
saliva, a biopsy sample, feces, tissue and a skin patch. In one
embodiment, the substance binds to and is internalized by the
minicell or is otherwise inactivated by selective absorption. In
one embodiment, the biological sample is returned to the organism
after being contacting to the minicell.
[0182] For a better understanding of the present invention,
reference is made to the accompanying detailed description and its
scope will be pointed out in the appended claims. All references
cited herein are hereby incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0183] FIG. 1 is a Western blot in which Edg-1-6.times.His and
Edg-3-6.times.His proteins expressed in minicells produced from
MC-T7 cells.
[0184] FIG. 2 shows induction of Ma1E(L)-NTR in isolated
minicells.
ABBREVIATIONS AND DEFINITIONS
[0185] For brevity's sake, the single-letter amino acid
abbreviations are used in some instances herein. Table 1 describes
the correspondence between the 1- and 3-letter amino acid
abbreviations.
TABLE-US-00001 TABLE 1 THREE- AND ONE-LETTER ABBREVIATIONS FOR
AMINO ACIDS Three-letter One-letter Amino acid abbreviation symbol
Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic Acid Asp D
Cysteine Cys C Glutamine Gln Q Glutamic acid Glu E Glycine Gly G
Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K
Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S
Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V
[0186] A "conjugatable compound" or "attachable compound" is
capable of being attached to another compound. The terms
"conjugated to" and "cross-linked with" indicate that the
conjugatable compound is in the state of being attached to another
compound. A "conjugate" is the compound formed by the attachment of
a conjugatable compound or conjugatable moiety to another
compound.
[0187] "Culturing" signifies incubating a cell or organism under
conditions wherein the cell or organism can carry out some, if not
all, biological processes. For example, a cell that is cultured may
be growing or reproducing, or it may be non-viable but still
capable of carrying out biological and/or biochemical processes
such as replication, transcription, translation, etc.
[0188] An agent is said to have been "purified" if its
concentration is increased, and/or the concentration of one or more
undesirable contaminants is decreased, in a composition relative to
the composition from which the agent has been purified.
Purification thus encompasses enrichment of an agent in a
composition and/or isolation of an agent therefrom.
[0189] A "solid support" is any solid or semisolid composition to
which an agent can be attached or contained within. Common forms of
solid support include, but are not limited to, plates, tubes, and
beads, all of which could be made of glass or another suitable
material, e.g., polystyrene, nylon, cellulose acetate,
nitrocellulose, and other polymers. Semisolids and gels that
minicells are suspended within are also considered to be solid
supports. A solid support can be in the form of a dipstick,
flow-through device, or other suitable configuration.
[0190] A "mutation" is a change in the nucleotide sequence of a
gene relative to the sequence of the "wild-type" gene. Reference
wild-type eubacterial strains are those that have been cultured in
vitro by scientists for decades; for example, a wild-type strain of
Escherichia coli iss E. coli K-12. Mutations include, but are not
limited to, point mutations, deletions, insertions and
translocations.
[0191] A "trans-acting regulatory domain" is a regulatory part of a
protein that is expressed from a gene that is not adjacent to the
site of regulatory effect. Trans-acting domains can activate or
stimulate (transactivate), or limit or block (transrepress) the
gene in question.
[0192] A "reporter gene" refers to a gene that is operably linked
to expression sequences, and which expresses a gene product,
typically a detectable polypeptide, the production and detection of
which is used as a measure of the robustness and/or control of
expression.
[0193] A "detectable compound" or "detectable moiety" produces a
signal that can be detected by spectroscopic, photochemical,
biochemical, immunochemical, electromagnetic, radiochemical, or
chemical means such as fluorescence, chemifluoresence, or
chemiluminescence, or any other appropriate means. A "radioactive
compound" or "radioactive composition" has more than the natural
(environmental) amount of one or more radioisotopes.
[0194] By "displayed" it is meant that a portion of the membrane
protein is present on the surface of a cell or minicell, and is
thus in contact with the external environment of the cell or
minicell. The external, displayed portion of a membrane protein is
an "extracellular domain" or a "displayed domain." A membrane
protein may have more than one displayed domain, and a minicell of
the invention may display more than one membrane protein.
[0195] A "domain" or "protein domain" is a region of a molecule or
structure that shares common physical and/or chemical features.
Non-limiting examples of protein domains include hydrophobic
transmembrane or peripheral membrane binding regions, globular
enzymatic or receptor regions, and/or nucleic acid binding
domains.
[0196] A "transmembrane domain" spans a membrane, a "membrane
anchoring domain" is positioned within, but does not traverse, a
membrane. An "extracellular" or "displayed" domain is present on
the exterior of a cell, or minicell, and is thus in contact with
the external environment of the cell or minicell.
[0197] A "eukaryote" is as the term is used in the art. A eukaryote
may, by way of non-limiting example, be a fungus, a unicellular
eukaryote, a plant or an animal. An animal may be a mammal, such as
a rat, a mouse, a rabbit, a dog, a cat, a horse, a cow, a pig, a
simian or a human.
[0198] A "eukaryotic membrane" is a membrane found in a eukaryote.
A eukaryotic membrane may, by way of non-limiting example, a
cytoplasmic membrane, a nuclear membrane, a nucleolar membrane, a
membrane of the endoplasmic reticulum (ER), a membrane of a Golgi
body, a membrane of a lysosome a membrane of a peroxisome, a
caveolar membrane, or an inner or outer membrane of a
mitochondrion, chloroplast or plastid.
[0199] The term "endogenous" refers to something that is normally
found in a cell as that cell exists in nature.
[0200] The term "exogenous" refers to something that is not
normally found in a cell as that cell exists in nature.
[0201] A "gene" comprises (a) nucleotide sequences that either (i)
act as a template for a nucleic acid gene product, or (ii) that
encode one or more open reading frames (ORFs); and (b) expression
sequences operably linked to (1) or (2). When a gene comprises an
ORF, it is a "structural gene."
[0202] By "immunogenic," it is meant that a compound elicits
production of antibodies or antibody derivatives and, additionally
or alternatively, a T-cell mediated response, directed to the
compound or a portion thereof. The compound is an "immunogen."
[0203] A "ligand" is a compound, composition or moiety that is
capable of specifically bound by a binding moiety, including
without limitation, a receptor and an antibody or antibody
derivative.
[0204] A "membrane protein" is a protein found in whole or in part
in a membrane. Typically, a membrane protein has (1) at least one
membrane anchoring domain, (2) at least one transmembrane domain,
or (3) at least one domain that interacts with a protein having (1)
or (2).
[0205] An "ORF" or "open reading frame" is a nucleotide sequence
that encodes an amino acid sequence of a known, predicted or
hypothetical polypeptide. An ORF is bounded on its 5' end by a
start codon (usually ATG) and on its 3' end by a stop codon (i.e.,
TAA or TGA). An ORF encoding a 10 amino acid sequence comprises 33
nucleotides (3 for each of 10 amino acids and 3 for a stop codon).
ORFs can encode amino acid sequences that comprise from 10, 25, 50,
125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800,
900 or more amino acids
[0206] The terms "Eubacteria" and"prokaryote" are used herein as
these terms are used by those in the art. The terms "eubacterial"
and "prokaryotic" encompasses Eubacteria, including both
gram-negative and gram-positive bacteria, prokaryotic viruses
(e.g., bacteriophage), and obligate intracellular parasites (e.g.,
Rickettsia, Chlamydia, etc.).
[0207] An "active site" is any portion or region of a molecule
required for, or that regulates, an activity of the molecule. In
the case of a protein, an active site can be a binding site for a
ligand or a substrate, an active site of enzyme, a site that
directs or undergoes conformational change in response to a signal,
or a site of post-translational modification of a protein.
[0208] In a poroplast, the eubacterial outer membrane (OM) and LPS
have been removed. In a spheroplast, portions of a disrupted
eubacterial OM and/or disrupted cell wall either may remain
associated with the inner membrane of the minicell, but the
membrane is nonetheless porous because the permeability of the
disrupted OM has been increased. A membrane is the to be
"disrupted" when the membrane's structure has been treated with an
agent, or incubated under conditions, that leads to the partial
degradation of the membrane, thereby increasing the permeability
thereof. In contrast, a membrane that has been "degraded" is
essentially, for the applicable intents and purposes, removed. In
preferred embodiments, irrespective of the condition of the OM and
cell wall, the eubacterial inner membrane is not disrupted, and
membrane proteins displayed on the inner membrane are accessible to
compounds that are brought into contact with the minicell,
poroplast, spheroplast, protoplast or cellular poroplast, as the
case may be.
[0209] Host cells (and/or minicells) harboring an expression
construct are components of expression systems.
[0210] An "expression vector" is an artificial nucleic acid
molecule into which an exogenous ORF encoding a protein, or a
template of a bioactive nucleic acid can be inserted in such a
manner so as to be operably linked to appropriate expression
sequences that direct the expression of the exogenous gene.
Preferred expression vectors are episomal vectors that can
replicate independently of chromosomal replication.
[0211] By the term "operably linked" it is meant that the gene
products encoded by the non-vector nucleic acid sequences are
produced from an expression element in vivo.
[0212] The term "gene product" refers to either a nucleic acid (the
product of transcription, reverse transcription, or replication) or
a polypeptide (the product of translation) that is produced using
the non-vector nucleic acid sequences as a template.
[0213] An "expression construct" is an expression vector into which
a nucleotide sequence of interest has been inserted in a manner so
as to be positioned to be operably linked to the expression
sequences present in the expression vector. Preferred expression
constructs are episomal.
[0214] An "expression element" is a nucleic acid having nucleotide
sequences that are present in an expression construct but not its
cognate expression vector. That is, an expression element for a
polypeptide is a nucleic acid that comprises an ORF operably linked
to appropriate expression sequences. An expression element can be
removed from its expression construct and placed in other
expression vectors or into chromosomal DNA.
[0215] "Expression sequences" are nucleic acid sequences that bind
factors necessary for the expression of genes that have been
inserted into an expression vector. An example of an expression
sequence is a promoter, a sequence that binds RNA polymerase, which
is the enzyme that produces RNA molecules using DNA as a template.
An example of an expression sequence that is both inducible and
repressible is L-arabinose operon (araC). See Schleif R. Regulation
of the L-arabinose operon of Escherichia coli. Trends Genet. 2000
December; 16(12):559-65.
[0216] In the present disclosure, "a nucleic acid" or "the nucleic
acid" refers to a specific nucleic acid molecule. In contrast, the
term "nucleic acid" refers to any collection of diverse nucleic
acid molecules, and thus signifies that any number of different
types of nucleic acids are present.
[0217] By way of non-limiting example, a nucleic acid may be a DNA,
a dsRNA, a tRNA (including a rare codon usage tRNA), an mRNA, a
ribosomal RNA (rRNA), a peptide nucleic acid (PNA), a DNA:RNA
hybrid, an antisense oligonucleotide, a ribozyme, or an
aptamer.
DETAILED DESCRIPTION OF THE INVENTION
[0218] The invention described herein is drawn to compositions and
methods for the production of achromosomal archeabacterial,
eubacterial and anucleate eukaryotic cells that are used for
diagnostic and therapeutic applications, for drug discovery, and as
research tools.
[0219] The general advantage of minicells over cell-based
expression systems (e.g., eucaryotic cells or bacterial expression
systems) is that one may express heterologous membrane bound
proteins or over express endogenous membrane bound proteins,
cytoplasmic or secreted soluble proteins, or small molecules on the
cytoplasmic or extracellular surfaces of the minicells that would
otherwise be toxic to live cells. Minicells are also advantageous
for proteins that require a particular lipid environment for proper
functioning because it is very manipulatable in nature. Other
advantages include the stability of the minicells due to the lack
of toxicity, the high level of expression that can be achieved in
the minicell, and the efficient flexible nature of the minicell
expression system. Such minicells could be used for in vivo
targeting or for selective absorption (i.e., molecular "sponges")
and that these molecules can be expressed and "displayed" at high
levels. Minicells can also be used to display proteins for low,
medium, high, and ultra high throughput screening, crystal
formation for structure determination, and for in vitro research
use only applications such as transfection. Minicells expressing
proteins or small molecules, radioisotopes, image-enhancing
reagents can be used for in vivo diagnostics and for in vitro
diagnostic and assay platforms. Also, soluble and/or membrane
associated signaling cascade elements may be reconstituted in
minicells producing encapsulated divices to follow extracellular
stimulation events using cytoplasmic reporter events, e.g.
transactivation resulting from dimerization of dimerization
dependant transcriptional activation or repression of said
reporter.
[0220] Regarding protein expression, minicells can be engineered to
express one or more recombinant proteins in order to produce more
protein per surface area of the particle (at least 10.times. more
protein per unit surface area of protein). The proteins or small
molecules that are "displayed" on the minicell surfaces can have
therapeutic, discovery or diagnostic benefit either when injected
into a patient or used in a selective absorption mode during
dialysis. In vitro assays include drug screening and discovery,
structural proteomics, and other functional proteomics
applications. Proteins that are normally soluble can be tethered to
membrane anchoring domains or membrane proteins can be expressed
for the purpose of displaying these proteins on the surfaces of the
minicell particle in therapeutic, discovery, and diagnostic modes.
The types of proteins that can be displayed include but are not
limited to receptors (e.g., GPCRs, sphingolipid receptors,
neurotransmitter receptors, sensory receptors, growth factor
receptors, hormone receptors, chemokine receptors, cytokine
receptors, immunological receptors, and complement receptors, FC
receptors), channels (e.g., potassium channels, sodium channels,
calcium channels), pores (e.g., nuclear pore proteins, water
channels), ion and other pumps (e.g., calcium pumps, proton pumps),
exchangers (e.g., sodium/potassium exchangers, sodium/hydrogen
exchangers, potassium/hydrogen exchangers), electron transport
proteins (e.g., cytochrome oxidase), enzymes and kinases (e.g.,
protein kinases, ATPases, GTPases, phosphatases, proteases),
structural/linker proteins (e.g., Caveolins, clathrin), adapter
proteins (e.g., TRAD, TRAP, FAN), chemotactic/adhesion proteins
(e.g., ICAM11, selectins, CD34, VCAM-1, LFA-1, VLA-1), and
chimeric/fusion proteins (e.g., proteins in which a normally
soluble protein is attached to a transmembrane region of another
protein). As a non-limiting example, the small molecules that can
be tethered and displayed on the surfaces of the minicells can be
carbohydrates (e.g., monosaccharides), bioactive lipids (e.g.,
lysosphingolipids, PAF, lysophospholipids), drugs (e.g.,
antibiotics, ion channel activators/inhibitors, ligands for
receptors and/or enzymes), nucleic acids (e.g., synthetic
oligonucleotides), fluorophores, metals, or inorganic and organic
small molecules typically found in combinatorial chemistry
libraries. Minicells may either contain (encapsulate) or display on
their surfaces radionuclides or image-enhancing reagents both of
which could be used for therapeutic and/or diagnostic benefit in
vivo or for in vitro assays and diagnostic platforms.
[0221] For in vivo therapeutic uses, minicells can express proteins
and/or display small molecules on their surfaces that would either
promote an immune response and passage through the RES system
(e.g., to eliminate the minicell and its target quickly), or to
evade the RES (e.g., to increase the bioavailability of the
minicell). Toxicity is reduced or eliminated because the
therapeutic agent is not excreted or processed by the liver and
thus does not damage the kidneys or liver, because the
minicell-based therapeutic is not activated until entry into the
target cell (e.g., in the case of cancer therapeutics or gene
therapy). Minicells are of the appropriate size (from about 0.005,
0.1, 0.15 or 0.2 micrometers to about 0.25, 0.3, 0.35, 0.4, 0.45 or
0.5 micrometers) to facilitate deep penetration into the lungs in
the cases where administration of the minicell-based therapeutic or
diagnostic is via an inhalant (Strong, A. A., et al. 1987. An
aerosol generator system for inhalation delivery of pharmacological
agents. Med. Instrum. 21:189-194). This is due to the fact that
minicells can be aerosolized. Without being limited to the
following examples, inhalant therapeutic uses of minicells could be
applied to the treatment of anaphylactic shock, viral infection,
inflammatory reactions, gene therapy for cystic fibrosis, treatment
of lung cancers, and fetal distress syndrome.
[0222] Minicells can also display expressed proteins that are
enzymes that may have therapeutic and/or diagnostic uses. The
enzymes that are displayed may be soluble enzymes that are
expressed as fusion proteins with a transmembrane domain of another
protein. Display of such enzymes could be used for in vitro assays
or for therapeutic benefit.
[0223] Gene therapy applications afforded by minicells generally
involve the ability of minicells to deliver DNA to target cells
(either for replacement therapy, modifation of cell function or to
kill cells). Expression plasmids can be delivered to target cells
that would encode proteins that could be cytoplasmic or could have
intracellular signal sequences that would target the protein to a
particular organelle (e.g., mitochondria, nuclei, endoplasmic
reticulum, etc.). In the case where minicells are engulfed by the
target cell, the minicells themselves could have these
intracellular targeting sequences expressed on their surfaces so
that the minicells could be `delivered` to intracellular
targets.
[0224] Minicells used for the following therapeutic, discovery, and
diagnostic applications can be prepared as described in this
application and then stored and/or packaged by a variety of ways,
including but not limited to lyophilization, freezing, mixing with
preservatives (e.g., antioxidants, glycerol), or otherwise stored
and packaged in a fashion similar to methods used for liposome and
proteoliposome formulations.
[0225] The small size of minicells (from about 0.005, 0.1, 0.15 or
0.2 micrometers to about 0.25, 0.3, 0.35, 0.4, 0.45 or 0.5
micrometers) makes them suitable for many in vitro diagnostic
platforms, including the non-limiting examples of lateral flow,
ELISA, HTS, especially those applications requiring microspheres or
nanospheres that display many target proteins or other molecules.
The use of protoplast or poroplast minicells may be especially
useful in this regard. Assay techniques are dependent on cell or
particle size, protein (or molecule to be tested) amount displayed
on the surface of the cell or particle, and the sensitivity of the
assay being measured. In current whole-cell systems, the expression
of the protein of interest is limiting, resulting in the higher
cell number requirement to satisfy the sensitivity of most assays.
However, the relatively large size of cells prevents the
incorporation of large numbers of cells in these assays, e.g. 96,
384, and smaller well formats. In contrast, minicells, protoplasts,
and poroplasts are smaller in size and can be manipulated to
express high levels of the preselected protein, and can be
incorporated into small well assay formats.
[0226] I. Types of Minicells
[0227] Minicells are derivatives of cells that lack chromosomal DNA
and which are sometimes referred to as anucleate cells. Because
eubacterial and achreabacterial cells, unlike eukaryotic cells, do
not have a nucleus (a distinct organelle that contains
chromosomes), these non-eukaryotic minicells are more accurately
described as being "without chromosomes" or "achromosomal," as
opposed to "anucleate." Nonetheless, those skilled in the art often
use the term "anucleate" when referring to bacterial minicells in
addition to other minicells. Accordingly, in the present
disclosure, the term "minicells" encompasses derivatives of
eubacterial cells that lack a chromosome; derivatives of
archeabacterial cells that lack their chromosome(s) (Laurence et
al., Nucleoid Structure and Partition in Methanococcus jannaschii:
An Archaeon With Multiple Copies of the Chromosome, Genetics
152:1315-1323, 1999); and anucleate derivatives of eukaryotic
cells. It is understood, however, that some of the relevant art may
use the terms "anucleate minicells" or anucleate cells" loosely to
refer to any of the preceeding types of minicells.
[0228] I.A. Eubacterial Minicells
[0229] One type of minicell is a eubacterial minicell. For reviews
of eubacterial cell cycle and division processes, see Rothfield et
al., Bacterial Cell Division, Annu. Rev. Genet., 33:423-48, 1999;
Jacobs et al., Bacterial cell division: A moveable feast, Proc.
Natl. Acad. Sci. USA, 96:5891-5893, May, 1999; Koch, The
Bacterium's Way for Safe Enlargement and Division, Appl. and Envir.
Microb., Vol. 66, No. 9, pp. 3657-3663; Bouche and Pichoff, On the
birth and fate of bacterial division sites. Mol Microbiol, 1998.
29: 19-26; Khachatourians et al., Cell growth and division in
Escherichia coli: a common genetic control involved in cell
division and minicell formation. J Bacteriol, 1973. 116: 226-229;
Cooper, The Escherichia coli cell cycle. Res Microbiol, 1990. 141:
17-29; and Danachie and Robinson, "Cell Division: Parameter Values
and the Process," in: Escherichia Coli and Salmonella Typhimurium:
Cellular and Molecular Biology, Neidhardt, Frederick C., Editor in
Chief, American Society for Microbiology, Washington, D.C., 1987,
Volume 2, pages 1578-1592, and references cited therein; and
Lutkenhaus et al., "Cell Division," Chapter 101 in: Escherichia
coli and Salmonella typhimurium: Cellular and Molecular Biology,
2.sup.nd Ed., Neidhardt, Frederick C., Editor in Chief, American
Society for Microbiology, Washington, D.C., 1996, Volume 2, pages
1615-1626, and references cited therein. When DNA replication
and/or chromosomal partitioning is altered, membrane-bounded
vesicles "pinch off" from parent cells before transfer of
chromosomal DNA is completed. As a result of this type of
dysfunctional division, minicells are produced which contain an
intact outer membrane, inner membrane, cell wall, and all of the
cytoplasm components but do not contain chromosomal DNA. See Table
2.
[0230] I.B. Eukaryotic Minicells
[0231] The term "eukaryote" is defined as is used in the art, and
includes any organism classified as Eucarya that are usually
classified into four kingdoms: plants, animals, fungi and protists.
The first three of these correspond to phylogenetically coherent
groups. However, the eucaryotic protists do not form a group, but
rather are comprised of many phylogenetically disparate groups
(including slime molds, multiple groups of algae, and many distinct
groups of protozoa). See, e.g., Olsen, G.,
http://www.bact.wisc.edu/microtextbook/. A type of animal of
particular interest is a mammal, including, by way of non-limiting
example a rat, a mouse, a rabbit, a dog, a cat, a horse, a cow, a
pig, a simian and a human.
[0232] Chromosomeless eukaryotic minicells (i.e., anucleate cells)
are within the scope of the invention. Platelets are a non-limiting
example of eukaryotic minicells. Platelets are anucleate cells with
little or no capacity for de novo protein synthesis. The tight
regulation of protein synthesis in platelets (Smith et al.,
Platelets and stroke, Vasc Med 4:165-72, 1999) may allow for the
over-production of exogenous proteins and, at the same time,
under-production of endogenous proteins. Thrombin-activated
expression elements such as those that are associated with Bcl-3
(Weyrich et al., Signal-dependent translation of a regulatory
protein, Bcl-3, in activated human platelets, Cel Biology
95:5556-5561, 1998) may be used to modulate the expression of
exogenous genes in platelets.
[0233] As another non-limiting example, eukaryotic minicells are
generated from tumor cell lines (Gyongyossy-Issa and
Khachatourians, Tumour minicells: single, large vesicles released
from cultured mastocytoma cells (1985) Tissue Cell 17:801-809;
Melton, Cell fusion-induced mouse neuroblastomas HPRT revertants
with variant enzyme and elevated HPRT protein levels (1981) Somatic
Cell Genet. 7: 331-344).
[0234] Yeast cells are used to generate fungal minicells. See,
e.g., Lee et al., Ibd1p, a possible spindle pole body associated
protein, regulates nuclear division and bud separation in
Saccharomyces cerevisiae, Biochim Biophys Acta 3:239-253, 1999;
Kopecka et al., A method of isolating anucleated yeast protoplasts
unable to synthesize the glucan fibrillar component of the wall J
Gen Microbiol 81:111-120, 1974; and Yoo et al., Fission yeast Hrp1,
a chromodomain ATPase, is required for proper chromosome
segregation and its overexpression interferes with chromatin
condensation, Nucl Acids Res 28:2004-2011, 2000. Cell division in
yeast is reviewed by Gould and Simanis, The control of septum
formation in fission yeast, Genes & Dev 11:2939-51, 1997).
[0235] I.C. Archeabacterial Minicells
[0236] The term "archeabacterium" is defined as is used in the art
and includes extreme thermophiles and other Archaea. Woese, C. R.,
L. Magrum. G. Fox. 1978. Archeabacteria. Journal of Molecular
Evolution. 11:245-252. Three types of Archeabacteria are
halophiles, thermophiles and methanogens. By physiological
definition, the Archaea (informally, archaes) are single-cell
extreme thermophiles (including thermoacidophiles), sulfate
reducers, methanogens, and extreme halophiles. The thermophilic
members of the Archaea include the most thermophilic organisms
cultivated in the laboratory. The aerobic thermophiles are also
acidophilic; they oxidize sulfur in their environment to sulfuric
acid. The extreme halophiles are aerobic or microaerophilic and
include the most salt tolerant organisms known. The
sulfate-reducing Archaea reduce sulfate to sulfide in extreme
environment. Methanogens are strict anaerobes, yet they gave rise
to at least two separate aerobic groups: the halophiles and a
thermoacidophilic lineage (Olsen, G.,
http://www.bact.wisc.edu/microtextbook/). Non-limiting examples of
halophiles include Halobacterium cutirubrum and Halogerax
mediterranei. Non-limiting examples of methanogens include
Methanococcus voltae; Methanococcus vanniela; Methanobacterium
thermoautotrophicum; Methanococcus voltae; Methanothermus fervidus;
and Methanosarcina barkeri. Non-limiting examples of thermophiles
include Azotobacter vinelandii; Thermoplasma acidophilum;
Pyrococcus horikoshii; Pyrococcus furiosus; and Crenarchaeota
(extremely thermophilic archaebacteria) species such as Sulfolobus
solfataricus and Sulfolobus acidocaldarius.
[0237] Archeabacterial minicells are within the scope of the
invention. Archeabacteria have homologs of eubacterial minicell
genes and proteins, such as the MinD polypeptide from Pyrococcus
furiosus (Hayashi et al., EMBO J. 2001 20:1819-28, Structural and
functional studies of MinD ATPase: implications for the molecular
recognition of the bacterial cell division apparatus). It is thus
possible to create Archeabacterial minicells by methods such as, by
way of non-limiting example, overexpressing the product of a min
gene isolated from a prokaryote or an archeabacterium; or by
disrupting expression of a min gene in an archeabacterium of
interest by, e.g., the introduction of mutations thereof or
antisense molecules thereto. See, e.g., Laurence et al., Nucleoid
Structure and Partition in Methanococcus jannaschii: An Archaeon
With Multiple Copies of the Chromosome, Genetics 152:1315-1323,
1999.
[0238] In one aspect, the invention is drawn to archael minicells.
By physiological definition, the Archaea (informally, archaes) are
single-cell extreme thermophiles (including thermoacidophiles),
sulfate reducers, methanogens, and extreme halophiles. The
thermophilic members of the Archaea include the most thermophilic
organisms cultivated in the laboratory. The aerobic thermophiles
are also acidophilic; they oxidize sulfur in their environment to
sulfuric acid. The extreme halophiles are aerobic or
microaerophilic and include the most salt tolerant organisms known.
The sulfate-reducing Archaea reduce sulfate to sulfide in extreme
environment. Methanogens are strict anaerobes, yet they gave rise
to at least two separate aerobic groups: the halophiles and a
thermoacidophilic lineage (Olsen, G.,
http://www.bact.wisc.edu/microtextbook/).
[0239] I.D. Minicells Produced from Diverse Organisms
[0240] There are genes that can be disrupted to cause minicell
production that are conserved among the three Kingdoms. For
example, SMC (structural maintenance of chromosomes) proteins are
conserved among prokaryotes, archeabacteria and eukaryotes (Hirano,
SMC-mediated chromosome and mechanics: a conserved scheme from
bacteria to vertebrates, Genes and Dev. 13:11-19, 1999; Holmes et
al., Closing the ring: Links between SMC proteins and chromosome
partitioning, condensation, and supercoiling, PNAS 97:1322-1324,
2000; Michiko and Hiranol, EMBO J. 17:7139-7148, 1998,
ATP-dependent aggregation of single-stranded DNA by a bacterial SMC
homodimer, 1998). Mutations in B. subtilis smc genes result in the
production of minicells (Britton et al., Characterization of a
eubacterial smc protein involved in chromosome partitioning, Genes
and Dev. 12:1254-1259, 1998; Moriya et al., A Bacillus subtilis
gene-encoding protein homologous to eukaryotic SMC motor protein is
necessary for chromosome partition Mol Microbiol 29:179-87, 1998).
Disruption of smc genes in various cells is predicted to result in
minicell production therefrom.
[0241] As another example, mutations in the yeast genes encoding
TRF topoisomerases result in the production of minicells, and a
human homolog of yeast TRF genes has been stated to exist (Castano
et al., A novel family of TRF (DNA topoisomerase I-related
function) genes required for proper nuclear segregation, Nucleic
Acids Res 24:2404-10, 1996). Mutations in a yeast chromodomain
ATPase, Hrp1, result in abnormal chromosomal segregation; (Yoo et
al., "Fission yeast Hrp1, a chromogomain ATPase, is required for
proper chromosome segregation and its overexpression interferes
with chromatin condensation," Nuc. Acids Res. 28:2004-2001).
Disruption of TRF and/or Hrp1 function is predicted to cause
minicell production in various cells. Genes involved in septum
formation in fission yeast (see, e.g., Gould et al., "The control
of septum formation in fission yeast," Genes and Dev. 11:2939-2951,
1997) can be used in like fashion.
[0242] As another example, mutations in the divIVA gene of Bacillus
subtilis results in minicell production (Table 2). When expressed
in E. coli or the yeast Schizosaccharomyces pombe, a B. subtilis
DivIVA-GFP protein is targeted to cell division sites therein, even
though clear homologs of DivIVA do not seem to exist in E. coli or
S. pombe (David et al., Promiscuous targeting of Bacillus subtilis
cell division protein DivIVA to division sites in Escherichia coli
and fission yeast, EMBO J. 19:2719-2727, 2000.) Over- or
under-expression of B. subtilis DivIVA or a homolog thereof may be
used to reduce minicell production in a variety of cells.
[0243] II. Production of Minicells
[0244] Eubacterial minicells are produced by parent cells having a
mutation in, and/or overexpressing, or under expressing a gene
involved in cell division and/or chromosomal partitioning, or from
parent cells that have been exposed to certain conditions, that
result in abberant fission of bacterial cells and/or partitioning
in abnormal chromosomal segregation during cellular fission
(division). The term "parent cells" or "parental cells" refers to
the cells from which minicells are produced. Minicells, most of
which lack chromosomal DNA (Mulder et al., The Escherichia coli
minB mutation resembles gyrB in Defective nucleoid segregation and
decreased negative supercoiling of plasmids. Mol Gen Genet, 1990,
221: 87-93), are generally, but need not be, smaller than their
parent cells. Typically, minicells produced from E. coli cells are
generally spherical in shape and are about 0.1 to about 0.3 um in
diameter, whereas whole E. coli cells are about from about 1 to
about 3 um in diameter and from about 2 to about 10 um in length.
Micrographs of E. coli cells and minicells that have been stained
with DAPI (4:6-diamidino-z-phenylindole), a compound that binds to
DNA, show that the minicells do not stain while the parent E. coli
are brightly stained. Such micrographs demonstrate the lack of
chromosomal DNA in minicells. (Mulder et al., Mol. Gen. Genet.
221:87-93, 1990).
[0245] As shown in Table 2, minicells are produced by several
different mechanisms such as, by way of non-limiting example, the
over expression of genes involved in chromosomal replication and
partitioning, mutations in such genes, and exposure to various
environmental conditions. "Overexpression" refers to the expression
of a polypeptide or protein encoded by a DNA introduced into a host
cell, wherein the polypeptide or protein is either not normally
present in the host cell, or wherein the polypeptide or protein is
present in the host cell at a higher level than that normally
expressed from the endogenous gene encoding the polypeptide or
protein. For example, in E. coli cells that overexpress the gene
product FtsZ (The FtsZ gene encodes a protein that is involved in
regulation of divisions; see Cook and Rothfield, Early stages in
development of the Escherichia coli cell-division site. Mol
Microbiol, 1994. 14: p. 485-495; and Lutkenhaus, Regulation of cell
division in E. coli. Trends Genet, 1990. 6: p. 22-25), there is an
increase in the formation of minicells (Begg et al., Roles of FtsA
and FtsZ in the activation of division sites. J. Bacteriology,
1997. 180: 881-884). Minicells are also produced by E. coli cells
having a mutation in one or more genes of the min locus, which is a
group of genes that encode proteins that are involved in cell
division (de Boer et al., Central role for the Escherichia coli
minC gene product in two different cell division-inhibition
systems. Proc. Natl. Acad. Sci. USA, 1990. 87: 1129-33; Akerlund et
al., Cell division in Escherichia coli minB mutants. Mol Microbiol,
1992. 6: 2073-2083).
[0246] Prokaryotes that have been shown to produce minicells
include species of Escherichia, Shigella, Bacillus, Lactobacillus,
and Campylobacter. Bacterial minicell-producing species of
particular interest are E. coli and Bacillus subtilis. E. coli is
amenable to manipulation by a variety of molecular genetic methods,
with a variety of well-characterized expression systems, including
many episomal expression systems, factors and elements useful in
the present invention. B. subtilis, also amenable to genetic
manipulation using episomal expression elements, is an important
industrial organism involved in the production of many of the
world's industrial enzymes (proteases, amylases, etc.), which it
efficiently produces and secretes.
[0247] In the case of other eubacterial species, homologs of E.
coli or B. subtilis genes that cause minicell production therein
are known or can be identified and characterized as is known in the
art. For example, the min regions of the chromosome of
Streptococcus pneumoniae and Neisseria gonorrhoeae have been
characterized (Massidda et al., Unconventional organization of the
division and cell wall gene cluster of Streptococcus pneumoniae,
Microbiology 144:3069-78, 1998; and Ramirez-Arcos et al.,
Microbiology 147:225-237, 2001 and Szeto et al., Journal of
Bacteria 183(21):6253, 2001, respectively). Those skilled in the
art are able to isolate minicell producing (min) mutants, or
prepare compounds inhibitory to genes that induce a minicell
production (e.g., antisense to min transcripts).
TABLE-US-00002 TABLE 2 Eubacterial Strains, Mutations and
Conditions that Promote Minicell Formation Species Strain Notes
References Campylobacter jejuni may occur naturally late in growth
Brock et al., 1987 cycle Bacillus subtilis Mutations in divIVB
locus (inc. Barak et al., 1999 minC, minD ripX mutations Sciochetti
et al., 1999; Lemon et al., 2001 smc mutations Moriya et al., 1998;
Britton et al., 1998 oriC deletions Moriya et al., 1997; Hassan et
al., 1997 prfA mutations Pederson and Setlow, 2001 Mutations in
divIVA locus Cha et al., 1997 B.s. 168 ts initiation mutation
TsB143 Sargent, 1975 Bacillus cereus WSBC Induced by exposure to
long-chain Maier et al., 1999 10030 polyphosphate Shigella flexneri
(2a) MC-1 Gemski et al., 1980 S. dysenteriae (1) MC-V Gemski et
al., 1980 Lactobacillus spp. Variant minicell-producing strains
Pidoux et al., 1990 isolated from grains Neisseria gonorrhoeae
deletion or overepression of min Ramirez-Arcos et al., 2001;
homologues Szeto et al., 2001 Escherichia coli MinA mutations
Frazer et al., 1975; Cohen et al. 1976 MinB mutations and deletions
Adler et al., 1967; Davie et al., 1984; Schaumberg et al.; 1983;
Jaffe et al., 1988; Akerlund et al., 1992 CA8000 cya, crp mutations
Kumar et al.; 1979 MukA1 mutation Hiraga et al., 1996 MukE, mukF
mutations Yamanaka et al., 1996 hns mutation Kaidow et al., 1995
DS410 Heighway et al., 1989 .chi.1972, .chi. 1776 and .chi. 2076
Curtiss, 1980 P678-54 Temperature-sensitive cell division Adler et
al. 1967; Allen et al., mutations 1972; Hollenberg et al., 1976
Induced by overexpression of minB De Boer et al., 1988 protein
Induced by overexpression of minE Pichoff et al., 1995 protein or
derivatives Induced by oveproduction of ftsZ Ward et al., 1985 gene
Induced by overexpression of sdiA Wang et al., 1991 gene Induced by
overexpression of min Ramirez-Arcos et al., 2001; genes from
Neisseria gonorrhoeae Szeto et al., 2001 Induced by exposure to
EGTA Wachi et al., 1999 Legionella Pneumophila Induced by exposure
to ampicillin Elliot et al., 1985 Citations for Table 2: Adler et
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Bacteriol. 179: 1671-1683 (1997) Cohen et al., Genetics 56: 550-551
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Gen. Genet. 250: 241-251 (1996)
[0248] II.A. Optimized Minicell Construction
[0249] Minicells are produced by several different eubacterial
strains and mechanisms including the overexpression of endogenous
or exogenous genes involved in cell division, chromosomal
replication and partitioning, mutations in such genes, and exposure
to various chemical and/or physical conditions. For example, in E.
coli cells that overexpress the gene product FtsZ (the ftsZ gene
encodes a protein that is involved in regulation of cell division;
see Cook and Rothfield, Early stages in development of the
Escherichia coli cell-division site. Mol Microbiol, 1994. 14: p.
485-495; and Lutkenhaus, Regulation of cell division in E. coli.
Trends Genet, 1990. 6: p. 22-25), there is an increase in the
formation of minicells (Begg et al., Roles of FtsA and FtsZ in the
activation of division sites. J. Bacteriology, 1997. 180: 881-884).
Minicells are also produced by E. coli cells having a mutation in
one or more genes of the min locus, which is a group of genes that
encode proteins that are involved in cell division (de Boer et al.,
Central role for the Escherichia coli minC gene product in two
different cell division-inhibition systems. Proc. Natl. Acad. Sci.
USA, 1990. 87: 1129-33; Akerlund et al., Cell division in
Escherichia coli minB mutants. Mol Microbiol, 1992. 6:
2073-2083).
[0250] Eubacterial cells that have been shown to produce minicells
include, but are not limited to species of Escherichia, Shigella,
Bacillus, Lactobacillus, Legionella and Campylobacter. Bacterial
minicell-producing species of particular interest are E. coli and
Bacillus subtilis. These organisms are amenable to manipulation by
a variety of molecular and genetic methods, with a variety of
well-characterized expression systems, including many episomal and
chromosomal expression systems, as well as other factors and
elements useful in the present invention.
[0251] The following sections describe genes that may be
manipulated so as to stimulate the production of minicells. The
invention may include any of these non-limiting examples for the
purpose of preparing minicells. Furthermore, these genes and gene
products and conditions, may be used in methodologies to identify
other gene(s), gene products, biological events, biochemical
events, or physiological events that induce or promote the
production of minicells. These methodologies include, but are not
limited to genetic selection, protein, nucleic acid, or
combinatorial chemical library screen, one- or two-hybrid analysis,
display selection technologies, e.g. phage or yeast display,
hybridization approaches, e.g. array technology, and other high- or
low-throughput approaches.
[0252] II.A.1. Homologs
[0253] Homologs of these genes and gene products from other
organisms may also be used. As used herein, a "homolog" is defined
is a nucleic acid or protein having a nucleotide sequence or amino
acid sequence, respectively, that is "identical," "essentially
identical," "substantially identical," "homologous" or "similar"
(as described below) to a reference sequence which may, by way of
non-limiting example, be the sequence of an isolated nucleic acid
or protein, or a consensus sequence derived by comparison of two or
more related nucleic acids or proteins, or a group of isoforms of a
given nucleic acid or protein. Non-limiting examples of types of
isoforms include isoforms of differing molecular weight that result
from, e.g., alternate RNA splicing or proteolytic cleavage; and
isoforms having different post-translational modifications, such as
glycosylation; and the like.
[0254] Two sequences are said to be "identical" if the two
sequences, when aligned with each other, are exactly the same with
no gaps, substitutions, insertions or deletions.
[0255] Two sequences are said to be "essentially identical" if the
following criteria are met. Two amino acid sequences are
"essentially identical" if the two sequences, when aligned with
each other, are exactly the same with no gaps, insertions or
deletions, and the sequences have only conservative amino acid
substitutions. Conservative amino acid substitutions are as
described in Table 3.
TABLE-US-00003 TABLE 3 CONSERVATIVE AMINO ACID SUBSTITUTIONS Type
of Amino Groups of Amino Acids that Are Conservative Acid Side
Chain Substitutions Relative to Each Other Short side chain
Glycine, Alanine, Serine, Threonine and Methionine Hydrophobic
Leucine, Isoleucine and Valine Polar Glutamine and Asparagine
Acidic Glutamic Acid and Aspartic Acid Basic Arginine, Lysine and
Histidine Aromatic Phenylalanine, Tryptophan and Tyrosine
[0256] Two nucleotide sequences are "essentially identical" if they
encode the identical or essentially identical amino acid sequence.
As is known in the art, due to the nature of the genetic code, some
amino acids are encoded by several different three base codons, and
these codons may thus be substituted for each other without
altering the amino acid at that position in an amino acid sequence.
In the genetic code, TTA, TTG, CTT, CTC, CTA and CTG encode Leu;
AGA, AGG, CGT, CGC, CGA and CGG encode Arg; GCT, GCC, GCA and GCG
encode Ala; GGT, GGC, GGA and GGG encode Gly; ACT, ACC, ACA and ACG
encode Thr; GTT, GTC, GTA and GTG encode Val; TCT, TCC, TCA and TCG
encode Ser; CCT, CCC, CCA and CCG encode Pro; ATA, ATC and ATA
encode Ile; GAA and GAG encode Glu; CAA and CAG encode Gln; GAT and
GAC encode Asp; AAT and AAC encode Asn; AGT and AGC encode Ser; TAT
and TAC encode Tyr; TGT and TGC encode Cys; AAA and AAG encode Lys;
CAT and CAC encode His; TTT and TTC encode Phe, TGG encodes Trp;
ATG encodes Met; and TGA, TAA and TAG are translation stop
codons.
[0257] Two amino acid sequences are "substantially identical" if,
when aligned, the two sequences are, (i) less than 30%, preferably
.ltoreq.20%, more preferably .ltoreq.15%, most preferably
.ltoreq.10%, of the identities of the amino acid residues vary
between the two sequences; (ii) the number of gaps between or
insertions in, deletions of and/or substitutions of, is
.ltoreq.10%, more preferably .ltoreq.5%, more preferably
.ltoreq.3%, most preferably .ltoreq.1%, of the number of amino acid
residues that occur over the length of the shortest of two aligned
sequences.
[0258] Two sequences are said to be "homologous" if any of the
following criteria are met. The term "homolog" includes without
limitation orthologs (homologs having genetic similarity as the
result of sharing a common ancestor and encoding proteins that have
the same function in different species) and paralog (similar to
orthologs, yet gene and protein similarity is the result of a gene
duplication).
[0259] One indication that nucleotide sequences are homologous is
if two nucleic acid molecules hybridize to each other under
stringent conditions. Stringent conditions are sequence dependent
and will be different in different circumstances. Generally,
stringent conditions are selected to be about 5.degree. C. lower
than the thermal melting point (Tm) for the specific sequence at a
defined ionic strength and pH. The Tm is the temperature (under
defined ionic strength and pH) at which 50% of the target sequence
hybridizes to a perfectly matched probe. Typically, stringent
conditions will be those in which the salt concentration is about
0.02 M at pH 7 and the temperature is at least about 60.degree.
C.
[0260] Another way by which it can be determined if two sequences
are homologous is by using an appropriate algorithm to determine if
the above-described criteria for substantially identical sequences
are met. Sequence comparisons between two (or more) polynucleotides
or polypeptides are typically performed by algorithms such as, for
example, the local homology algorithm of Smith and Waterman (Adv.
Appl. Math. 2:482, 1981); by the homology alignment algorithm of
Needleman and Wunsch (J. Mol. Biol. 48:443, 1970); by the search
for similarity method of Pearson and Lipman (Proc. Natl. Acad. Sci.
U.S.A. 85:2444, 1988); and by computerized implementations of these
algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, version 10.2 Genetics Computer Group
(GCG), 575 Science Dr., Madison, Wis.); BLASTP, BLASTN, and FASTA
(Altschul et al., J. Mol. Biol. 215:403-410, 1990); or by visual
inspection.
[0261] Optimal alignments are found by inserting gaps to maximize
the number of matches using the local homology algorithm of Smith
and Waterman (1981) Adv. Appl. Math. 2:482-489. "Gap" uses the
algorithm of Needleman and Wunsch (1970 J. Mol. Biol. 48:443-453)
to find the alignment of two complete sequences that maximizes the
number of matches and minimizes the number of gaps. In such
algorithms, a "penalty" of about 3.0 to about 20 for each gap, and
no penalty for end gaps, is used.
[0262] Homologous proteins also include members of clusters of
orthologous groups of proteins (COGs), which are generated by
phylogenetic classification of proteins encoded in complete
genomes. To date, COGs have been delineated by comparing protein
sequences encoded in 43 complete genomes, representing 30 major
phylogenetic lineages. Each COG consists of individual proteins or
groups of paralogs from at least 3 lineages and thus corresponds to
an ancient conserved domain (see Tatusov et al., A genomic
perspective on protein families. Science, 278: 631-637, 1997;
Tatusov et al., The COG database: new developments in phylogenetic
classification of proteins from complete genomes, Nucleic Acids
Res. 29:22-28, 2001; Chervitz et al., Comparison of the Complete
Sets of Worm and Yeast: Orthology and Divergence, Science
282:2022-2028, 1998; and http://www.ncbi.nlm.nih.gov/COG/).
[0263] The entirety of two sequences may be identical, essentially
identical, substantially identical, or homologous to one another,
or portions of such sequences may be identical or substantially
identical with sequences of similar length in other sequences. In
either case, such sequences are similar to each other. Typically,
stretches of identical or essentially within similar sequences have
a length of .gtoreq.12, preferably .gtoreq.24, more preferably
.gtoreq.48, and most preferably .gtoreq.96 residues.
[0264] II.A.2. Escherichia Coli Genes
[0265] Exemplary genes and gene products from E. coli the
expression and/or sequence of which can be manipulated so as to
stimulate minicell production in E. coli or any other organism, as
can homologs thereof from any species, include without limitation,
the bolA gene (Aldea, M., et al. 1988. Identification, cloning, and
expression of bolA, an ftsZ-dependent morphogene of Escherichia
coli. J. Bacteriol. 170:5196-5176; Aldea, M., et al. 1990. Division
genes in Escherichia coli are expressed coordinately to cell septum
requirements by gearbox promoters. EMBO J. 9:3787-3794); the chpA
gene (Masuda, Y., et al. 1993. chpA and chpB, Escherichia coli
chromosomal homologs of the pem locus responsible for stable
maintenance of plasmid R100. J. Bacteriol. 175:6850-6856); the chpB
gene (Masuda, Y., et al. 1993. chpA and chpB, Escherichia coli
chromosomal homologs of the pem locus responsible for stable
maintenance of plasmid R100. J. Bacteriol. 175:6850-6856); the chpR
(chpAI) gene (Masuda, Y., et al. 1993. chpA and chpB, Escherichia
coli chromosomal homologs of the pem locus responsible for stable
maintenance of plasmid R100. J. Bacteriol. 175:6850-6856); the chpS
(chpBI)gene (Masuda, Y., et al. 1993. chpA and chpB, Escherichia
coli chromosomal homologs of the pem locus responsible for stable
maintenance of plasmid R100. J. Bacteriol. 175:6850-6856); the crg
gene (Redfield, R. J., and A. M. Campbell. 1987. Structure of
cryptic lambda prophages. J. Mol. Biol. 198:393-404); the crp gene
(Kumar, S., et al. 1979. Control of minicell producing cell
division by cAMP-receptor protein complex in Escherichia coli. Mol.
Gen. Genet. 176:449-450); the cya gene (Kumar, S., et al. 1979.
Control of minicell producing cell division by cAMP-receptor
protein complex in Escherichia coli. Mol. Gen. Genet. 176:449-450);
the dicA gene (Labie, C., et al. 1989. Isolation and mapping of
Escherichia coli mutations conferring resistance to division
inhibition protein DicB. J. Bacteriol. 171:4315-4319); the dicB
gene (Labie, C., et al. 1989. Isolation and mapping of Escherichia
coli mutations conferring resistance to division inhibition protein
DicB. J. Bacteriol. 171:4315-4319; Labie, C., et al. 1990.
Minicell-forming mutants of Escherichia coli: suppression of both
DicB- and MinD-dependent division inhibition by inactivation of the
minC gene product. J. Bacteriol. 1990. 172:5852-5858); the dicC
gene (Bejar, S., et al. 1988. Cell division inhibition gene dicB is
regulated by a locus similar to lambdoid bacteriophage immunity
loci. Mol. Gen. Genet. 212:11-19); the dicF gene (Tetart, F., and
J. P. Bouche. 1992. Regulation of the expression of the cell-cycle
gene ftsZ by DicF antisense RNA. Division does not require a fixed
number of FtsZ molecules. Mol. Microbiol. 6:615-620); the dif gene
(Kuempel, P. L., et al. 1991. dif, a recA-independent recombination
site in the terminus region of the chromosome of Escherichia coli.
New Biol. 3:799-811); the dksA gene (Yamanaka, K., et al. 1994.
Cloning, sequencing, and characterization of multicopy suppressors
of a mukB mutation in Escherichia coli. Mol. Microbiol.
13:301-312); the dnaK gene (Paek, K. H., and G. C. Walker. 1987.
Escherichia coli dnaK null mutants are invariable at high
temperature. J. Bacteriol. 169:283-290); the dnaJ gene (Hoffman, H.
J., et al. 1992. Activity of the Hsp70 chaperone complex--DnaK,
DnaJ, and GrpE--in initiating phage lambda DNA replication by
sequestering and releasing lambda P protein. Proc. Natl. Acad. Sci.
89:12108-12111); the fcsA gene (Kudo, T., et al. 1977.
Characteristics of a cold-sensitive cell division mutant
Escherichia coli K-12. Agric. Biol. Chem. 41:97-107); the fic gene
(Utsumi, R., et al. 1982. Involvement of cyclic AMP and its
receptor protein in filamentation of an Escherichia coli fic
mutant. J. Bacteriol. 151:807-812; Komano, T., et al. 1991.
Functional analysis of the fic gene involved in regulation of cell
division. Res. Microbiol. 142:269-277); the fis gene
(Spaeny-Dekking, L. et al. 1995. Effects of N-terminal deletions of
the Escherichia coli protein F is on the growth rate, tRNA (2Ser)
expression and cell morphology. Mol. Gen. Genet. 246:259-265); the
ftsA gene (Bi, E., and J. Lutkenhaus. 1990. Analysis of ftsZ,
mutations that confer resistance to the cell division inhibitor
SulA (SfiA). J. Bacterial. 172:5602-5609; Dai, K, and J.
Lutkenhaus. 1992. The proper ration of FtsZ to FtsA is required for
cell division to occur in Escherichia coli. J. Bacteriol.
174:6145-6151); the ftsE gene (Taschner, P. E. et al. 1988.
Division behavior and shape changes in isogenic ftsZ, ftsQ, ftsA,
pbpB, and ftsE cell division mutants of Escherichia coli during
temperature shift experiments. J. Bacteriol. 170:1533-1540); the
ftsH gene (Ogura, T. et al. 1991. Structure and function of the
ftsH gene in Escherichia coli. Res. Microbiol. 142:279-282); the
ftsI gene (Begg, K. J., and W. D. Donachie. 1985. Cell shape and
division in Escherichia coli: experiments with shape and division
mutants. J. Bacteriol. 163:615-622); the ftsJ gene (Ogura, T. et
al. 1991. Structure and function of the ftsH gene in Escherichia
coli. Res. Microbiol. 142:279-282); the ftsL gene (Guzman, et al.
1992. FtsL, an essential cytoplasmic membrane protein involved in
cell division in Escherichia coli. J. Bacteriol. 174:7716-7728);
the ftsN gene (Dai, K. et al. 1993. Cloning and characterization of
ftsN, an essential cell division gene in Escherichia coli isolated
as a multicopy suppressor of ftsA12(Ts). J. Bacteriol.
175:3790-3797); the ftsQ gene (Wang, X. D. et al. 1991. A factor
that positively regulates cell division by activating transcription
of the major cluster of essential cell division genes of
Escherichia coli. EMBO J. 10:3362-3372); the ftsW gene (Khattar, M.
M. et al. 1994. Identification of FtsW and characterization of a
new ftsW division mutant of Escherichia coli. J. Bacteriol.
176:7140-7147); the ftsX (ftsS) gene (Salmond, G. P. and S.
Plakidou. 1984. Genetic analysis of essential genes in the ftsE
region of the Escherichia coli genetic map and identification of a
new cell division gene, ftsS. Mol. Gen. Genet. 197:304-308); the
ftsY gene (Gill, D. R. and G. P. Salmond. 1990. The identification
of the Escherichia coli ftsY gene product: an unusual protein. Mol.
Microbiol. 4:575-583); the ftsZ gene (Ward, J. E., and J.
Lutkenhaus. 1985. Overproduction of FtsZ induces minicell
formation. Cell. 42:941-949; Bi, E., and J. Lutkenhaus. 1993. Cell
division inhibitors SulA and MinCD prevent formation of the FtsZ
ring. J. Bacteriol. 175:1118-1125); the gyrB gene (Mulder, E., et
al. 1990. The Escherichia coli minB mutation resembles gyrB in
defective nucleoid segregation and decreased negative supercoiling
of plasmids. Mol. Gen. Genet. 221:87-93); the hlfB (ftsH)gene
(Herman, C., et al. 1993. Cell growth and lambda phage development
controlled by the same essential Escherichia coli gene, ftsH/hflB.
Proc. Natl. Acad. Sci. 90:10861-10865); the hfq gene (Takada, A.,
et al. 1999. Negative regulatory role of the Escherichia coli hfq
gene in cell division. Biochem. Biophys. Res. Commun. 266:579-583;
the hipA gene (Scherrer, R., and H. S. Moyed. 1988. Conditional
impairment of cell division and altered lethality in hipA mutants
of Escherichia coli K-12. J. Bacteriol. 170:3321-3326); the hipB
gene (Hendricks, E. C., et al. 2000. Cell division, guillotining of
dimer chromosomes and SOS induction in resolution mutants (dif,
xerC and xerD) of Escherichia coli. Mol. Microbiol. 36:973-981);
the hns gene (Kaidow, A., et al. 1995. Anucleate cell production by
Escherichia coli delta hns mutant lacking a histone-like protein,
H--NS. J. Bacteriol. 177:3589-3592); the htrB gene (Karow, M., et
al. 1991. Complex phenotypes of null mutations in the htr genes,
whole products are essential for Escherichia coli growth at
elevated temperatures. Res. Microbiol. 142:289-294); the lpxC
(envA)gene (Beall, B., and J. Lutkenhaus. 1987. Sequence analysis,
transcriptional organization, and insertional mutagenesis of the
envA gene of Escherichia coli. J. Bacteriol. 169:5408-5415; Young,
K., et al. 1995. The envA permeability/cell division gene of
Escherichia coli encodes the second enzyme of lipid A biosynthesis.
UDP-3-O--(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase. J.
Biol. Chem. 270:30384-30391); the malE gene (Pichoff, S., et al.
1997. MinCD-independent inhibition of cell division by a protein
that fuses MalE to the topological specificity factor MinE. J.
Bacteriol. 179:4616-4619); the minA gene (Davie, E., et al. 1984.
Genetic basis of minicell formation in Escherichia coli K-12. J.
Bacteriol. 158:1202-1203); the minB gene (Davie, E., et al. 1984.
Genetic basis of minicell formation in Escherichia coli K-12. J.
Bacteriol. 158:1202-1203); the minC gene (de Boer, P. A., et al.
1990. Central role for the Escherichia coli minC gene product in
two different cell division-inhibition systems. Proc. Natl. Acad.
Sci. 87:1129-1133); the minD gene (Labie, C., et al. 1990.
Minicell-forming mutants of Escherichia coli: suppression of both
DicB- and MinD-dependent division inhibition by inactivation of the
minC gene product. J. Bacteriol. 172:5852-5855; Hayashi, I., et al.
2001. Structural and functional studies of MinD ATPase:
implications for the molecular recognition of the bacterial cell
division apparatus. EMBO J. 20:1819-1828); the minE gene (de Boer,
P. A., et al. 1989. A division inhibitor and a topological
specificity factor coded for by the minicell locus determine proper
placement of the division septum in E. coli. Cell. 56:641-649); the
mreB gene (Doi, M., et al. 1988. Determinations of the DNA sequence
of the mreB gene and of the gene products of the mre region that
function in formation of the rod shape of Escherichia coli cells.
J. Bacteriol. 170:4619-4624); the mreC gene (Wachi, M., et al.
1989. New mre genes mreC and mreD, responsible for formation of the
rod shape of Escherichia coli cells. J. Bacteriol. 171:6511-6516);
the mreD gene (Wachi, M., et al. 1989. New mre genes mreC and mreD,
responsible for formation of the rod shape of Escherichia coli
cells. J. Bacteriol. 171:6511-6516); the mukA gene (Hiraga, S., et
al. 1989. Chromosome partitioning in Escherichia coli: novel
mutants producing anucleate cells. J. Bacteriol. 171:1496-1505);
the mukB gene (Hiraga, S., et al. 1991. Mutants defective in
chromosome partitioning in E. coli. Res. Microbiol. 142:189-194);
the mukE gene (Yamanaka, K., et al. 1996. Identification of two new
genes, mukE and mukF, involved in chromosome partitioning in
Escherichia coli. Mol. Gen. Genet. 250:241-251; Yamazoe, M., et al.
1999. Complex formation of MukB, MukE and MukF proteins involved in
chromosome partitioning in Escherichia coli. EMBO J. 18:5873-5884);
the mukF gene (Yamanaka, K., et al. 1996. Identification of two new
genes, mukE and mukF, involved in chromosome partitioning in
Escherichia coli. Mol. Gen. Genet. 250:241-251; Yamazoe, M., et al.
1999. Complex formation of MukB, MukE and MukF proteins involved in
chromosome partitioning in Escherichia coli. EMBO J. 18:5873-5884);
the parC gene (Kato, J., et al. 1988. Gene organization in the
region containing a new gene involved in chromosome partition in
Escherichia coli. J. Bacteriol. 170:3967-3977); the parE gene
(Roberts, R. C., et al. 1994. The parDE operon of the
broad-host-range plasmid RK2 specifies growth inhibition associated
with plasmid loss. J. Mol. Biol. 237:35-51); the pbpA gene
(Rodriguez, M. C., and M. A. de Pedro. 1990. Initiation of growth
in pbpAts and rodAts mutants of Escherichia coli. FEMS Microbiol.
Lett. 60:235-239); the pcnB gene (Makise, M., et al. 1999.
Identification of a high-copy-number plasmid suppressor of a lethal
phenotype caused by mutant DnaA protein which has decreased
intrinsic ATPase activity. Biol. Pharm. Bull. 22:904-909); the parF
(plsC in E. coli) gene product from Salmonella (Luttinger, A. L.,
et al. 1991. A cluster of genes that affects nucleoid segregation
in Salmonella typhimurium. New Biol. 3:687-697); the rpoS gene
(Cam, K., et al. 1995. Sigma S-dependent overexpression of ftsZ in
an Escherichia coli K-12 rpoB mutant that is resistant to the
division inhibitors DicB and DicF RNA. Mol. Gen. Genet.
248:190-194); the rcsB gene (Gervais, F. G., et al. 1992. The rcsB
gene, a positive regulator of colanic acid biosynthesis in
Escherichia coli, is also an activator of ftsZ expression. J.
Bacteriol. 174:3964-3971); the rcsF gene (Gervais, F. G., and G. R.
Drapeau. 1992. Identification, cloning, and characterization of
rcsF, a new regulator gene for exopolysaccharide synthesis that
suppresses the division mutation ftsZ84 in Escherichia coli K-12.
J. Bacteriol. 174:8016-8022); the rodA gene (Rodriguez, M. C., and
M. A. de Pedro. 1990. Initiation of growth in pbpAts and rodAts
mutants of Escherichia coli. FEMS Microbiol. Lett. 60:235-239); the
sdiA (sulB, sfiB) gene (Wang, X. D., et al. 1991. A factor that
positively regulates cell division by activating transcription of
the major cluster of essential cell division genes of Escherichia
coli. EMBO J. 10:3363-3372); the sefA (fabZ) gene (Mohan, S., et
al. 1994. An Escherichia coli gene (FabZ) encoding
(3R)-hydroxymyristoyl acyl carrier protein dehydrase. Relation to
fabA and suppression of mutations in lipid A biosynthesis. J. Biol.
Chem. 269:32896-32903); the sfiC gene (D' Ari, R., and O. Huisman.
1983. Novel mechanism of cell division inhibition associated with
the SOS response in Escherichia coli. J. Bacteriol. 156:243-250);
the sulA gene (Bi, E., and J. Lutkenhaus. 1990. Interaction between
the min locus and ftsZ. J. Bacteriol. 172:5610-5616; Bi, E., and J.
Lutkenhaus. 1993. Cell division inhibitors SulA and MinCD prevent
formation of the FtsZ ring. J. Bacteriol. 175:1118-1125); the stfZ
gene (Dewar, S. J., and W. D. Donachie. 1993. Antisense
transcription of the ftsZ-ftsA gene junction inhibits cell division
in Escherichia coli. J. Bacteriol. 175:7097-7101); the tolC gene
(Hiraga, S., et al. 1989. Chromosome partitioning in Escherichia
coli: novel mutants producing anucleate cells. J. Bacteriol.
171:1496-1505; Hiraga, S., et al. 1991. Mutants defective in
chromosome partitioning in E. coli. Res. Microbiol. 142:189-194);
and the zipA gene (Hale, C. A., and P. A. de Boer. 1997. Direct
binding of FtsZ to ZipA, an essential component of the septal ring
structure that mediates cell division in E. coli. Cell.
88:175-185).
[0266] The guanosine 5'-diphosphate 3' diphosphate (ppGpp) or
guanosine 5'-triphosphate 3' diphosphate (pppGpp) nucleotides,
collectively (p)ppGpp, found in E. coli or in other members of the
Eubacteria, Eucarya or Archaea may be employed to produce minicells
(Vinella, D., et al. 1993. Penicillin-binding protein 2
inactivation in Escherichia coli results in cell division
inhibition, which is relieved by FtsZ overexpression. J. Bacteriol.
175:6704-6710; Navarro, F., et al. Analysis of the effect of ppGpp
on the ftsQAZ operon in Escherichia coli. Mol. Microbiol.
29:815-823). The levels, or rate of production of (p)ppGpp may be
increased or decreased. By way of non-limiting example, increased
(p)ppGpp production results from induction of the stringent
response. The stringent response in E. coli is a physiological
response elicited by a failure of the capacity for tRNA
aminoacylation to keep up with the demands of protein synthesis.
This response can be provoked either by limiting the availability
of amino acids or by limiting the ability to aminoacylate tRNA even
in the presence of abundant cognate amino acids. Many features of
the stringent response behave as if they are mediated by
accumulation of (p)ppGpp. The accumulation of (p)ppGpp can also be
provoked by nutritional or other stress conditions in addition to a
deficiency of aminoacyl-tRNA. See Cashel et al., "The Stringent
Response," Chapter 92 in: Escherichia coli and Salmonella
typhimurium: Cellular and Molecular Biology, 2nd Ed., Neidhardt,
Frederick C., Editor in Chief, American Society for Microbiology,
Washington, D.C., 1996, Volume 1, pages 1458-1496, and references
cited therein.
[0267] By way of non-limiting example, factors that may provoke the
stringent response include the lyt gene or gene product (Harkness,
R. E., et al. 1992. Genetic mapping of the lytA and lytB loci of
Escherichia coli, which are involved in penicillin tolerance and
control of the stringent response. Can J. Microbiol. 38:975-978),
the relA gene or gene product (Vinella, D., and R. D'Ari. 1994.
Thermoinducible filamentation in Escherichia coli due to an altered
RNA polymerase beta subunit is suppressed by high levels of ppGpp.
J. Bacteriol. 176:96-972), the relB gene or gene product
(Christensen, S. K., et al. 2001. RelE, a global inhibitor of
translation, is activated during nutritional stress. Proc. Natl.
Acad. Sci. 98:14328-14333), the relC (rplK) gene or gene product
(Yang, X., and E. E. Ishiguro. 2001. Involvement of the N Terminus
of Ribosomal Protein L11 in Regulation of the RelA Protein of
Escherichia coli. J. Bacteriol. 183:6532-6537), the relX gene or
gene product (St. John, A. C., and A. L. Goldberg. 1980. Effects of
starvation for potassium and other inorganic ions on protein
degradation and ribonucleic acid synthesis in Escherichia coli. J.
Bacteriol. 143:1223-1233), the spoT gene or gene product (Vinella,
D., et al. 1996. Mecillinam resistance in Escherichia coli is
conferred by loss of a second activity of the AroK protein. J.
Bacteriol. 178:3818-3828), the gpp gene or gene product (Keasling,
J. D., et al. 1993. Guanosine pentaphosphate phosphohydrolase of
Escherichia coli is a long-chain exopolyphosphatase. Proc. Natl.
Acad. Sci. 90:7029-7033), the ndk gene or gene product (Kim, H. Y.,
et al. 1998. Alginate, inorganic polyphosphate, GTP and ppGpp
synthesis co-regulated in Pseudomonas aeruginosa: implications for
stationary phase survival and synthesis of RNA/DNA precursors. Mol.
Microbiol. 27:717-725), the rpoB gene or gene product (Vinella, D.,
and R. D' Ari. 1994. Thermoinducible filamentation in Escherichia
coli due to an altered RNA polymerase beta subunit is suppressed by
high levels of ppGpp. J. Bacteriol. 176:96-972), the rpoC gene or
gene product (Bartlett, M. S., et al. 1998. RNA polymerase mutants
that destabilize RNA polymerase-promoter complexes alter NTP--
sensing by rrn P1 promoters. J. Mol. Biol. 279:331-345), the rpoD
gene or gene product (Hernandez, V. J., and M. Cashel. 1995.
Changes in conserved region 3 of Escherichia coli sigma 70 mediate
ppGpp-dependent functions in vivo. 252:536-549), glnF gene or gene
product (Powell, B. S., and D. L. Court. 1998. Control of ftsZ
expression, cell division, and glutamine metabolism in
Luria-Bertani medium by the alarmone ppGpp in Escherichia coli. J.
Bacteriol. 180:1053-1062), or glnD gene or gene product (Powell, B.
S., and D. L. Court. 1998. Control of ftsZ expression, cell
division, and glutamine metabolism in Luria-Bertani medium by the
alarmone ppGpp in Escherichia coli. J. Bacteriol. 180:1053-1062).
These genes or gene products, and/or expression thereof, may be
manipulated to create minicells.
[0268] II.A.3. Bacillus Subtilis Genes
[0269] Exemplary genes and gene products from B. subtilis, the
expression and/or sequence of which can be manipulated so as to
stimulate minicell production in B. subtilis or any other organism,
as can homologs thereof from any species, include without
limitation, the divI (divD)gene (Van Alstyne, D., and M. I. Simon.
1971. Division mutants of Bacillus subtilis: isolation of PBS1
transduction of division-specific markers. J. Bacteriol.
108:1366-1379); the divIB (dds, ftsQ) gene (Harry, E. J., et al.
1993. Characterization of mutations in divIB of Bacillus subtilis
and cellular localization of the DivIB protein. Mol. Microbiol.
7:611-621; Harry E. J., et al. 1994. Expression of divIB of
Bacillus subtilis during vegetative growth. J. Bacteriol.
176:1172-1179); the divIC gene product from B. subtilis or
homologues of this gene or gene product found in other members of
the Eubacteria, Eucarya or Archaea may be employed to produce
minicells (Levin, P. A., and R. Losick. 1994. Characterization of a
cell division gene from Bacillus subtilis that is required for
vegetative and sporulation septum formation. J. Bacteriol.
176:1451-1459; Katis, V. L., et al. 1997. The Bacillus subtilis
division protein DivIC is a highly abundant membrane-bound protein
that localizes to the division site; the divII (divC) gene (Van
Alstyne, D., and M. I. Simon. 1971. Division mutations of Bacillus
subtilis: isolation and PBS1 transduction of division-specific
markers. J. Bacteriol. 108:1366-1379); the divIVA (divD) gene (Cha,
J.-H., and G. C. Stewart. 1997. The divIVA minicell locus of
Bacillus subtilis. J. Bacteriol. 179:1671-1683); the divIVC (divA)
gene (Van Alstyne, D., and M. I. Simon. 1971. Division mutations of
Bacillus subtilis: isolation and PBS1 transduction of
division-specific markers. J. Bacteriol. 108:1366-1379); the divV
(divB) gene (Van Alstyne, D., and M. I. Simon. 1971. Division
mutations of Bacillus subtilis: isolation and PBS1 transduction of
division-specific markers. J. Bacteriol. 108:1366-1379); the erzA
(ytwP) gene (Levin, P. A., et al. 1999. Identification and
regulation of a negative regulator of FtsZ ring formation in
Bacillus subtilis. Proc. Natl. Acad. Sci. 96:9642-9647); the ftsA
(spoIIN) gene (Feucht, A., et al. 2001. Cytological and biochemical
characterization of the FtsA cell division protein of Bacillus
subtilis. Mol. Microbiol. 40:115-125); the ftsE gene (Yoshida, K.,
et al. 1994. Cloning and nucleotide sequencing of a 15 kb region of
the Bacillus subtilis genome containing the iol operon.
Microbiology. 140:2289-2298); the ftsH gene (Deuerling. E., et al.
1995. The ftsH gene of Bacillus subtilis is transiently induced
after osmotic and temperature upshift. J. Bacteriol. 177:4105-4112;
Wehrl, W., et al. 2000. The FtsH protein accumulates at the septum
of Bacillus subtilis during cell division and sporulation. J.
Bacteriol. 182:3870-3873); the ftsK gene (Sciochetti, S. A., et al.
2001. Identification and characterization of the dif Site from
Bacillus subtilis. J. Bacteriol. 183:1058-1068); the ftsL
(yIID)gene (Daniel, R. A., et al. 1998. Characterization of the
essential cell division gene ftsL (yIID) of Bacillus subtilis and
its role in the assembly of the division apparatus. Mol. Microbiol.
29:593-604); the ftsW gene (Ikeda, M., et al. 1989. Structural
similarity among Escherichia coli FtsW and RodA proteins and
Bacillus subtilis SpoVE protein, which function in cell division,
cell elongation, and spore formation, respectively. J. Bacteriol.
171:6375-6378); the ftsX gene (Reizer, J., et al. 1998. A novel
protein kinase that controls carbon catabolite repression in
bacteria. Mol. Microbiol. 27:1157-1169); the ftsZ gene (Beall, B.,
and J. Lutkenhaus). FtsZ in Bacillus subtilis is required for
vegetative septation and for asymmetric septation during
sporulation. Genes and Dev. 5:447-45); the gcaD gene (Hove-Jensen,
B. 1992. Identification of tms-26 as an allele of the gcaD gene,
which encodes N-acetylglucosamine 1-phosphate uridyltransferase in
Bacillus subtilis. J. Bacteriol. 174:6852-6856); the gid (ylyC)
gene (Kunst, F., et al. 1997. The complete genome sequence of the
gram-positive bacterium Bacillus subtilis. Nature. 390:237-238);
the gidA gene (Ogasawara, N., and H. Yoshikawa. 1992. Genes and
their organization in the replication origin region of the
bacterial chromosome. Mol. Microbiol. 6:629-634; Nakayashiki, T.,
and H. Inokuchi. 1998. Novel temperature-sensitive mutants of
Escherichia coli that are unable to grow in the absence of
wild-type tRNA6Leu. J. Bacteriol. 180:2931-2935); the gidB gene
(Ogasawara, N., and H. Yoshikawa. 1992. Genes and their
organization in the replication origin region of the bacterial
chromosome. Mol. Microbiol. 6:629-634; Nakayashiki, T., and H.
Inokuchi. 1998. Novel temperature-sensitive mutants of Escherichia
coli that are unable to grow in the absence of wild-type tRNA6Leu.
J. Bacteriol. 180:2931-2935); the lytC (cwlB) gene (Blackman, S.
A., et al. 1998. The role of autolysins during vegetative growth of
Bacillus subtilis 168. Microbiology. 144:73-82); the lytD (cwlG)
gene (Blackman, S. A., et al. 1998. The role of autolysins during
vegetative growth of Bacillus subtilis 168. Microbiology.
144:73-82); the lytE (cwlF) gene (Ishikawa, S., et al. 1998.
Regulation of a new cell wall hydrolase gene, cwlF, which affects
cell separation in Bacillus subtilis. J. Bacteriol.
180:23549-2555); the lytF (cwlE, yhdD) gene (Ohnishi, R., et al.
1999. Peptidoglycan hydrolase lytF plays a role in cell separation
with CwlF during vegetative growth of Bacillus subtilis. J.
Bacteriol. 181:3178-1384); the maf gene (Butler, Y. X., et al.
1993. Amplification of the Bacillus subtilis maf gene results in
arrested septum formation. J. Bacteriol. 175:3139-3145); the minC
gene (Varley, A. W., and G. C. Stewart. 1992. The divIVB region of
the Bacillus subtilis chromosome encodes homologs of Escherichia
coli septum placement (minCD) and cell shape (mreBCD) determinants.
J. Bacteriol. 174:6729-6742; Barak, I., et al. 1998. MinCD proteins
control the septation process during sporulation of Bacillus
subtilis. J. Bacteriol. 180:5327-5333); the minD gene (Varley, A.
W., and G. C. Stewart. 1992. The divIVB region of the Bacillus
subtilis chromosome encodes homologs of Escherichia coli septum
placement (minCD) and cell shape (mreBCD) determinants. J.
Bacteriol. 174:6729-6742; Barak, I., et al. 1998. MinCD proteins
control the septation process during sporulation of Bacillus
subtilis. J. Bacteriol. 180:5327-5333); the pbpB gene (Daniel, R.
A., and J. Errington. 2000. Intrinsic instability of the essential
cell division protein FtsL of Bacillus subtilis and a role for
DivIB protein FtsL turnover. Mol. Microbiol. 35:278-289); the ponA
gene (Pederson, L. B., et al. Septal localization of
penicillin-binding protein 1 in Bacillus subtilis. J. Bacteriol.
181:3201-3211); the prfA gene (Popham, D. L., and P. Setlow. 1995.
Cloning, nucleotide sequence, and mutagenesis of the Bacillus
subtilis ponA operon, which codes for penicillin-binding protein
(PBP) 1 and a PBP-related factor. J. Bacteriol. 177:326-335); the
rodB gene (Burdett, I. D. 1979. Electron microscope study of the
rod-to-coccus shape change in a temperature-sensitive rod-mutant of
Bacillus subtilis. J. Bacteriol. 137:1395-1405; Burdett, I. D.
1980. Quantitative studies of rod--coccus morphogenesis in a
temperature-sensitive rod-mutant of Bacillus subtilis. J. Gen.
Microbil. 121:93-103); the secA gene (Sadaie, Y., et al. 1991.
Sequencing reveals similarity of the wild-type div+ gene of
Bacillus subtilis to the Escherichia coli secA gene. Gene.
98:101-105); the smc gene (Britton, R. A., et al. 1998.
Characterization of a prokaryotic SMC protein involved in
chromosome partitioning. Genes Dev. 12:1254-1259; Moriya, S., et
al. 1998. A Bacillus subtilis gene-encoding protein homologous to
eukaryotic SMC motor protein is necessary for chromosome partition.
Mol. Microbiol. 29:179-187; Hirano, M., and T. Hirano. 1998.
ATP-dependent aggregation of single-stranded DNA by a bacterial SMC
homodimer. EMBO J. 17:7139-7148); the spoIIE gene (Feucht, a., et
al. 1996. Bifunctional protein required for asymmetric cell
division and cell-specific transcription in Bacillus subtilis.
Genes Dev. 10:794-803; Khvorova, A., et al. 1998. The spoIIE locus
is involved in the Spo0A-dependent switch in the localization of
FtsZ rings in Bacillus subtilis. J. Bacteriol. 180:1256-1260;
Lucet, I., et al. 2000. Direct interaction between the cell
division protein FtsZ and the cell differentiation protein SpoIIE.
EMBO J. 19:1467-1475); the spo0A gene (Ireton, K., et al. 1994.
spo0J is required for normal chromosome segregation as well as the
initiation of sporulation in Bacillus subtilis. J. Bacteriol.
176:5320-5329); the spoIVF gene (Lee, S., and C. W. Price. 1993.
The minCD locus of Bacillus subtilis lacks the minE determinant
that provides topological specificity to cell division. Mol.
Microbiol. 7:601-610); the spo0J gene (Lin, D.C., et el. 1997.
Bipolar localization of a chromosome partition protein in Bacillus
subtilis. Proc. Natl. Acad. Sci. 94:4721-4726; Yamaichi, Y., and H.
Niki. 2000. Active segregation by the Bacillus subtilis
partitioning system in Escherichia coli. Proc. Natl. Acad. Sci.
97:14656-14661); the smc gene (Moriya, S., et al. 1998. A Bacillus
subtilis gene-encoding protein homologous to eukaryotic SMC motor
protein is necessary for chromosome partition. Mol. Microbiol.
29:179-187); the ripX gene (ciochetti, S. A. et al. 1999. The ripX
locus of Bacillus subtilis encodes a site-specific recombinase
involved in proper chromosome partitioning. J. Bacteriol.
181:6053-6062); and the spoIIIE gene (Wu, L. J., and J. Errington.
1994. Bacillus subtilis spoIIIE protein required for DNA
segregation during asymmetric cell division. Science. 264:572-575);
the gene corresponding to the B. subtilis mutant alleal ts-31
(Errington, J., and A. D. Richard. Cell division during growth and
sporulation. In A. L. Sonenshein, J. A. Hoch., and R. Losick
(eds.). Bacillus subtilis and its closest relatives: from genes to
cells. American Society for Microbiology, Washington D.C.); the
gene corresponding to the B. subtilis mutant alleal ts-526 (Id.);
the yacA gene (Kunst, F., et al. 1997. The complete genome sequence
of the gram-positive bacterium Bacillus subtilis. Nature.
390:237-238); the yfhF gene (Kunst, F., et al. 1997. The complete
genome sequence of the gram-positive bacterium Bacillus subtilis.
Nature. 390:237-238); the yfhK gene (Kunst, F., et al. 1997. The
complete genome sequence of the gram-positive bacterium Bacillus
subtilis. Nature. 390:237-238); the yjoB gene (Kunst, F., et al.
1997. The complete genome sequence of the gram-positive bacterium
Bacillus subtilis. Nature. 390:237-238); and the ywbG gene (Smith,
T. J., et al. 2000. Autolysins of Bacillus subtilis: multiple
enzymes with multiple functions. Microbiology. 146:249-262).
[0270] II.A.3. Saccharomyes Cervisiae Genes
[0271] Exemplary genes and gene products from S. cerevisiae the
expression and/or sequence of which can be manipulated so as to
stimulate minicell production in any organism, as can homologs
thereof from any species, include without limitation, the trf gene
product family (TRF1, TRF2, TRF3, TRF4, and TRF5) from
Saccharomyces cerevisiae (Sadoff, B. U., et al. 1995. Isolation of
mutants of Saccharomyces cerevisiae requiring DNA topoisomerase I.
Genetics. 141:465-479; Castano, I. B., et al. 1996. A novel family
of TRF (DNA topoisomerase I-related function) genes required for
proper nuclear segregation. Nucleic Acids Res. 2404-2410); the 1BD1
gene product from Saccharomyces cerevisiae (Lee, J., et al. 1999.
Ibd1p, a possible spindle pole body associated protein, regulates
nuclear division and bud separation in Saccharomyces cerevisiae.
Biochim. Biophys. Acta. 1449:239-253); the plo1 gene product from
Saccharomyces cerevisiae (Cullen, C. F., et al. 2000. A new genetic
method for isolating functionally interacting genes: high
plo1(+)-dependent mutants and their suppressors define genes in
mitotic and septation pathways in fission yeast. Genetics.
155:1541-1534); the cdc7 locus product(s) from Saccharomyces
cerevisiae or homologues of this found in other members of the
Eubacteria, Eucarya or Archaea may be employed to produce minicells
(Biggins, s. et al. 2001. Genes involved in sister chromatid
separation and segregation in the budding yeast Saccharomyces
cerevisiae. Genetics. 159:453-470); the cdc15 locus product(s) from
Saccharomyces cerevisiae or homologues of this found in other
members of the Eubacteria, Eucarya or Archaea may be employed to
produce minicells (Mah, A. S., et al. 2001. Protein kinase Cdc15
activates the Dbf2-Mob1 kinase complex. Proc. Natl. Acad. Sci.
98:7325-7330); the cdc11 locus product(s) from Saccharomyces
cerevisiae or homologues of this found in other members of the
Eubacteria, Eucarya or Archaea may be employed to produce minicells
(Fares, H., et al. 1996. Identification of a developmentally
regulated septin and involvement of the septins in spore formation
in Saccharomyces cerevisiae. J. Cell Biol. 132:399-411); the spg1
locus product(s) from Saccharomyces cerevisiae or homologues of
this found in other members of the Eubacteria, Eucarya or Archaea
may be employed to produce minicells (Cullen, C. F., et al. 2000. A
new genetic method for isolating functionally interacting genes:
high plo1(+)-dependent mutants and their suppressors define genes
in mitotic and septation pathways in fission yeast. Genetics.
155:1521-1534); the sid2 locus product(s) from Saccharomyces
cerevisiae or homologues of this found in other members of the
Eubacteria, Eucarya or Archaea may be employed to produce minicells
(Cullen, C. F., et al. 2000. A new genetic method for isolating
functionally interacting genes: high plo1(+)-dependent mutants and
their suppressors define genes in mitotic and septation pathways in
fission yeast. Genetics. 155:1521-1534); the cdc8 gene product from
Saccharomyces cerevisiae (Cullen, C. F., et al. 2000. A new genetic
method for isolating functionally interacting genes: high
plo1(+)-dependent mutants and their suppressors define genes in
mitotic and septation pathways in fission yeast. Genetics.
155:1521-1534); the rho1 gene product from Saccharomyces cerevisiae
(Cullen, C. F., et al. 2000. A new genetic method for isolating
functionally interacting genes: high plo1(+)-dependent mutants and
their suppressors define genes in mitotic and septation pathways in
fission yeast. Genetics. 155:1521-1534); the mpd1 gene product from
Saccharomyces cerevisiae (Cullen, C. F., et al. 2000. A new genetic
method for isolating functionally interacting genes: high
plo1(+)-dependent mutants and their suppressors define genes in
mitotic and septation pathways in fission yeast. Genetics.
155:1521-1534); the mpd2 gene product from Saccharomyces cerevisiae
(Cullen, C. F., et al. 2000. A new genetic method for isolating
functionally interacting genes: high plo1(+)-dependent mutants and
their suppressors define genes in mitotic and septation pathways in
fission yeast. Genetics. 155:1521-1534); the smy2 gene product from
Saccharomyces cerevisiae (Cullen, C. F., et al. 2000. A new genetic
method for isolating functionally interacting genes: high
plo1(+)-dependent mutants and their suppressors define genes in
mitotic and septation pathways in fission yeast. Genetics.
155:1521-1534); the cdc16 gene product from Saccharomyces
cerevisiae (Heichman, K. A., and J. M. Roberts. 1996. The yeast
CDC16 and CDC27 genes restrict DNA replication to once per cell
cycle. Cell. 85:39-48); the dma1 gene product from Saccharomyces
cerevisiae (Murone, M., and V. Simanis. 1996. The fission yeast
dma1 gene is a component of the spindle assembly checkpoint,
required to prevent septum formation and premature exit from
mitosis if spindle function is compromised. EMBO J. 15:6605-6616);
the plo1 gene product from Saccharomyces cerevisiae (Cullen, C. F.,
et al. 2000. A new genetic method for isolating functionally
interacting genes: high plo1(+)-dependent mutants and their
suppressors define genes in mitotic and septation pathways in
fission yeast. Genetics. 155:1521-1534); the byr3 gene product from
Saccharomyces cerevisiae (Cullen, C. F., et al. 2000. A new genetic
method for isolating functionally interacting genes: high
plo1(+)-dependent mutants and their suppressors define genes in
mitotic and septation pathways in fission yeast. Genetics.
155:1521-1534); the byr4 gene product from Saccharomyces cerevisiae
(Cullen, C. F., et al. 2000. A new genetic method for isolating
functionally interacting genes: high plo1(+)-dependent mutants and
their suppressors define genes in mitotic and septation pathways in
fission yeast. Genetics. 155:1521-1534); the pds1 gene product from
Saccharomyces cerevisiae (Yamamoto, A., et al. 1996. Pds1p, an
inhibitor of anaphase in budding yeast, plays a critical role in
the APC and checkpoint pathway(s). J. Cell Biol. 133:99-110); the
esp1 gene product from Saccharomyces cerevisiae (Rao, H., et al.
2001. Degradation of a cohesin subunit by the N-end rule pathway is
essential for chromosome stability. Nature. 410:955-999); the ycs4
gene product from Saccharomyces cerevisiae (Biggins, S., et al.
2001. Genes involved in sister chromatid separation and segregation
in the budding yeast Saccharomyces cerevisiae. Genetics.
159:453-470); the cse4 gene product from Saccharomyces cerevisiae
(Stoler, S. et al. 1995. A mutation in CSE4, an essential gene
encoding a novel chromatin-associated protein in yeast, causes
chromosome nondisjunction and cell cycle arrest at mitosis. Genes
Dev. 9:573-586); the ipl1 gene product from Saccharomyces
cerevisiae (Biggins, S., and A. W. Murray. 2001. The budding yeast
protein kinase Ipl1/Aurora allows the absence of tension to
activate the spindle checkpoint. Genes Dev. 15:3118-3129); the smt3
gene product from Saccharomyces cerevisiae (Takahashi, Y., et al.
1999. Smt3, a SUMO-1 homolog, is conjugated to Cdc3, a component of
septin rings at the mother-bud neck in budding yeast. Biochem.
Biophys. Res. Commun. 259:582-587); the prp16 gene product from
Saccharomyces cerevisiae (Hotz, H. R., and B. Schwer. 1998.
Mutational analysis of the yeast DEAH-box splicing factor Prp16.
Genetics. 149:807-815); the prp19 gene product from Saccharomyces
cerevisiae (Chen, C. H., et al. 2001. Identification and
characterization of two novel components of the Prp19p-associated
complex, Ntc30p and Ntc20p. J. Biol. Chem. 276:488-494); the wss1
gene product from Saccharomyces cerevisiae (Biggins, S., et al.
2001. Genes involved in sister chromatid separation and segregation
in the budding yeast Saccharomyces cerevisiae. Genetics.
159:453-470); the histone H4 gene product from Saccharomyces
cerevisiae (Smith, M. M., et al. 1996. A novel histone H4 mutant
defective in nuclear division and mitotic chromosome transmission.
Mol. Cell. Biol. 16:1017-1026); the histone H3 gene product from
Saccharomyces cerevisiae (Smith, M. M., et al. 1996. A novel
histone H4 mutant defective in nuclear division and mitotic
chromosome transmission. Mol. Cell. Biol. 16:1017-1026); the cse4
gene product from Saccharomyces cerevisiae (Stoler, S., et al.
1995. A mutation in CSE4, an essential gene encoding a novel
chromatin-associated protein in yeast, causes chromosome
nondisjunction and cell cycle arrest at mitosis. Genes Dev.
9:573-586); the spt4 gene product from Saccharomyces cerevisiae
(Basrai, M. A., et al. 1996. Faithful chromosome transmission
requires Spt4p, a putative regulator of chromatin structure in
Saccharomyces cerevisiae. Mol. Cell. Biol. 16:2838-2847); the spt5
gene product from Saccharomyces cerevisiae (Yamaguchi, Y., et al.
2001. SPT genes: key players in the regulation of transcription,
chromatin structure and other cellular processes. J. Biochem.
(Tokyo). 129:185-191); the spt6 gene product from Saccharomyces
cerevisiae (Clark-Adams, C. D., and F. Winston. 1987. The SPT6 gene
is essential for growth and is required for delta-mediated
transcription in Saccharomyces cerevisiae. Mol. Cell. Biol.
7:679-686); the ndc10 gene product from Saccharomyces cerevisiae
(Chiang, P. W., et al. 1998. Isolation of murine SPT5 homologue:
completion of the isolation and characterization of human and
murine homologues of yeast chromatin structural protein complex
SPT4, SPT5, and SPT6. Genomics. 47:426-428); the ctf13 gene product
from Saccharomyces cerevisiae (Doheny et al., Identification of
essential components of the S. cerevisiae kinetochore, Cell
73:761-774, 1993); the spo1 gene product from Saccharomyces
cerevisiae (Tavormina et al. 1997. Differential requirements for
DNA replication in the activation of mitotic checkpoints in
Saccharomyces cerevisiae. Mol. Cell. Biol. 17:3315-3322); the cwp1
gene product from Saccharomyces cerevisiae (Tevzadze, G. G., et al.
2000. Spot, a phospholipase B homolog, is required for spindle pole
body duplication during meiosis in Saccharomyces cerevisiae.
Chromosoma. 109:72-85); the dhp1 gene product from
Schizosaccharomyces pombe (Shobuike, T., et al. 2001. The dhp1(+)
gene, encoding a putative nuclear 5'.fwdarw.3' exoribonuclease, is
required for proper chromosome segregation in fission yeast.
Nucleic Acids Res. 29:1326-1333); the rat1 gene product from
Saccharomyces cerevisiae (Shobuike, T., et al. 2001. The dhp1(+)
gene, encoding a putative nuclear 5'.fwdarw.3' exoribonuclease, is
required for proper chromosome segregation in fission yeast.
Nucleic Acids Res. 29:1326-1333); the hsk1 gene product from
Saccharomyces cerevisiae (Masai, H., et al. 1995. hsk1+, a
Schizosaccharomyces pombe gene related to Saccharomyces cerevisiae
CDC7, is required for chromosomal replication. EMBO J.
14:3094-3104); the dfp1 gene product from Saccharomyces cerevisiae
(Takeda, T., et al. 1999. A fission yeast gene, him1(+)/dfp1(+),
encoding a regulatory subunit for Hsk1 kinase, plays essential
roles in S-phase initiation as well as in S-phase checkpoint
control and recovery from DNA damage. Mol. Cell. Biol.
19:5535-5547); the dbf4 gene product from Saccharomyces cerevisiae
(Weinreich, M., and B. Stillman. 1999. Cdc7p-Dbf4p kinase binds to
chromatin during S phase and is regulated by both the APC and the
RAD53 checkpoint pathway. EMBO J. 18:5334-5346); the rad53 gene
product from Saccharomyces cerevisiae (Sun, Z., et al. Spk1/Rad53
is regulated by Mec1-dependent protein phosphorylation in DNA
replication and damage checkpoint pathways. Genes Dev. 10:395-406);
the ibd1 gene product from Saccharomyces cerevisiae (Lee, J., et
al. 1999. Ibd1p, a possible spindle pole body associated protein,
regulates nuclear division and bud separation in Saccharomyces
cerevisiae. Biochim. Biophys. Acta. 1449:239-253); and the hrp1
gene product from Saccharomyces cerevisiae (Henry, M., et al. 1996.
Potential RNA binding proteins in Saccharomyces cerevisiae
identified as suppressors of temperature-sensitive mutations in
NPL3. Genetics. 142:103-115).
[0272] II.B. Gene Expression in Minicells
[0273] II.B.1. In General
[0274] In some aspects of the invention, it may be desirable to
alter the expression of a gene and the production of the
corresponding gene product. As is known in the art, and is used
herein, a "gene product" may be a protein (polypeptide) or nucleic
acid. Gene products that are proteins include without limitation
enzymes, receptors, transcription factors, termination factors,
expression factors, DNA-binding proteins, proteins that effect
nucleic acid structure, or subunits of any of the preceding. Gene
products that are nucleic acids include, but are not limited to,
ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), antisense RNAs,
nucleases (including but not limited to catalytic RNAs,
ribonucleases, and the like).
[0275] Depending on the function of a gene product, and on the type
of application of the invention, it may be desirable to increase
protein production, decrease protein production, increase protein
nucleic acid production and/or increase nucleic acid production.
Provided herein are non-limiting examples of genes and gene
products that may be manipulated, individually or in combination,
in order to modulate the expression of gene products to be included
into minicells or parent strains from which minicells are derived.
The expression elements so modulated may be chromosomal and/or
episomal, and may be expressed constitutively or in a regulated
fashion, i.e., repressible and/or inducible. Furthermore, gene
products under the regulation may be either monocistronic or
polycistronic with other genes or with themselves.
[0276] II.B.2. Protein Production
[0277] By way of non-limiting example, increased protein production
may occur through increased gene dosage (increased copy number of a
given gene under the control of the native or artificial promotor
where this gene may be on a plasmid or in more than one copy on the
chromosome), modification of the native regulatory elements,
including, but not limited to the promotor or operator region(s) of
DNA, or ribosomal binding sites on RNA, relevant
repressors/silencers, relevant activators/enhancers, or relevant
antisense nucleic acid or nucleic acid analog, cloning on a plasmid
under the control of the native or artificial promotor, and
increased or decreased production of native or artificial promotor
regulatory element(s) controlling production of the gene or gene
product
[0278] By way of non-limiting example, decreased protein production
may occur through modification of the native regulatory elements,
including, but not limited to the promotor or operator region(s) of
DNA, or ribosomal binding sites on RNA, relevant
repressors/silencers, relevant activators/enhancers, or relevant
antisense nucleic acid or nucleic acid analog, through cloning on a
plasmid under the control of the native regulatory region
containing mutations or an artificial promotor, either or both of
which resulting in decreased protein production, and through
increased or decreased production of native or artificial promotor
regulatory element(s) controlling production of the gene or gene
product.
[0279] As used herein with regards to proteins, "intramolecular
activity" refers to the enzymatic function or structure-dependent
function. By way of non-limiting example, alteration of
intramolecular activity may be accomplished by mutation of the
gene, in vivo or in vitro chemical modification of the protein,
inhibitor molecules against the function of the protein, e.g.
competitive, non-competitive, or uncompetitive enzymatic
inhibitors, inhibitors that prevent protein-protein,
protein-nucleic acid, or protein-lipid interactions, e.g.
expression or introduction of dominant-negative or
dominant-positive protein or other protein fragment(s),
carbohydrate(s), fatty acid(s), lipid(s), and nucleic acid(s) that
may act directly or allosterically upon the protein, and/or
modification of protein, carbohydrate, fatty acid, lipid, or
nucleic acid moieties that modify the gene or gene product to
create the functional protein.
[0280] As used herein with regards to proteins, "intermolecular
function" refers to the effects resulting from an intermolecular
interaction between the protein or nucleic acid and another
protein, carbohydrate, fatty acid, lipid, nucleic acid, or other
molecule(s) in or on the cell or the action of a product or
products resulting from such an interaction. By way of non-limiting
example, intermolecular or intramolecular function may be the act
or result of intermolecular phosphorylation, biotinylation,
methylation, acylation, glycosylation, and/or other signaling
event; this function may be the result of a protein-protein,
protein-nucleic acid, or protein-lipid complex, and/or carrier
function, e.g. the capacity to bind, either covalently or
non-covalently small organic or inorganic molecules, protein(s),
carbohydrate(s), fatty acid(s), lipid(s), and nucleic acid(s); this
function may be to interact with the membrane to recruit other
molecules to this compartment of the cell; this function may be to
regulate the transcription and/or translation of the gene, other
protein, or nucleic acid; and this function may be to stimulate the
function of another process that is not yet described or
understood.
[0281] II.B.3. Nucleic Acid Production
[0282] By way of non-limiting example, increased nucleic acid
production may occur through increased gene dosage (increased copy
number of a given gene under the control of the native or
artificial promotor where this gene may be on a plasmid or in more
than one copy on the chromosome), modification of the native
regulatory elements, including, but not limited to the promotor or
operator region(s) of DNA, or ribosomal binding sites on RNA,
relevant repressors/silencers, relevant activators/enhancers, or
relevant antisense nucleic acid or nucleic acid analog, cloning on
a plasmid under the control of the native or artificial promotor,
and increased or decreased production of native or artificial
promotor regulatory element(s) controlling production of the gene
or gene product.
[0283] By way of non-limiting example, decreased nucleic acid
production may occur through modification of the native regulatory
elements, including, but not limited to the promotor or operator
region(s) of DNA, or ribosomal binding sites on RNA, relevant
repressors/silencers, relevant activators/enhancers, or relevant
antisense nucleic acid or nucleic acid analog, through cloning on a
plasmid under the control of the native regulatory region
containing mutations or an artificial promotor, either or both of
which resulting in decreased protein production, and through
increased or decreased production of native or artificial promotor
regulatory element(s) controlling production of the gene or gene
product.
[0284] As used herein with regards to nucleic acids,
"intramolecular activity" refers to a structure-dependent function.
By way of non-limiting example, alteration of intramolecular
activity may be accomplished by mutation of the gene, in vivo or in
vitro chemical modification of the nucleic acid, inhibitor
molecules against the function of the nucleic acid, e.g.
competitive, non-competitive, or uncompetitive enzymatic
inhibitors, inhibitors that prevent protein-nucleic acid
interactions, e.g. expression or introduction of dominant-negative
or dominant-positive protein or other nucleic acid fragment(s), or
other carbohydrate(s), fatty acid(s), and lipid(s) that may act
directly or allosterically upon the nucleic acid or nucleic
acid-protein complex, and/or modification of nucleic acid moieties
that modify the gene or gene product to create the functional
nucleic acid.
[0285] As used herein with regards to nucleic acids,
"intermolecular function" refers to the effects resulting from an
intermolecular interaction between the nucleic acid and another
nucleic acid, protein, carbohydrate, fatty acid, lipid, or other
molecule(s) in or on the cell or the action of a product or
products resulting from such an interaction. By way of non-limiting
example, intermolecular function may be the act or result of
intermolecular or intramolecular phosphorylation, biotinylation,
methylation, acylation, glycosylation, and/or other signaling
event; this function may be the result of a protein-nucleic acid,
and/or carrier function, e.g. the capacity to bind, either
covalently or non-covalently small organic or inorganic molecules,
protein(s), carbohydrate(s), fatty acid(s), lipid(s), and other
nucleic acid(s); this function may be to interact with the membrane
to recruit other molecules to this compartment of the cell; this
function may be to regulate the transcription and/or translation of
the gene, other nucleic acid, or protein; and this function may be
to stimulate the function of another process that is not yet
described or understood.
[0286] II.C. Genes and Gene Products for Regulation of
Expression
[0287] As is known in the art, a variety of genes, gene products
and expression elements may be manipulated, individually or in
combination, in order to modulate the expression of genes and/or
production gene products. These include, by way of non-limiting
example, RNA polymerases, ribosomes (ribosomal proteins and
ribosomal RNAs), transfer RNAs (tRNAs), amino transferases,
regulatory elements and promoter regions, transportation of
inducible and inhibitory compounds, catabolite repression, general
deletions and modifications, cytoplasmic redox state,
transcriptional terminators, mechanisms for ribosomal targeting,
proteases, chaperones, export apparatus and membrane targeting, and
mechanisms for increasing stability and solubility. Each of these
is discussed in more detail in the following sections.
[0288] II.C.1. RNA Polymerases
[0289] Included in the design of the invention are techniques that
increase the efficiency of gene expression and protein production
in minicells. By way of non-limiting example, these techniques may
include modification of an endogenous and/or introduction of an
exogenous RNA polymerase. A rpo gene, or any other gene that
encodes a RNA polymerase subunit product from E. coli, or homologs
of this gene or its gene product found in other prokaryotes,
eukaryotes, archaebacteria or organelles (mitochondria,
chloroplasts, plastids and the like) may be employed to increase
the efficiency of gene expression and protein production in parent
cells prior to minicell formation and/or in segregated
minicells.
[0290] The production or activity of a desired gene product may be
increased by increasing the level and/or activity of an RNA
polymerase that transcribes the gene product's cognate gene. The
production or activity of a desired protein gene product may be
increased by decreasing the level and/or activity of an RNA
polymerase that transcribes a gene product that inhibits the
production or function of the desired gene product.
[0291] As one example, manipulation of the rpoA (phs, sez) gene or
gene product from E. coli, or homologs of this gene or gene product
found in other members of the Prokaryotes, Eukaryotes,
Archaebacteria and/or organelles (e.g., mitochondria, chloroplasts,
plastids and the like) may be employed to increase the efficiency
of gene expression and protein production in parent cells prior to
minicell formation and/or in segregated minicells. In addition to
rpoA, E. coli. genes that encode RNA polymerase subunits include
rpoB (ftsR, groN, nitB, rif, ron, stl, sty, tabD, sdgB, mbrD), rpoC
(tabD), rpoD (alt), rpoE, rpoH (fam, hin, htpR), rpoN (glnF, ntrA),
rpoS (abrD, dpeB, katF, nur), and rpoZ (spoS). See Berlyn et al.,
"Linkage Map of Escherichia coli K-12, Edition 9," Chapter 109 in:
Escherichia coli and Salmonella typhimurium: Cellular and Molecular
Biology, 2nd Ed., Neidhardt, Frederick C., Editor in Chief,
American Society for Microbiology, Washington, D.C., 1996, Volume
2, pages 1715-1902, and references cited therein; and Sanderson et
al., "Linkage Map of Salmonella typhimurium, Edition VIII" Chapter
110 in: Escherichia coli and Salmonella typhimurium: Cellular and
Molecular Biology, 2nd Ed., Neidhardt, Frederick C., Editor in
Chief, American Society for Microbiology, Washington, D.C., 1996,
Volume 2, pages 1903-1999, and references cited therein.
[0292] Production of a desired gene product may be preferentially
or selectively enhanced by the introduction of an exogenous RNA
polymerase that specifically recognizes expression sequences that
are operably linked to the corresponding gene. By way of
non-limiting example, the use of a T7 RNA polymerase to selectively
express genes present on expression elements that segregate into
minicells is described herein.
[0293] II.C.2. Ribosomes
[0294] Included in the design of the invention are techniques that
increase the efficiency of gene expression and protein production
in minicells. By way of non-limiting example, these techniques may
include modification of endogenous, and/or addition of exogenous,
ribosomes or ribosomal subunits. The techniques may be employed to
increase the efficiency of gene expression and protein production
in parent cells prior to minicell formation and/or in segregated
minicells.
[0295] As is known in the art, a ribosome includes both proteins
(polypeptides) and RNA (rRNA). Thus, in the case of a gene that
encodes a component of a ribosome, the gene product may be a
protein or an RNA. For a review, see Noller et al., "Ribosomes,"
Chapter 13 in: Escherichia coli and Salmonella typhimurium:
Cellular and Molecular Biology, 2nd Ed., Neidhardt, Frederick C.,
Editor in Chief, American Society for Microbiology, Washington,
D.C., 1996, Volume 1, pages 167-186, and references cited therein.
For the sake of convenience, both ribosomal proteins and rRNAs are
encompassed by the term "ribosomal subunits."
[0296] The production or activity of a desired protein gene product
may be increased by increasing the level and/or activity of a
ribosomal subunit that causes or enhances the translation of the
desired protein. The production or activity of a desired protein
gene product may be increased by decreasing the level and/or
activity of a ribosomal subunit that causes or enhances translation
of a protein that has a negative impact on the production or
activity of the desired protein.
[0297] Exemplary ribosomal genes and gene products that may be
manipulated include without limitation the E. coli genes rimB,
rimC, rimD, rimE, rimF (res), rimG, rimH, rimI, rimf (tcp), rimK,
rimL; rplA, rplB, rplC, rplD, rplE, rplF, rplI, rplJ, rplK, rplL,
rplM, rplN, rplO, rplP, rplQ, rplR, rplS, rplT, rplU, rplV, rplW,
rplX, rplY; rpsA, rpsB, rpsC, rpsE (eps, spc, spcA), rpsF (sdgH),
rpsG, rpsH, rpsl, rpsJ (nusE), rpsK, rpsL (strA), rpsM, rpsN, rpsO,
rpsP, rpsQ, rpsR, rpsS, rpsT, rpsU, rpsV; rrfA, rrfB, rrfC, rrfD,
rrfE, rrfF (rrfDbeta, rrvD), rrfG, rrfH; rrlA, rrlB, rrlC, rrlD,
rrlE, rrlG, rrlH; rrnA, rrnB (csqE, rrnB1), rrnC (cqsB), rrnD
(cqsD), rrnE (rrnD1), rrnG, rrnH; rrsA, rrsB, rrsC, rrsD, rrsE,
rrsG, rrsH, and their cognate gene products.
[0298] Homologs of ribosomal genes or gene products found in other
members of the Prokaryotes, Eukaryotes, Archaebacteria and
organelles (including but not limited to mitochondria,
chloroplasts, plastids, and the like) may be employed to increase
the efficiency of gene expression and protein production in parent
cells prior to minicell formation and/or segregated minicells. See,
for example, Barkan, A. and M. Goldschmidt-Clermont, Participation
of nuclear genes in chloroplast gene expression, (2000) Biochimie
82:559-572; Willhoeft, U., H. Bu.beta., and E. Tannich, Analysis of
cDNA Expressed sequence tags from Entamoeba histolytica:
Identification of two highly abundant polyadenylated transcripts
with no overt open reading frames, (March 1999) Protist 150:61-70;
Emelyanov, V., Evolutionary relationship of Rickettsiae and
mitochondria (February 2001) FEBS Letters 501:11-18; and Gray, M.,
G. Burger and B. Lang, Mitochondrial Evolution (March 1999) Science
283:1476-1481. Ribosomal RNA sequences from a multitude of
organisms and organelles are available through the Ribosomal
Database Project (Maidak et al., A new version of the RDP
(Ribosomal Database Project) (1999) Nucleic Acids Research
27:171-173). An index of ribosomal proteins classified by families
on the basis of sequence similarities is available on-line at
http://www.expasy.ch/cgi-bin/lists?ribosomp.txt; see also
(Ramakrishnan et al., Ribosomal protein structures: insights into
the architecture, machinery and evolution of the ribosome, TIBS
23:208-212, 1998.
[0299] II.C.3. Transfer RNAs (tRNAs)
[0300] Included in the design of the invention are techniques that
increase the efficiency of gene expression and protein production
in minicells. By way of non-limiting example, these techniques may
include utilization and/or modification of endogenous and/or
exogenous transfer RNAs (tRNAs). Manipulation of the tRNA genes or
gene products from E. coli, or homologs of tRNA genes or gene
products found in other members of the Prokaryotes, Eukaryotes,
Archaebacteria and organelles (including but not limited to
mitochondria, chloroplasts, plastids, and the like) may be employed
to increase the efficiency of gene expression and protein
production in parent cells prior to minicell formation and/or in
segregated minicells.
[0301] Exemplary E. coli tRNA genes include, but are not limited
to, the alaT (talA) gene, the alaU (talD) gene, the alaV gene, the
alaW (alaW.alpha.) gene, the alaX (alaW.beta.) gene, the argQ
(alaV.delta.) gene, the argU (dnaY, pin) gene, the alaU (talD)
gene, the argV(argV2) gene, the argW gene, the argX gene, the argY
(argV.beta.) gene, the argZ (argV.alpha.) gene, the asnT gene, the
asnU gene, the asnV gene, the aspT gene, the aspU gene, the cysT
gene, the glnU (supB) gene, the glnV (supE) gene, the glnW (supB)
gene, the gltT (tgtB) gene, the gltU (tgtC) gene, the gltV (tgtE)
gene, the gltW gene, the glyT (sumA) gene, the glyU (sufD, sumA,
sumB, supT) gene, the glyV (ins, mutA) gene, the glyW (ins, mutC)
gene, the glyX gene, the glyY gene, the hisR (hisT) gene, the ileT
gene, the ileU gene, the ileV gene, the ileX gene, the leuP
(leuV.beta.) gene, the leuQ (leuV.delta.) gene, the leuQ
(leuV.delta.) gene, the leuT gene, the leuU gene, the leuV
(leuV.alpha.) gene, the leuW (feeB) gene, the leuX (supP) gene, the
leuZ gene, the lysT gene, the lysV (supN) gene, the lysW gene, the
metT (metT.alpha.) gene, the metU (metT.beta.) gene, the metV
(metZ.beta.) gene, the metW gene, the metY gene, the pheU (pheR,
pheW) gene, the pheV gene, the proK (proV) gene, the proL (proW)
gene, the proM (proU) gene, the serT (divE) gene, the serU (ftsM,
supD, supH) gene, the serV (supD) gene, the serW gene, the serX
(serW) gene, the thrT gene, the thrU gene, the thrV gene, the thrW
gene, the trpT (supU) gene, the tyrT (supC) gene, the tyrU (supM)
gene, the atyrV (tyrT, tyrT.beta.) gene, the valT gene, the valU
(valU.alpha.) gene, the valV (val) gene, the valW (val) gene, the
valX gene, and the valX gene (Komine et al., Genomic Organization
and Physical Mapping of the Transfer RNA Genes in Escherichia coli
K12. J. Mol. Biol. 212:579-598, 1990; Berlyn et al., "Linkage Map
of Escherichia coli K-12, Edition 9," Chapter 109 in: Escherichia
Coli and Salmonella Typhimurium: Cellular and Molecular Biology,
2.sup.nd Ed., Neidhardt, Frederick C., Editor in Chief, American
Society for Microbiology, Washington, D.C., 1996, Volume 2, pages
1715-1902, and references cited therein; Sanderson et al., "Linkage
Map of Salmonella typhimurium, Edition VIII" Chapter 110, Id.,
pages 1903-1999, and references cited therein; and Hershey,
"Protein Synthesis," Chapter 40 in: Escherichia Coli and Salmonella
Typhimurium: Cellular and Molecular Biology, Neidhardt, Frederick
C., Editor in Chief, American Society for Microbiology, Washington,
D.C., 1987, Volume 2, pages 613-647, and references cited
therein).
[0302] Also included in the modification of transfer RNA molecules
are the transfer RNA processing enzymes. Exemplary E. coli genes
encoding tRNA processing enzymes include, but are not limited to
the rnd gene (Blouin R T, Zaniewski R, Deutscher M P. Ribonuclease
D is not essential for the normal growth of Escherichia coli or
bacteriophage T4 or for the biosynthesis of a T4 suppressor tRNA, J
Biol. Chem. 258:1423-1426, 1983) and the rnpAB genes (Kirsebom L A,
Baer M F, Altman S., Differential effects of mutations in the
protein and RNA moieties of RNase P on the efficiency of
suppression by various tRNA suppressors, J Mol. Biol. 204:879-888,
1988).
[0303] Also included in the modification of transfer RNA molecules
are modifications in endogenous tmRNAs and/or the introduction of
exogenous tmRNAs to minicells and/or their parent cells. The tmRNA
(a.k.a. 10S RNA) molecules have properties of tRNAs and mRNAs
combined in a single molecule. Examples of tmRNAs are described in
Zwieb et al. (Survey and Summary: Comparative Sequence Analysis of
tmRNA, Nucl. Acids Res. 27:21063-2071, 1999).
[0304] II.C.4. Aminoacyl Synthetases
[0305] Included in the design of the invention are techniques that
increase the efficiency of gene expression and protein production
in minicells. By way of non-limiting example, these techniques may
include utilization and/or modification of endogenous and/or
exogenous aminoacyl synthetases and proteins that effect their
production and/or activity. Aminoacyl synthetases are involved in
"charging" a tRNA molecule, i.e., attaching a tRNA to its cognate
amino acid. (Martinis et al., Aminoacyl-tRNA Synthetases: General
Features and Relationships. Chapter 58 in: Escherichia coli and
Salmonella typhimurium: Cellular and Molecular Biology, 2nd Ed.,
Neidhardt, Frederick C., Editor in Chief, American Society for
Microbiology, Washington, D.C., 1996, Volume 1, pages 887-901) and
references cited therein; (Grunberg-Manago, Regulation of the
Expression of Aminoacyl-tRNA Synthetases and Translation. Chapter
91 in: Escherichia coli and Salmonella typhimurium: Cellular and
Molecular Biology, 2nd Ed., Neidhardt, Frederick C., Editor in
Chief, American Society for Microbiology, Washington, D.C., 1996,
Volume 1, pages 1432-1457), and references cited therein; and
(Hershey, "Protein Synthesis," Chapter 40 in: Escherichia Coli and
Salmonella Typhimurium: Cellular and Molecular Biology, Neidhardt,
Frederick C., Editor in Chief, American Society for Microbiology,
Washington, D.C., 1987, Volume 1, pages 613-647), and references
cited therein.
[0306] By way of non-limiting example, manipulation of the aat gene
or gene product from E. coli, or homologs of this gene or gene
product found in other members of the Prokaryotes, Eukaryotes,
Archaebacteria and/or organelles (e.g., mitochondria, chloroplasts,
plastids and the like) may be employed to increase the efficiency
of gene expression and protein production in parent cells prior to
minicell formation and/or in segregated minicells (Bochner, B. R.,
and Savageau, M. A. 1979. Inhibition of growth by imidazol(on)e
propionic acid: evidence in vivo for coordination of histidine
catabolism with the catabolism of other amino acids. Mol. Gen.
Genet. 168(1):87-95).
[0307] In addition to aat, other exemplary E. coli genes encoding
aminoacyl synthestases include alaS (act, ala-act, lovB) (Buckel et
al., Suppression of temperature-sensitive aminoacyl-tRNA synthetase
mutations by ribosomal mutations: a possible mechanism. Mol. Gen.
Genet. 149:51-61, 1976); argS (lovB) (Eriani et al., Isolation and
characterization of the gene coding for Escherichia coli
arginyl-tRNA synthetase. Nucleic Acids Res. 17:5725-36, 1989); asnS
(lcs, tss) (Yamamoto et al., Identification of a
temperature-sensitive asparaginyl-transfer ribonucleic acid
synthetase mutant of Escherichia coli. J. Bacteriol. 132:127-31,
1977); aspS (tls) (Eriani et al., Aspartyl-tRNA synthetase from
Escherichia coli: cloning and characterisation of the gene,
homologies of its translated amino acid sequence with asparaginyl-
and lysl-tRNA syntheases. Nucleic Acids Res. 18:7109-18, 1990);
cysS (Eriani et al., Cysteinyl-tRNA synthetase: determination of
the last E. coli aminoacyl-tRNA synthetase primary structure.
Nucleic Acids Res. 19:265-9, 1991); glnS (Yamao et al., Escherichia
coli glutaminyl-tRNA synthetase. I. Isolation and DNA sequence of
the glnS gene. J. Biol. Chem. 257:11639-43, 1982); gltE (Lapointe
et al., Thermosensitive mutants of Escherichia coli K-12 altered in
the catalytic Subunit and in a Regulatory factor of the
glutamy-transfer ribonucleic acid synthetase. J. Bacteriol.
122:352-8, 1975); gltM (Lapointe et al., Thermosensitive mutants of
Escherichia coli K-12 altered in the catalytic Subunit and in a
Regulatory factor of the glutamy-transfer ribonucleic acid
synthetase. J. Bacteriol. 122:352-8, 1975); gltX (Lapointe et al.,
Thermosensitive mutants of Escherichia coli K-12 altered in the
catalytic Subunit and in a Regulatory factor of the
glutamy-transfer ribonucleic acid synthetase. J. Bacteriol.
122:352-8, 1975); glyQ (glySa) (Webster et al., Primary structures
of both subunits of Escherichia coli glycyl-tRNA synthetase, J.
Biol. Chem. 252:10637-41, 1983); glyS (act, gly, glySB) (Id.); hisS
(Parker et al., Mapping hisS, the structural gene for
histidyl-transfer ribonucleic acid synthetase, in Escherichia coli.
J. Bacteriol. 138:264:7, 1979); ileS (Singer et al., Synthesis of
the isoleucyl- and valyl-tRNA synthetases and the isoleucine-valine
biosythetic enzymes in a threonine deaminase regulatory mutant of
Escherichia coli K-12. J. Mol. Biol. 175:39-55, 1984); leuS (Morgan
et al., Regulation of biosythesis of aminoacyl-transfer RNA
synthestases and of transfer-RNA in Escherichia coli. Arch. Biol.
Med. Exp. (Santiago.) 12:415-26, 1979); lysS (herC, asaD) (Clark et
al., Roles of the two lysyl-tRNA synthetases of Escherichia coli:
analysis of nucleotide sequences and mutant behavior. J. Bacteriol.
172:3237-43, 1990); lysU (Clark et al., Roles of the two lysyl-tRNA
synthetases of Escherichia coli: analysis of nucleotide sequences
and mutant behavior, J. Bacteriol. 172:3237-43, 1990); metG (Dardel
et al., Molecular cloning and primary structure of the Escherichia
coli methionyl-tRNA synthetase gene. J. Bacteriol. 160:1115-22,
1984); pheS (phe-act) (Elseviers et al., Molecular cloning and
regulation of expression of the genes for initiation factor 3 and
two aminoacyl-tRNA synthetases, J. Bacteriol. 152:357-62, 1982);
pheT (corner et al., Genes for the alpha and beta subunits of the
phenylalanyl-transfer ribonucleic acid synthetase of Escherichia
coli. J. Bacteriol. 127:923-33, 1976); proS (drp) (Bohman et al., A
temperature-sensitive mutant in prolinyl-tRNA ligase of Escherichia
coli K-12 Mo. Gen. Genet. 177:603-5, 1980); serS (Hartlein et al.,
Cloning and characterization of the gene for Escherichia coli
seryl-tRNA synthetase. Nucleic Acids Res. 15:1005-17, 1987); thrS
(Frohler et al., Genetic analysis of mutations causing borrelidin
resistance by overproduction of threonyl-transfer ribonucleic acid
synthetase. J. Bacteriol. 143:1135-41, 1980); trpS (Hall et al.,
Cloning and characterization of the gene for Escherichia coli
tryptophanyl-transfer ribonucleic acid synthetase. J. Bacteriol.
148:941-9, 1981); tyrS (Buonocore et al., Properties of tyrosyl
transfer ribonucleic acid synthetase from two tyrS mutants of
Escherichia coli K-12. J. Biol. Chem. 247:4843-9, 1972); and valS
(Baer et al., Regulation of the biosynthesis of aminoacyl-transfer
ribonucleic acid synthetases and of transfer ribonucleic acid in
Escherichia coli. V. Mutants with increased levels of
valyl-transfer ribonucleic acid synthetase. J. Bacteriol.
139:165-75, 1979).
[0308] II.C.5. Regulatory Elements and Promoter Regions
[0309] Included in the design of the invention are techniques that
increase the efficiency of gene expression and protein production
in minicells. By way of non-limiting example, these techniques may
include utilization and/or modification of regulatory elements and
promoter regions. Such manipulations may result in increased or
decreased production, and/or changes in the intramolecular and
intermolecular functions, of a protein in a segregated minicell or
its parent cell prior to minicell formation; in the latter
instance, the protein may be one that is desirably retained in
segregated minicells.
[0310] The production or activity of a desired gene product may be
increased by increasing the level and/or activity of a promoter or
other regulatory region that acts to stimulate or enhance the
production of the desired gene product. The production or activity
of a desired gene product may be increased by decreasing the level
and/or activity of a promoter or other regulatory region that acts
to stimulate or enhance the production of a gene product that acts
to reduce or eliminate the level and/or activity of the desired
gene product.
[0311] II.C.5.a. Escherichia Coli
[0312] Regulatory elements, promoters and other expression elements
and expression factors from E. coli include but are not limited to
acrR (Ma, D., et al. 1996. The local repressor AcrR plays a
modulating role in the regulation of acrAB genes of Escherichia
coli by global stress signals. Mol. Microbiol. 19:101-112); ampD
(Lindquist, S., et al. 1989. Signalling proteins in enterobacterial
AmpC beta-lactamase regulation. Mol. Microbiol. 3:1091-1102;
Holtje, J. V., et al. 1994. The negative regulator of
beta-lactamase induction AmpD is a
N-acetyl-anhydromuramyl-L-alanine amidase. FEMS Microbiol. Lett.
122:159-164); appR (Diaz-Guerra, L., et al. 1989. appR gene product
activates transcription of microcin C7 plasmid genes. J. Bacteriol.
171:2906-2908; Touati, E., et al. 1991. Are appR and katF the same
Escherichia coli gene encoding a new sigma transcription initiation
factor? Res. Microbiol. 142:29-36); appY (Atlung, T., et al. 1989.
Isolation, characterization, and nucleotide sequence of appY, a
regulatory gene for growth-phase-dependent gene expression in
Escherichia coli. J. Bacteriol. 171:1683-1691); araC (Casadaban, M.
J., et al. 1976. Regulation of the regulatory gene for the
arabinose pathway, araC. J. Mol. Biol. 104:557-566); arcA (Iuchi,
S., and E. C. Lin. 1988. arcA (dye), a global regulatory gene in
Escherichia coli mediating repression of enzymes in aerobic
pathways. Proc. Natl. Acad. Sci. 85:1888-1892; Iuchi, S., et al.
1989. Differentiation of arcA, arcB, and cpxA mutant phenotypes of
Escherichia coli by sex pilus formation and enzyme regulation. J.
Bacteriol. 171:2889-2893); argR (xerA, Rarg) (Kelln, R. A., and V.
L. Zak. 1978. Arginine regulon control in a Salmonella
typhimurium--Escherichia coli hybrid merodiploid. Mol. Gen. Genet.
161:333-335; Vogel, R. H., et al. 1978. Evidence for translational
repression of arginine biosynthetic enzymes in Escherichia coli:
altered regulation in a streptomycin-resistant mutant. Mol. Gen.
Genet. 162:157-162); ascG (Hall, B. G., and L. Xu. Nucleotide
sequence, function, activation, and evolution of the cryptic asc
operon of Escherichia coli K12. Mol. Biol. Evol. 9:688-706); aslB
(Bennik, M. H., et al. 2000. Defining a rob regulon in Escherichia
coli by using transposon mutagenesis. J. Bacteriol. 182:3794-3801);
asnC (Kolling, R., and H. Lother. 1985. AsnC: an autogenously
regulated activator of asparagine synthetase A transcription in
Escherichia coli. J. Bacteriol. 164:310-315); atoC (Jenkins, L. S.,
and W. D. Nunn. 1987. Regulation of the ato operon by the atoC gene
in Escherichia coli. J. Bacteriol. 169:2096-2102); baeR (Nagasawa,
S., et al. 1993. Novel members of the two-component signal
transduction genes in Escherichia coli. J. Biochem. (Tokyo).
114:350-357); baeS (Id.Id.); barA (Nagasawa, S., et al. 1992. A
novel sensor-regulator protein that belongs to the homologous
family of signal-transduction proteins involved in adaptive
responses in Escherichia coli. Mol. Microbiol. 6:799-807; Ishige,
K., et al. 1994. A novel device of bacterial signal transducers.
EMBO J. 13:5195-5202); basS (Nagasawa, S., et al. 1993. Novel
members of the two-component signal transduction genes in
Escherichia coli. J. Biochem. (Tokyo). 114:350-357); betI (Lamark,
T., et al. 1996. The complex bet promoters of Escherichia coli:
regulation by oxygen (ArcA), choline (BetI), and osmotic stress. J.
Bacteriol. 178:1655-1662); bglG (bglC, bglS) (Schnetz, K., and B.
Rak. 1988. Regulation of the bgl operon of Escherichia coli by
transcriptional antitermination. EMBO J. 7:3271-3277; Schnetz, K.,
and B. Rak. 1990. Beta-glucoside permease represses the bgl operon
of Escherichia coli by phosphorylation of the antiterminator
protein and also interacts with glucose-specific enzyme III, the
key element in catabolite control. Proc. Natl. Acad. Sci.
87:5074-5078); birA (bioR, dhbB) (Barker, D. F., and A. M.
Campbell. 1981. Genetic and biochemical characterization of the
birA gene and its product: evidence for a direct role of biotin
holoenzyme synthetase in repression of the biotin operon in
Escherichia coli. J. Mol. Biol. 146:469-492; Barker, D. F., and A.
M. Campbell. 1981. The birA gene of Escherichia coli encodes a
biotin holoenzyme synthetase. J. Mol. Biol. 146:451-467; Howard, P.
K., et al. 1985. Nucleotide sequence of the birA gene encoding the
biotin operon repressor and biotin holoenzyme synthetase functions
of Escherichia coli. Gene. 35:321-331); btuR (Lundrigan, M. D., et
al. 1987. Separate regulatory systems for the repression of metE
and btuB by vitamin B12 in Escherichia coli. Mol. Gen. Genet.
206:401-407; Lundrigan, M. D., and R. J. Kadner. 1989. Altered
cobalamin metabolism in Escherichia coli btuR mutants affects btuB
gene regulation. J. Bacteriol. 171:154-161); cadC (Watson, N., et
al. 1992. Identification of elements involved in transcriptional
regulation of the Escherichia coli cad operon by external pH. J.
Bacteriol. 174:530-540); celD (Parker, L. L., and B. G. Hall. 1990.
Characterization and nucleotide sequence of the cryptic cel operon
of Escherichia coli K12. Genetics. 124:455-471); chaB (Berlyn, M.
K. B., et al. 1996. Linkage map of Escherichia coli K-12, Edition
9. In F. C. Neidhardt, R. Curtiss, J. L. Ingraham, E. C. C. Lin, K.
B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and
H. E. Umbarger (eds.). Escherichia coli and Salmonella typhimurium:
cellular and molecular biology, 2nd ed. American Society for
Microbiology, Washington D.C.); chaC (Berlyn, M. K. B., et al.
1996. Linkage map of Escherichia coli K-12, Edition 9. In F. C.
Neidhardt, R. Curtiss, J. L. Ingraham, E. C. C. Lin, K. B. Low, B.
Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E.
Umbarger (eds.). Escherichia coli and Salmonella typhimurium:
cellular and molecular biology, 2nd ed. American Society for
Microbiology, Washington D.C.); cpxR (Danese, P. N., et al. 1995.
The Cpx two-component signal transduction pathway of Escherichia
coli regulates transcription of the gene specifying the
stress-inducible periplasmic protease, DegP. Genes Dev. 9:387-398);
crl (Arnqvist, A., et al. 1992. The Cr1 protein activates cryptic
genes for curli formation and fibronectin binding in Escherichia
coli HB101. Mol. Microbiol. 6:2443-2452); cspA (Bae, W., et al.
1999. Characterization of Escherichia coli cspE, whose product
negatively regulates transcription of cspA, the gene for the major
cold shock protein. Mol. Microbiol. 31:1429-1441); cspE (Id.); csrA
(Liu, M. Y., et al. 1995. The product of the pleiotropic
Escherichia coli gene csrA modulates glycogen biosynthesis via
effects on mRNA stability. J. Bacteriol. 177:2663-2672); cynR
(Anderson, P. M., et al. 1990. The cyanase operon and cyanate
metabolism. FEMS Microbiol. Rev. 7:247-252; Sung, Y. C., and J. A.
Fuchs. 1992. The Escherichia coli K-12 cyn operon is positively
regulated by a member of the lysR family. J. Bacteriol.
174:3645-3650); cysB (Jagura-Burdzy, G., and D. Hulanicka. 1981.
Use of gene fusions to study expression of cysB, the regulatory
gene of the cysteine regulon. J. Bacteriol. 147:744-751); cytR
(Hammer-Jespersen, K., and A. Munch-Ptersen. 1975. Multiple
regulation of nucleoside catabolizing enzymes: regulation of the
deo operon by the cytR and deoR gene products. Mol. Gen. Genet.
137:327-335); dadQ (alnR) (Wild, J., and B. Obrepalska. 1982.
Regulation of expression of the dadA gene encoding D-amino acid
dehydrogenase in Escherichia coli: analysis of dadA-lac fusions and
direction of dadA transcription. Mol. Gen. Genet. 186:405-410);
dadR (alnR) (Wild, J., et al. 1985. Identification of the dadX gene
coding for the predominant isozyme of alanine racemase in
Escherichia coli K12. Mol. Gen. Genet. 198:315-322); deoR (nucR,
tsc, nupG) (Hammer-Jespersen, K., and A. Munch-Ptersen. 1975.
Multiple regulation of nucleoside catabolizing enzymes: regulation
of the deo operon by the cytR and deoR gene products. Mol. Gen.
Genet. 137:327-335); dgoR (Berlyn, M. K. B., et al. 1996. Linkage
map of Escherichia coli K-12, Edition 9. In F. C. Neidhardt, R.
Curtiss, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W.
S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (eds.).
Escherichia coli and Salmonella typhimurium: cellular and molecular
biology, 2nd ed. American Society for Microbiology, Washington
D.C.); dicA (Bejar, S., et al. 1988. Cell division inhibition gene
dicB is regulated by a locus similar to lambdoid bacteriophage
immunity loci. Mol. Gen. Genet. 212:11-19); dnaK (gro, groP,
groPAB, groPC, groPF, grpC, grpF, seg) (Bochner, B. R., et al.
1986. Escherichia coli DnaK protein possesses a 5'-nucleotidase
activity that is inhibited by AppppA. J. Bacteriol. 168:931-935);
dniR (Kajie, S., et al. 1991. Molecular cloning and DNA sequence of
dniR, a gene affecting anaerobic expression of the Escherichia coli
hexaheme nitrite reductase. FEMS Microbiol. Lett. 67:205-211); dsdC
(Heincz, M. C., and E. McFall. 1978. Role of the dsdC activator in
regulation of D-serine deaminase synthesis. J. Bacteriol.
136:96-103); ebgR (Hall, B. G., and N. D. Clarke. 1977. Regulation
of newly evolved enzymes. III Evolution of the ebg repressor during
selection for enhanced lactase activity. Genetics. 85:193-201);
envY (Lundrigan, M. D., and C. F. Earhart. 1984. Gene envY of
Escherichia coli K-12 affects thermoregulation of major porin
expression. J. Bacteriol. 157:262-268); envZ (ompB, perA, tpo)
(Russo, F. D, and T. J. Silhavy. 1991. EnvZ controls the
concentration of phosphorylated OmpR to mediate osmoregulation of
the porin genes. J. Mol. Biol. 222:567-580); evgA (Nishino, K., and
A. Yamaguichi. 2001. Overexpression of the response regulator evgA
of the two-component signal transduction system modulates multidrug
resistance conferred by multidrug resistance transporters. J.
Bacteriol. 183:1455-1458); evgS (Id.); exuR (Portalier, R., et al.
1980. Regulation of Escherichia coli K-12 hexuronate system genes:
exu regulon. J. Bacteriol. 143:1095-1107); fadR (dec, ole, thdB)
(Simons, R. W., et al. 1980. Regulation of fatty acid degradation
in Escherichia coli: isolation and characterization of strains
bearing insertion and temperature-sensitive mutations in gene fadR.
J. Bacteriol. 142:621-632); fecI (Van Hove, B., et al. 1990. Novel
two-component transmembrane transcription control: regulation of
iron dicitrate transport in Escherichia coli K-12. J. Bacteriol.
172:6749-6758); fecR (Id.); fhlA (Maupin, J. A., and K. T.
Shanmugam. 1990. Genetic regulation of formate hydrogenlyase of
Escherichia coli: role of the fhlA gene product as a
transcriptional activator for a new regulatory gene, fhlB. J.
Bacteriol. 172:4798-4806; Rossmann, R., et al. 1991. Mechanism of
regulation of the formate-hydrogenlyase pathway by oxygen, nitrate,
and pH: definition of the formate regulon. Mol. Microbiol.
5:2807-2814); fhlB (Maupin, J. A., and K. T. Shanmugam. 1990.
Genetic regulation of formate hydrogenlyase of Escherichia coli:
role of the fhlA gene product as a transcriptional activator for a
new regulatory gene, fhlB. J. Bacteriol. 172:4798-4806); fimB (pil)
(Pallesen, L., et al. 1989. Regulation of the phase switch
controlling expression of type 1 fimbriae in Escherichia coli. Mol.
Microbiol. 3:925-931); fimE (pilH) (Id.); fliA (flat) (Liu, X., and
P. Matsumura. 1994. The FlhD/FlhC complex, a transcriptional
activator of the Escherichia coli flagellar class II operons. J.
Bacteriol. 176:7345-7351); flhD (flhB) (Id.); fliA (flaD, rpoF)
(Komeda, Y., et al. 1986. Transcriptional control of flagellar
genes in Escherichia coli K-12. J. Bacteriol. 168:1315-1318); fnr
(frdB, nirA, nirR) (Jones, H. M., and R. P. Gunsalus. 1987.
Regulation of Escherichia coli fumarate reductase (frdABCD) operon
expression by respiratory electron acceptors and the fnr gene
product. J. Bacteriol. 169:3340-3349); fruR (fruC, shl) (Geerse, R.
H., at al. The PEP: fructose phosphotransferase system in
Salmonella typhimurium: FPr combines enzyme IIIFru and pseudo-HPr
activities. Mol. Gen. Genet. 216:517-525); fucR (Zhu, Y., and E. C.
Lin. 1986. An evolvant of Escherichia coli that employs the
L-fucose pathway also for growth on L-galactose and D-arabinose. J.
Mol. Evol. 23:259-266); fur (Bagg, A., and J. B. Neilands. 1987.
Ferric uptake regulation protein acts as a repressor, employing
iron (II) as a cofactor to bind the operator of an iron transport
operon in Escherichia coli. Biochemistry 26:5471-5477); gadR gene
product from Lactococcus lactis (Sanders, J. W., et al. 1997. A
chloride-inducible gene expression cassette and its use in induced
lysis of Lactococcus lactis. Appl. Environ. Microbiol.
63:4877-4882); galR (von Wilcken-Bergmann, B., and B. Muller-Hill.
1982. Sequence of galR gene indicates a common evolutionary origin
of lac and gal repressor in Escherichia coli. Proc. Natl. Acad.
Sci. 79:2427-2431); galS (mglD) (Weickert., M. J., and S. Adhya.
1992. Isorepressor of the gal regulon in Escherichia coli. J. Mol.
Biol. 226:69-83); galU (Berlyn, M. K. B., et al. 1996. Linkage map
of Escherichia coli K-12, Edition 9. In F. C. Neidhardt, R.
Curtiss, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W.
S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (eds.).
Escherichia coli and Salmonella typhimurium: cellular and molecular
biology, 2nd ed. American Society for Microbiology, Washington
D.C.); gatR (Nobelmann, B., and J. W. Lengeler. 1996. Molecular
analysis of the gat genes from Escherichia coli and of their roles
in galactitol transport and metabolism. J. Bacteriol.
178:6790-6795); gcvA (Wilson, R. L., et al. 1993. Positive
regulation of the Escherichia coli glycine cleavage enzyme system.
J. Bacteriol. 175:902-904); glgS (Hengge-Aronis, R., et al. 1993.
Osmotic regulation of rpoS-dependent genes in Escherichia coli. J.
Bacteriol. 175:259-265; Yang, H., et al. 1996. Coordinate genetic
regulation of glycogen catabolism and biosynthesis in Escherichia
coli via the CsrA gene product. J. Bactgeriol. 178:1012-1017); glnB
(Bueno, R., et al. 1985. Role of glnB and glnD gene products in
regulation of the glnALG operon of Escherichia coli. J. Bacteriol.
164:816-822); glnG (gln, ntrC) (Pahel, G., and B. Tyler. 1979. A
new glnA-linked regulatory gene for glutamine synthetase in
Escherichia coli. Proc. Natl. Acad. Sci. 76:4544-4548); glnL (glnR,
ntrB) (MacNeil, T., et al. The products of glnL and glnG are
bifunctional regulatory proteins. Mol. Gen. Genet. 188:325-333);
glpR (Silhavy, T. J., et al. 1976. Periplasmic protein related to
the sn-glycerol-3-phosphate transport system of Escherichia coli.
J. Bacteriol. 126:951-958); gltF (Castano, I., et al. gltF, a
member of the gltBDF operon of Escherichia coli, is involved in
nitrogen-regulated gene expression. Mol. Microbiol. 6:2733-2741);
gntR (Peekhaus, N., and T. Conway. 1998. Positive and negative
transcriptional regulation of the Escherichia coli gluconate
regulon gene gntT by GntR and the cyclic AMP (cAMP)-cAMP receptor
protein complex. J. Bacteriol. 180:1777-1785); hha (Neito, J. M.,
et al. The hha gene modulates haemolysin expression in
Escherichia coli. Mol. Microbiol. 5:1285-1293); himD (hip) (Goosen,
N., et al. 1984. Regulation of Mu transposition. II. The
Escherichia coli HimD protein positively controls two repressor
promoters and the early promoter of bacteriophage Mu. Gene.
32:419-426); hrpB gene product from Pseudomonas solanacearum (Van
Gijsegem, F., et al. 1995. The hrp gene locus of Pseudomonas
solanacearum, which controls the production of a type III secretion
system, encodes eight proteins related to components of the
bacterial flagellar biogenesis complex. Mol. Microbiol.
15:1095-1114); hybF (Berlyn, M. K. B., et al. 1996. Linkage map of
Escherichia coli K-12, Edition 9. In F. C. Neidhardt, R. Curtiss,
J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S.
Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (eds.).
Escherichia coli and Salmonella typhimurium: cellular and molecular
biology, 2nd ed. American Society for Microbiology, Washington
D.C.); hycA (Hopper, S., et al. 1994. Regulated expression in vitro
of genes coding for formate hydrogenlyase components of Escherichia
coli. J. Biol. Chem. 269:19597-19604); hydG (Leonhartsberger, S. et
al. 2001. The hydH/G genes from Escherichia coli code for a zinc
and lead responsive two-component regulatory system. J. Mol. Biol.
307:93-105); hydH (Id.); iciA (Thony, B., et al. 1991. iciA, an
Escherichia coli gene encoding a specific inhibitor of chromosomal
initiation of replication in vitro. Proc. Natl. Acad. Sci.
88:4066-4070); iclR (Maloy, S. R., and W. D. Nunn. 1982. Genetic
regulation of the glyoxylate shunt in Escherichia coli K-12. J.
Bacteriol. 149:173-180); ileR (avr, firA) (Johnson, D. I., and R.
L. Somerville. 1984. New regulatory genes involved in the control
of transcription initiation at the thr and ilv promoters of
Escherichia coli K-12. Mol. Gen. Genet. 195:70-76); ilvR (Id.);
ilvU (Fayerman, J. T., et al. 1979. ilvU, a locus in Escherichia
coli affecting the derepression of isoleucyl-tRNA synthetase and
the RPC-5 chromatographic profiles of tRNAIle and tRNAVal. J. Bio.
Chem. 254:9429-9440); ilvY (Wek, R. C., and G. W. Hatfield. 1988.
Transcriptional activation at adjacent operators in the
divergent-overlapping ilvY and ilvC promoters of Escherichia coli.
J. Mol. Biol. 203:643-663); inaA (White, S., et al. 1992. pH
dependence and gene structure of inaA in Escherichia coli. J.
Bacteriol. 174:1537-1543); inaR (Id.); kdgR (Nemoz, G., et al.
1976. Physiological and genetic regulation of the aldohexuronate
transport system in Escherichia coli. J. Bacteriol. 127:706-718);
lad (Riggs, A. D, and S. Bourgeois. 1968. On the assay, isolation
and characterization of the lac repressor. J. Mol. Biol.
34:361-364); leuO (Shi, X., and G. N. Bennett. 1995. Effects of
multicopy LeuO on the expression of the acid-inducible lysine
decarboxylase gene in Escherichia coli. J. Bacteriol. 177:810-814;
Klauck, E., et al. 1997. The LysR-like regulator LeuO in
Escherichia coli is involved in the translational regulation of
rpoS by affecting the expression of the small regulatory DsrA-RNA.
Mol. Microbiol. 25:559-569); leuR (Theall, G., et al. 1979.
Regulation of the biosynthesis of aminoacyl-tRNA synthetases and of
tRNA in Escherichia coli. IV. Mutants with increased levels of
leucyl- or seryl-tRNA synthetase. Mol. Gen. Genet. 169:205-211);
leuY (Morgan, S., et al. 1979. Regulation of biosynthesis of
aminoacyl-transfer RNA synthetases and of transfer-RNA in
Escherichia coli. Arch. Biol. Med. Exp. (Santiago) 12:415-426);
lexA (Mount, D. W. 1977. A mutant of Escherichia coli showing
constitutive expression of the lysogenic induction and error-prone
DNA repair pathways. Proc. Natl. Acad. Sci. 74:300-304; Little, J.
W., et al. 1980. Cleavage of the Escherichia coli lexA protein by
the recA protease. Proc. Natl. Acad. Sci. 77:3225-3229); lldR
(lctR) (Dong, J. M., et al. 1993. Three overlapping lct genes
involved in L-lactate utilization by Escherichia coli. J.
Bacteriol. 175:6671-6678); lpp (Brosius, J. Expression vectors
employing lambda-, trp-, lac-, and lpp-derived promoters. 1988.
Biotechnology. 10:205-225); lrhA (genR) (Bongaerts, J., et al.
1995. Transcriptional regulation of the proton translocating NADH
dehydrogenase genes (nuoA-N) of Escherichia coli by electron
acceptors, electron donors and gene regulators. Mol. Microbiol.
16:521-534); lrp (ihb, livR, lss, lstR, oppI, rblA, mbf) (Ito, K.,
et al. Multiple control of Escherichia coli lysyl-tRNA synthetase
expression involves a transcriptional repressor and a translational
enhancer element. Proc. Natl. Acad. Sci. 90:302-306); lysR
(Gicquel-Sanzey, B. and P. Cossart. 1982. Homologies between
different procaryotic DNA-binding regulatory proteins and between
their sites of action. EMBO J. 1:591-595; Stragier, P., et al.
1983. Regulation of diaminopimelate decarboxylase synthesis in
Escherichia coli. II. Nucleotide sequence of the lysA gene and its
regulatory region. J. Mol. Biol. 168:321-331); malI (Reidl, J., et
al. 1989. MalI, a novel protein involved in regulation of the
maltose system of Escherichia coli, is highly homologous to the
repressor proteins GalR, CytR, and LacI. J. Bacteriol.
171:4888-4499); malT (malA) (Bebarbouille, M., and M. Schwartz.
Mutants which make more malT product, the activator of the maltose
regulon in Escherichia coli. Mol. Gen. Genet. 178:589-595); marA
(cpxB, soxQ) (Ariza, R. R., et al. Repressor mutations in the
marRAB operon that activate oxidative stress genes and multiple
antibiotic resistance in Escherichia coli. J. Bacteriol.
176:143-148); marB (Berlyn, M. K. B., et al. 1996. Linkage map of
Escherichia coli K-12, Edition 9. In F. C. Neidhardt, R. Curtiss,
J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S.
Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (eds.).
Escherichia coli and Salmonella typhimurium: cellular and molecular
biology, 2nd ed. American Society for Microbiology, Washington
D.C.); marR (Ariza, R. R., et al. Repressor mutations in the marRAB
operon that activate oxidative stress genes and multiple antibiotic
resistance in Escherichia coli. J. Bacteriol. 176:143-148); melR
(Williams, J., et al. 1994. Interactions between the Escherichia
coli MelR transcription activator protein and operator sequences at
the melAB promoter. Biochem. J. 300:757-763); metJ (Smith, A. A.,
et al. 1985. Isolation and characterization of the product of the
methionine-regulatory gene metJ of Escherichia coli K-12. Proc.
Natl. Acad. Sci. 82:6104-6108; Shoeman, R., et al. 1985. Regulation
of methionine synthesis in Escherichia coli: effect of metJ gene
product and S-adenosylmethionine on the in vitro expression of the
metB, metL and metJ genes. Biochem. Biophys. Res. Commun.
133:731-739); metR (Maxon, M. E., et al. 1989. Regulation of
methionine synthesis in Escherichia coli: effect of the MetR
protein on the expression of the metE and metR genes. Proc. Natl.
Acad. Sci. 86:85-89); mglR(R-MG) (Berlyn, M. K. B., et al. 1996.
Linkage map of Escherichia coli K-12, Edition 9. In F. C.
Neidhardt, R. Curtiss, J. L. Ingraham, E. C. C. Lin, K. B. Low, B.
Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E.
Umbarger (eds.). Escherichia coli and Salmonella typhimurium:
cellular and molecular biology, 2nd ed. American Society for
Microbiology, Washington D.C.); mhpR (Ferrandez, A., et al. 1997.
Genetic characterization and expression in heterologous hosts of
the 3-(3-hydroxyphenyl)propionate catabolic pathway of Escherichia
coli K-12. J. Bacteriol. 179:2573-2581); mhpS (Berlyn, M. K. B., et
al. 1996. Linkage map of Escherichia coli K-12, Edition 9. In F. C.
Neidhardt, R. Curtiss, J. L. Ingraham, E. C. C. Lin, K. B. Low, B.
Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E.
Umbarger (eds.). Escherichia coli and Salmonella typhimurium:
cellular and molecular biology, 2nd ed. American Society for
Microbiology, Washington D.C.); micF (stc) (Aiba, H., et al. 1987.
Function of micF as an antisense RNA in osmoregulatory expression
of the ompF gene in Escherichia coli. J. Bacteriol. 169:3007-3012);
mprA (emrR) (del Castillo, I., et al. 1990. mprA, an Escherichia
coli gene that reduces growth-phase-dependent synthesis of
microcins B17 and C7 and blocks osmoinduction of proU when cloned
on a high-copy-number plasmid. J. Bacteriol. 172:437-445); mt1R
(Figge, R. M., et al. 1994. The mannitol repressor (Mt1R) of
Escherichia coli. J. Bacteriol. 176:840-847); nagC (nagR)
(Plumbridge, J. A. 1991. Repression and induction of the nag
regulon of Escherichia coli K-12: the roles of nagC and nagA in
maintenance of the uninduced state. Mol. Microbiol. 5:2053-3062);
narL (frdR, narR) (Stewart, V. 1982. Requirement of Fnr and NarL
functions for nitrate reductase expression in Escherichia coli
K-12. J. Bacteriol. 151:1320-1325; Miller, J. B., et al. 1987.
Molybdenum-sensitive transcriptional regulation of the chlD locus
of Escherichia coli. J. Bacteriol. 169:1853-1860; Iuchi, S., and E.
C. Lin. 1987. Molybdenum effector of fumarate reductase repression
and nitrate reductase induction in Escherichia coli. J. Bacteriol.
169:3720-3725); narP (Rabin, R. S., and V. Stewart. 1993. Dual
response regulators (NarL and NarP) interact with dual sensors
(NarX and NarQ) to control nitrate- and nitrite-regulated gene
expression in Escherichia coli K-12. J. Bacteriol. 175:3259-3268);
nhaR (gene product from E. coli (Rahav-Manor, 0., et al. 1992.
NhaR, a protein homologous to a family of bacterial regulatory
proteins (LysR), regulates nhaA, the sodium proton antiporter gene
in Escherichia coli. J. Biol. Chem. 267:10433-10438); ompR (cry,
envZ, ompB) (Taylor, R. K., et al. Identification of OmpR: a
positive regulatory protein controlling expression of the major
outer membrane matrix porin proteins of Escherichia coli K-12. J.
Bacteriol. 147:255-258); oxyR (mor, momR) (VanBogelen, R. A, et al.
1987. Differential induction of heat shock, SOS, and oxidation
stress regulons and accumulation of nucleotides in Escherichia
coli. J. Bacteriol. 169:26-32); pdhR (Haydon, D. J., et al. A
mutation causing constitutive synthesis of the pyruvate
dehydrogenase complex in Escherichia coli is located within the
pdhR gene. FEBS Lett. 336:43-47); phnF (Wanner, B. L., and W. W.
Metcalf. 1992. Molecular genetic studies of a 10.9-kb operon in
Escherichia coli for phosphonate uptake and biodegradation. FEMS
Microbiol. Lett. 79:133-139); phoB (phoRc, phoT) (Pratt, C. 1980.
Kinetics and regulation of cell-free alkaline phosphatase
synthesis. J. Bacteriol. 143:1265-1274); phoP (Kasahara, M., et al.
1992. Molecular analysis of the Escherichia coli phoP-phoQ operon.
J. Bacteriol. 174:492-498); phoQ (Id.); phoR(R1pho, nmpB, phoR1)
(Bracha, M., and E. Yagil. 1969. Genetic mapping of the phoR
regulator gene of alkaline phosphatase in Escherichia coli. J. Gen.
Microbiol. 59:77-81); phoU (phoT) (Nakata, A., et al. 1984.
Regulation of the phosphate regulon in Escherichia coli K-12:
regulation of the negative regulatory gene phoU and identification
of the gene product. J. Bacteriol. 159:979-985); poaR (Berlyn, M.
K. B., et al. 1996. Linkage map of Escherichia coli K-12, Edition
9. In F. C. Neidhardt, R. Curtiss, J. L. Ingraham, E. C. C. Lin, K.
B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and
H. E. Umbarger (eds.). Escherichia coli and Salmonella typhimurium:
cellular and molecular biology, 2nd ed. American Society for
Microbiology, Washington D.C.); poxA (Chang, Y. Y., and J. E.
Cronan Jr. 1982. Mapping nonselectable genes of Escherichia coli by
using transposon Tn10: location of a gene affecting pyruvate
oxidase. J. Bacteriol. 151:1279-1289); proQ (Milner, J. L., and J.
M. Wood. 1989. Insertion proQ220::Tn5 alters regulation of proline
porter II, a transporter of proline and glycine betaine in
Escherichia coli. J. Bacteriol. 171:947-951); pspA (Weiner, L., et
al. 1991. Stress-induced expression of the Escherichia coli phage
shock protein operon is dependent on sigma 54 and modulated by
positive and negative feedback mechanisms. Genes Dev. 5:1912-1923);
pspB (Weiner, L., et al. 1991. Stress-induced expression of the
Escherichia coli phage shock protein operon is dependent on sigma
54 and modulated by positive and negative feedback mechanisms.
Genes Dev. 5:1912-1923); pspC (Weiner, L., et al. 1991.
Stress-induced expression of the Escherichia coli phage shock
protein operon is dependent on sigma 54 and modulated by positive
and negative feedback mechanisms. Genes Dev. 5:1912-1923); pssR
(Sparrow, C. P., and C. R. Raetz. 1983. A trans-acting regulatory
mutation that causes overproduction of phosphatidylserine synthase
in Escherichia coli. J. Biol. Chem. 258:9963-9967); purR (Meng, L.
M., et al. 1990. Autoregulation of PurR repressor synthesis and
involvement of purR in the regulation of purB, purC, purL, purMN
and guaBA expression in Escherichia coli. Eur. J. Biochem.
187:373-379); putA (poaA) gene product from Salmonella enterica
serotype Typhimurium (Menzel, R., and J. Roth. 1981. Regulation of
the genes for proline utilization in Salmonella typhimurium:
autogenous repression by the putA gene product. J. Mol. Biol.
148:21-44); pyrI (Cunin, R., et al. 1985. Structure-function
relationship in allosteric aspartate carbamoyltransferase from
Escherichia coli. I. Primary structure of a pyrI gene encoding a
modified regulatory subunit. J. Mol. Biol. 186:707-713); rbsR
(Lopilato, J. E., et al. 1984. D-ribose metabolism in Escherichia
coli K-12: genetics, regulation, and transport. J. Bacteriol.
158:665-673); rcsA (Gottesman, S., et al. 1985. Regulation of
capsular polysaccharide synthesis in Escherichia coli K-12:
characterization of three regulatory genes. J. Bacteriol.
162:1111-1119); rcsB (Id.); rcsC (Id.); rcsF (Grevais, F. G., and
G. R. Drapeau. 1992. Identification, cloning, and characterization
of rcsF, a new regulator gene for exopolysaccharide synthesis that
suppresses the division mutation ftsZ84 in Escherichia coli K-12.
J. Bacteriol. 174:8016-8022); relB (Christensen, S. K., et al.
2001. RelE, a global inhibitor of translation, is activated during
nutritional stress. Proc Natl. Acad. Sci. 98:14328-14333); rfaH
(sfrB) (Pradel, E., and C. A. Schnaitman. 1991. Effect of rfaH
(sfrB) and temperature on expression of rfa genes of Escherichia
coli K-12. J. Bacteriol. 173:6428-6431); rhaR (Tobin, J. F., and R.
F. Schleif. 1987. Positive regulation of the Escherichia coli
L-rhamnose operon is mediated by the products of tandemly repeated
regulatory genes. J. Mol. Biol. 196:789-799); rhaS (Id.); rnk
(Berlyn, M. K. B., et al. 1996. Linkage map of Escherichia coli
K-12, Edition 9. In F. C. Neidhardt, R. Curtiss, J. L. Ingraham, E.
C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M.
Schaechter, and H. E. Umbarger (eds.). Escherichia coli and
Salmonella typhimurium: cellular and molecular biology, 2nd ed.
American Society for Microbiology, Washington D.C.); rob (Skarstad,
K., et al. A novel binding protein of the origin of the Escherichia
coli
chromosome. J. Biol. Chem. 268:535-5370); rseA (mclA) (Missiakas,
D., et al. 1997. Modulation of the Escherichia coli sigmaE (RpoE)
heat-shock transcription-factor activity by the RseA, RseB and RseC
proteins. Mol. Microbiol. 24:355-371; De Las Penas, A. 1997. The
sigmaE-mediated response to extracytoplasmic stress in Escherichia
coli is transduced by RseA and RseB, two negative regulators of
sigmaE. Mol. Microbiol. 24:373-385); rseB (Id.); rseC (Id.); rspA
(Huisman, G. W., and T. Kolter. 1994. Sensing starvation: a
homoserine lactone--dependent signaling pathway in Escherichia
coli. Science. 265:537-539); rspB (Shafqat, J., et al. An
ethanol-inducible MDR ethanol dehydrogenase/acetaldehyde reductase
in Escherichia coli: structural and enzymatic relationships to the
eukaryotic protein forms. Eur. J. Biochem. 263:305-311); rssA
(Berlyn, M. K. B., et al. 1996. Linkage map of Escherichia coli
K-12, Edition 9. In F. C. Neidhardt, R. Curtiss, J. L. Ingraham, E.
C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M.
Schaechter, and H. E. Umbarger (eds.). Escherichia coli and
Salmonella typhimurium: cellular and molecular biology, 2nd ed.
American Society for Microbiology, Washington D.C.); rssB (Muffler,
A., et al. 1996. The response regulator RssB controls stability of
the sigma(S) subunit of RNA polymerase in Escherichia coli. EMBO J.
15:1333-1339); sbaA (Berlyn, M. K. B., et al. 1996. Linkage map of
Escherichia coli K-12, Edition 9. In F. C. Neidhardt, R. Curtiss,
J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S.
Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (eds.).
Escherichia coli and Salmonella typhimurium: cellular and molecular
biology, 2nd ed. American Society for Microbiology, Washington
D.C.); sdaC (Id.); sdiA (Sitnikov, D. M., et al. 1996. Control of
cell division in Escherichia coli: regulation of transcription of I
involves both rpoS and SdiA-mediated autoinduction. Proc. Natl.
Acad. Sci. 93:336-341); serR (Theall, G., et al. 1979. Regulation
of the biosynthesis of aminoacyl-tRNA synthetases and of tRNA in
Escherichia coli. IV. Mutants with increased levels of leucyl- or
seryl-tRNA synthetase. Mol. Gen. Genet. 169:205-211); sfsA (Takeda,
K., et al. 2001. Effects of the Escherichia coli sfsA gene on mal
genes expression and a DNA binding activity of SfsA. Biosci.
Biotechnol. Biochem. 65:213-217); sfsB (nlp, sfs1) (Berlyn, M. K.
B., et al. 1996. Linkage map of Escherichia coli K-12, Edition 9.
In F. C. Neidhardt, R. Curtiss, J. L. Ingraham, E. C. C. Lin, K. B.
Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H.
E. Umbarger (eds.). Escherichia coli and Salmonella typhimurium:
cellular and molecular biology, 2nd ed. American Society for
Microbiology, Washington D.C.); soxR (Tsaneva, I. R., and B. Weiss.
1990. soxR, a locus governing a superoxide response regulon in
Escherichia coli K-12. J. Bacteriol. 172:4197-4205); soxS (Wu, J.,
and B. Weiss. 1991. Two divergently transcribed genes, soxR and
soxS, control a superoxide response regulon of Escherichia coli. J.
Bacteriol. 173:2864-2871); sr1R (gutR) (Csonka, L. N., and A. J.
Clark. 1979. Deletions generated by the transposon Tn10 in the srl
recA region of the Escherichia coli K-12 chromosome. Genetics.
93:321-343); tdcA (Ganduri, Y. L., et al. 1993. TdcA, a
transcriptional activator of the tdcABC operon of Escherichia coli,
is a member of the LysR family of proteins. Mol. Gen. Genet.
240:395-402); tdcR (Hagewood, B. T., et al. 1994. Functional
analysis of the tdcABC promoter of Escherichia coli: roles of TdcA
and TdcR. J. Bacteriol. 176:6241-6220); thrS (Springer, M., et al.
1985. Autogenous control of Escherichia coli threonyl-tRNA
synthetase expression in vivo. J. Mol. Biol. 185:93-104); tor R
(Simon, G., et al. 1994. The tor R gene of Escherichia coli encodes
a response regulator protein involved in the expression of the
trimethylamine N-oxide reductase genes. J. Bacteriol.
176:5601-5606); treR (Horlacher, R., and W. Boos. 1997.
Characterization of TreR, the major regulator of the Escherichia
coli trehalose system. J. Biol. Chem. 272:13026-13032); trpR
(Gunsalus, R. P., and C. Yanofsky. 1980. Nucleotide sequence and
expression of Escherichia coli trpR, the structural gene for the
trp aporepressor. Proc. Natl. Acad. Sci. 77:7117-7121); tyrR
(Camakaris, H., and J. Pittard. 1973. Regulation of tyrosine and
phenylalanine biosynthesis in Escherichia coli K-12: properties of
the tyrR gene product. J. Bacteriol. 115:1135-1144); uhpA (Kadner,
R. J., and D. M. Shattuck-Eidens. 1983. Genetic control of the
hexose phosphate transport system of Escherichia coli: mapping of
deletion and insertion mutations in the uhp region. J. Bacteriol.
155:1052-1061); uidR (gusR) (Novel, M., and G. Novel. 1976.
Regulation of beta-glucuronidase synthesis in Escherichia coli
K-12: pleiotropic constitutive mutations affecting uxu and uidA
expression. J. Bacteriol. 127:418-432); uspA (Berlyn, M. K. B., et
al. 1996. Linkage map of Escherichia coli K-12, Edition 9. In F. C.
Neidhardt, R. Curtiss, J. L. Ingraham, E. C. C. Lin, K. B. Low, B.
Magasanik, W. S.
[0313] Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger
(eds.). Escherichia coli and Salmonella typhimurium: cellular and
molecular biology, 2nd ed. American Society for Microbiology,
Washington D.C.); uxuR (Novel, M., and G. Novel. 1976. Regulation
of beta-glucuronidase synthesis in Escherichia coli K-12:
pleiotropic constitutive mutations affecting uxu and uidA
expression. J. Bacteriol. 127:418-432); wrbA (Yang, W., et al.
1993. A stationary-phase protein of Escherichia coli that affects
the mode of association between the trp repressor protein and
operator-bearing DNA. Proc. Natl. Acad. Sci. 90:5796-5800); xapR
(pndR) (Seeger, C., et al. 1995. Identification and
characterization of genes (xapA, xapB, and xapR) involved in
xanthosine catabolism in Escherichia coli. J. Bacteriol.
177:5506-5516); and xylR (Inouye, S., et al. 1987. Expression of
the regulatory gene xylS on the TOL plasmid is positively
controlled by the xylR gene product. Proc. Natl. Acad. Sci.
84:5182-5186);
[0314] Regulatory elements, promoters and other expression elements
and factors from prokaryotes other than E. coli and B. subtilis
include without limitation ahyRl gene product from Aeromonas
hydrophila and Aeromonas salmonicida (Swift, S., et al. 1997.
Quorum sensing in Aeromonas hydrophila and Aeromonas salmonicida:
identification of the LuxRI homologs AhyRI and AsaRI and their
cognate N-acylhomoserine lactone signal molecules. J. Bacteriol.
179:5271-5281); angR gene product from Vibrio anguillarum (Salinas,
P. C., et al. 1989.
[0315] Regulation of the iron uptake system in Vibrio anguillarum:
evidence for a cooperative effect between two transcriptional
activators. Proc. Natl. Acad. Sci. 86:3529-3522); aphA gene product
from Vibrio cholerae (Kovacikova, G., and K. Skorupski. 2001.
Overlapping binding sites for the virulence gene regulators AphA,
AphB and cAMP-CRP at the Vibrio cholerae tcpPH promoter. Mol.
Microbiol. 41:393-407); aphB gene product from Vibrio cholerae
(Kovachikova, G., and K. Skorupski. 2000. Differential activation
of the tcpPH promoter by AphB determines biotype specificity of
virulence gene expression in Vibrio cholerae. J. Bacteriol.
182:3228-3238); comE gene product from Streptococcus pneumoniae
(Ween, 0., et al. 1999. Identification of DNA binding sites for
ComE, a key regulator of natural competence in Streptococcus
pneumoniae. Mol. Microbiol. 33:817-827); esaI gene product from
Pantoea stewartii subsp. stewartii (von Bodman, S. B., et al. 1998.
A negative regulator mediates quorum-sensing control of
exopolysaccharide production in Pantoea stewartii subsp. stewartii.
Proc. Natl. Acad. Sci. 95:7687-7692); esaR gene product from
Pantoea stewartii subsp. stewartii (Id.); expl gene product from
Erwinia chrysanthemi (Nasser, W., et al. 1998. Characterization of
the Erwinia chrysanthemi expl-expR locus directing the synthesis of
two N-acyl-homoserine lactone signal molecules. Mol. Microbiol.
29:1391-1405); expR gene product from Erwinia chrysanthemi (Id.);
gacA gene product from Pseudomonas aeruginosa (Pessi, G., and D.
Haas. 2001. Dual control of hydrogen cyanide biosynthesis by the
global activator GacA in Pseudomonas aeruginosa PAO1. FEMS
Microbiol. Lett. 200:73-78); hapR gene product from Vibrio cholerae
(Jobling, M. G., and R. K. Holmes. Characterization of hapR, a
positive regulator of the Vibrio cholerae HA/protease gene hap, and
its identification as a functional homologue of the Vibrio harveyi
luxR gene. Mol. Microbiol. 26:1023-1034); hlyR gene product from
Vibrio cholerae (von Mechow, S., et al. 1985. Mapping of a gene
that regulates hemolysin production in Vibrio cholerae. J.
Bacteriol. 163:799-802); hupR gene product from Vibrio vulnificus
(Litwin, C. M., and J. Quackenbush. 2001. Characterization of a
Vibrio vulnificus LysR homologue, HupR, which regulates expression
of the haem uptake outer membrane protein, HupA. Microb. Pathog.
31:295-307); lasR gene product from Pseudomonas aerugenosa
(Gambella, M. J., and B. H. Igleweski. 1991. Cloning and
characterization of the Pseudomonas aeruginosa lasR gene, a
transcriptional activator of elastase expression. J. Bacteriol.
173:3000-3009); leuO gene product from Salmonella enterica serovar
Typhimurium (Fang, M., and H. Y. Wu. 1998. A promoter relay
mechanism for sequential gene activation. J. Bacteriol.
180:626-633); luxI gene product from Vibrio cholerae (Engebrecht,
J., and M. Silverman. Nucleotide sequence of the regulatory locus
controlling expression of bacterial genes for bioluminescence.
Nucleic Acids Res. 15:10455-10467); luxO gene product from Vibrio
cholerae (Bassler, B. L., et al. 1994. Sequence and function of
LuxO, a negative regulator of luminescence in Vibrio harveyi. Mol.
Microbiol. 12:403-412); luxR gene product from Vibrio cholerae
(Engebrecht, J., and M. Silverman. Nucleotide sequence of the
regulatory locus controlling expression of bacterial genes for
bioluminescence. Nucleic Acids Res. 15:10455-10467); phzR gene
product from Pseudomonas aureofaciens (Pierson, L. S., et al. 1994.
Phenazine antibiotic biosynthesis in Pseudomonas aureofaciens 30-84
is regulated by PhzR in response to cell density. J. Bacteriol.
176:3966-3974); rhlR gene product from Pseudomonas aeruginosa
(Ochsner, U. A. et al. 1994. Isolation and characterization of a
regulatory gene affecting rhamnolipid biosurfactant synthesis in
Pseudomonas aeruginosa. J. Bacteriol. 176:2044-2054); rsmA gene
product from Erwinia carotovora subsp. carotovora (Cui, Y., et al.
1995. Identification of a global repressor gene, rsmA, of Erwinia
carotovora subsp. carotovora that controls extracellular enzymes,
N-(3-oxohexanoyl)-L-homoserine lactone, and pathogenicity in
soft-rotting Erwinia spp. J. Bacteriol. 177:5108-5115); rsmB gene
product from Erwinia carotovora subsp. carotovora (Cui, Y., et al.
1999. rsmC of the soft-rotting bacterium Erwinia carotovora subsp.
carotovora negatively controls extracellular enzyme and harpin(Ecc)
production and virulence by modulating levels of regulatory RNA
(rsmB) and RNA-binding protein (RsmA). J. Bacteriol.
181:6042-6052); sirA gene product from Salmonella enterica serovar
Typhimurium (Goodier, R. I., and B. M. Ahmer. 2001. SirA orthologs
affects both motility and virulence. J. Bacteriol. 183:2249-2258);
taf gene product from Vibrio cholerae (Salinas, P. C., et al. 1989.
Regulation of the iron uptake system in Vibrio anguillarum:
evidence for a cooperative effect between two transcriptional
activators. Proc. Natl. Acad. Sci. 86:3529-3522); tcpP gene product
from Vibrio cholerae (Hase, C. C., and J. J. Mekalanos. 1998. TcpP
protein is a positive regulator of virulence gene expression in
Vibrio cholerae. Proc. Natl. Acad. Sci. 95:730-734); toxR gene
product from Vibrio cholerae (Miller, V. L., and J. J. Mekalanos.
1984. Synthesis of cholera toxin is positively regulated at the
transcriptional level by toxR. Proc. Natl. Acad. Sci.
81:3471-4375); toxS gene product from Vibrio cholerae (Miller, V.
L., et al. 1989. Identification of toxS, a regulatory gene whose
product enhances toxR-mediated activation of the cholera toxin
promoter. J. Bacteriol. 171:1288-1293); toxT from Vibrio cholerae
(Kaufman, M. R., et al. 1993. Biogenesis and regulation of the
Vibrio cholerae toxin-coregulated pilus: analogies to other
virulence factor secretory systems. Gene. 126:43-49); traM gene
product from Agrobacterium tumefaciens (Faqua, C., et al. 1995.
Activity of the Agrobacterium Ti plasmid conjugal transfer
regulator TraR is inhibited by the product of the traM gene. J.
Bacteriol. 177:1367-1373); traR gene product from Agrobacterium
tumefaciens (Piper, K. R., et al. 1993. Conjugation factor of
Agrobacterium tumefaciens regulates Ti plasmid transfer by
autoinduction. Nature. 362:448-450); vicH gene product from Vibrio
cholerae (Tendeng, C., et al. 2000. Isolation and characterization
of vicH, encoding a new pleiotropic regulator in Vibrio cholerae.
J. Bacteriol. 182:2026-2032); vspR gene product from Vibrio
cholerae (Yildiz, F. H., et al. 2001. VpsR, a Member of the
Response Regulators of the Two-Component Regulatory Systems, Is
Required for Expression of vps Biosynthesis Genes and
EPS(ETr)-Associated Phenotypes in Vibrio choleraeO1 E1 Tor. J.
Bacteriol. 183:1716-1726).
[0316] II.C.5.b. Bacillus Subtilis
[0317] Regulatory elements, promoters and other expression elements
and expression elements from B. subtilis include but are not
limited to abrB (Perego, M., et al. 1988. Structure of the gene for
the transition state regulator, abrB: regulator synthesis is
controlled by the spo0A sporulation gene in Bacillus subtilis. Mol.
Microbiol. 2:698-699); acoR (Ali, N. 0., et al. 2001. Regulation of
the acetoin catabolic pathway is controlled by sigma L in Bacillus
subtilis. J. Bacteriol. 183:2497-2504); ahrC (Klinger, U., et al.
1995. A binding site for activation by the Bacillus subtilis AhrC
protein, a repressor/activator of arginine metabolism. Mol. Gen.
Genet. 248:329-340); alaR (Sohenshein, A. L., J. A. Hoch, and R.
Losick (eds.) 2002. Bacillus subtilis and its closest relatives:
from genes to cells. American Society for Microbiology, Washington
D.C.); alsR (Renna, M. C., et al. 1993. Regulation of the Bacillus
subtilis alsS, alsD, and alsR genes involved in
post-exponential-phase production of acetoin. J. Bacteriol.
175:3863-3875); ansR (Sun, D., and P. Setlow. 1993. Cloning and
nucleotide sequence of the Bacillus subtilis ansR gene, which
encodes a repressor of the ans operon coding for L-asparaginase and
L-aspartase. J. Bacteriol. 175:2501-2506); araR (Sa-Nogueira, I.,
and L. J. Mota. 1997. Negative regulation of L-arabinose metabolism
in Bacillus subtilis: characterization of the araR (araC) gene. J.
Bacteriol. 179:1598-1608); arfM (Marino, M., et al. 2001.
Modulation of anaerobic energy metabolism of Bacillus subtilis by
arfM (ywiD). J. Bacteriol. 183:6815-6821); arsR (Rosenstein, R., et
al. 1992. Expression and regulation of the antimonite, arsenite,
and arsenate resistance operon of Staphylococcus xylosus plasmid
pSX267. J. Bacteriol. 174:3676-3683); azlB (Belitsky, B. R., et al.
1997. An lrp-like gene of Bacillus subtilis involved in
branched-chain amino acid transport. J. Bacteriol. 179:54485457);
birA (Bower, S., et al. 1995. Cloning and characterization of the
Bacillus subtilis birA gene encoding a repressor of the biotin
operon. J. Bacteriol. 177:2572-2575); bkdR (Bebarbouille, M., et
al. 1999. Role of bkdR, a transcriptional activator of the
sigL-dependent isoleucine and valine degradation pathway in
Bacillus subtilis. J. Bacteriol. 181:2059-2066); bltR (Ahmed, M.,
et al. 1995. Two highly similar multidrug transporters of Bacillus
subtilis whose expression is differentially regulated. J.
Bacteriol. 177:3904-3910); bmrR (Ahmed, M., et al. 1994. A protein
that activates expression of a multidrug efflux transporter upon
binding the transporter substrates. J. Biol. Chem.
269:28506-28513); ccpA (Henkin, T. M., et al. 1991. Catabolite
repression of alpha-amylase gene expression in Bacillus subtilis
involves a trans-acting gene product homologous to the Escherichia
coli lacl and galR repressors. Mol. Microbiol. 5:575-584); ccpB
(Chauvaux, S., et al. 1998. CcpB, a novel transcription factor
implicated in catabolite repression in Bacillus subtilis. J.
Bacteriol. 180:491-497); ccpC (Jourlin-Castelli, C., et al. 2000.
CcpC, a novel regulator of the LysR family required for glucose
repression of the citB gene in Bacillus subtilis. J. Mol. Biol.
295:865-878); cggR (Fillinger, S., et al. 2000. Two
glyceraldehyde-3-phosphate dehydrogenases with opposite
physiological roles in a nonphotosynthetic bacterium. J. Biol.
Chem. 275:14031-14037); cheB (Bischoff, D. S., and G. W. Ordal.
1991. Sequence and characterization of Bacillus subtilis CheB, a
homolog of Escherichia coli CheY, and its role in a different
mechanism of chemotaxis. J. Biol. Chem. 266:12301-12305); cheY
(Bischoff, D. S., et al. 1993. Purification and characterization of
Bacillus subtilis CheY. Biochemistry 32:9256-9261); citR (Jin, S.,
and A. L. Sonenshein. 1994. Transcriptional regulation of Bacillus
subtilis citrate synthase genes. J. Bacteriol. 176:4680-4690); citT
(Yamamoto, H., et al. 2000. The CitST two-component system
regulates the expression of the Mg-citrate transporter in Bacillus
subtilis. Mol. Microbiol. 37:898-912); codY (Slack, F. J., et al.
1995. A gene required for nutritional repression of the Bacillus
subtilis dipeptide permease operon. Mol. Microbiol. 15:689-702);
comA (Nakano, M. M., and P. Zuber. 1989. Cloning and
characterization of srfB, a regulatory gene involved in surfactin
production and competence in Bacillus subtilis. J. Bacteriol.
171:5347-5353); comK (Msadek, T., et al. 1994. MecB of Bacillus
subtilis, a member of the ClpC ATPase family, is a pleiotropic
regulator controlling competence gene expression and growth at high
temperature. Proc. Natl. Acad. Sci. 91:5788-5792); comQ (Weinrauch,
Y., et al. 1991. Sequence and properties of comQ, a new competence
regulatory gene of Bacillus subtilis. J. Bacteriol. 173:5685-5693);
cssR (Hyyrylainen, H. L., et al. 2001. A novel two-component
regulatory system in Bacillus subtilis for the survival of severe
secretion stress. Mol. Microbiol. 41:1159-1172); ctsR (Kruger, E.,
and M. Hecker. 1998. The first gene of the Bacillus subtilis clpC
operon, ctsR, encodes a negative regulator of its own operon and
other class III heat shock genes. J. Bacteriol. 180:6681-6688);
dctR (Sohenshein, A. L., J. A. Hoch, and R. Losick (eds.) 2002.
Bacillus subtilis and its closest relatives: from genes to cells.
American Society for Microbiology, Washington D.C.); degA (Bussey,
L. B., and R. L. Switzer. 1993. The degA gene product accelerates
degradation of Bacillus subtilis phosphoribosylpyrophosphate
amidotransferase in Escherichia coli. J. Bacteriol. 175:6348-6353);
degU (Msadek, T., et al. 1990. Signal transduction pathway
controlling synthesis of a class of degradative enzymes in Bacillus
subtilis: expression of the regulatory genes and analysis of
mutations in degS and degU. J. Bacteriol. 172:824-834); deoR
(Saxild, H. H., et al. 1996. Dra-nupC-pdp operon of Bacillus
subtilis: nucleotide sequence, induction by deoxyribonucleosides,
and transcriptional regulation by the deoR-encoded DeoR repressor
protein. J. Bacteriol. 178:424-434); exuR (Sohenshein, A. L., J. A.
Hoch, and R. Losick (eds.) 2002. Bacillus subtilis and its closest
relatives: from genes to cells. American Society for Microbiology,
Washington D.C.); frn (Cruz Ramos, H., et al. 1995. Anaerobic
transcription activation in Bacillus subtilis: identification of
distinct FNR-dependent and -independent regulatory mechanisms. EMBO
J. 14:5984-5994); fruR (Saier, M. H. Jr. 1996. Cyclic
AMP-independent catabolite repression in bacteria. FEMS Microbiol.
Lett. 138:97-103); fur (Chen, L., et al. 1993. Metalloregulation in
Bacillus subtilis: isolation and characterization of two genes
differentially repressed by metal ions. J. Bacteriol.
175:5428-5437); gabR (Sohenshein, A. L., J. A. Hoch, and R. Losick
(eds.) 2002. Bacillus subtilis and its closest relatives: from
genes to cells. American Society for Microbiology, Washington
D.C.); gerE (Holand, S. K., et al. 1987. The possible DNA-binding
nature of the regulatory proteins, encoded by spo11D and gerE,
involved in the sporulation of Bacillus subtilis. J. Gen.
Microbiol. 133:2381-2391); glcR (Stulke, J., et al. 2001.
Characterization of glucose-repression-resistant mutants of
Bacillus subtilis: identification of the glcR gene. Arch.
Microbiol. 175:441-449); gleT (Paulsen, I. T., et al. 1998.
Characterization of glucose-specific catabolite
repression-resistant mutants of Bacillus subtilis: identification
of a novel hexose:H+ symporter. J. Bacteriol. 180:498-504); glnR
(Schreier, H. J., et al. 1989. Regulation of Bacillus subtilis
glutamine synthetase gene expression by the product of the glnR
gene. J. Mol. Biol. 210:51-63); glpP (Holmberg, C., and B. Rutberg.
1991. Expression of the gene encoding glycerol-3-phosphate
dehydrogenase (g1pD) in Bacillus subtilis is controlled by
antitermination. Mol. Microbiol. 5:2891-2900); gltC (Bohannon, D.
E. and A. L. Sonenshein. 1989. Positive regulation of glutamate
biosynthesis in Bacillus subtilis. J. Bacteriol. 171:4718-4727);
gltR (Belitsky, B. R., and A. L. Sonenshein. 1997. Altered
transcription activation specificity of a mutant form of Bacillus
subtilis G1tR, a LysR family member. J. Bacteriol. 179:1035-1043);
gntR (Fujita, Y., and T. Fujita. 1987. The gluconate operon gnt of
Bacillus subtilis encodes its own transcriptional negative
regulator. Proc. Natl. Acad. Sci. 84:4524-4528); gutR (Ye, R., et
al. 1994. Glucitol induction in Bacillus subtilis is mediated by a
regulatory factor, GutR. J. Bacteriol. 176:3321-3327); hpr (Perego,
M., and J. A. Hoch. 1988. Sequence analysis and regulation of the
hpr locus, a regulatory gene for protease production and
sporulation in Bacillus subtilis. J. Bacteriol. 170:2560-2567);
hrcA (Schulz, A., and W. Schumann. 1996. hrcA, the first gene of
the Bacillus subtilis dnaK operon encodes a negative regulator of
class I heat shock genes. J. Bacteriol. 178:1088-1093); hutP (Oda,
M., et al. 1992. Analysis of the transcriptional activity of the
but promoter in Bacillus subtilis and identification of a
cis-acting regulatory region associated with catabolite repression
downstream from the site of transcription. Mol. Microbiol.
6:2573-2582); hxlR (Sohenshein, A. L., J. A. Hoch, and R. Losick
(eds.) 2002. Bacillus subtilis and its closest relatives: from
genes to cells. American Society for Microbiology, Washington
D.C.); iolR (Yoshida, K. I., et al. 1999. Interaction of a
repressor and its binding sites for regulation of the Bacillus
subtilis iol divergon. J. Mol. Biol. 285:917-929); kdgR (Pujic, P.,
et al. 1998. The kdgRKAT operon of Bacillus subtilis: detection of
the transcript and regulation by the kdgR and ccpA genes.
Microbiology. 144:3111-3118); kipR (Sohenshein, A. L., J. A. Hoch,
and R. Losick (eds.) 2002. Bacillus subtilis and its closest
relatives: from genes to cells. American Society for Microbiology,
Washington D.C.); lacR (Errington, J., and C. H. Vogt. 1990.
Isolation and characterization of mutations in the gene encoding an
endogenous Bacillus subtilis beta-galactosidase and its regulator.
J. Bacteriol. 172:488-490); levR (Bebarbouille, M., et al. 1991.
The transcriptional regulator LevR of Bacillus subtilis has domains
homologous to both sigma 54- and phosphotransferase
system-dependent regulators. Proc. natl. Acad. Sci. 88:2212-2216);
lexA (Lovett, C. M. Jr., and J. W. Roberts. 1985. Purification of a
RecA protein analogue from Bacillus subtilis. J. Biol. Chem.
260:3305-3313); licR (Tobisch, S., et al. 1997. Identification and
characterization of a new beta-glucoside utilization system in
Bacillus subtilis. J. Bacteriol. 179:496-506); licT (Le Coq, D., et
al. 1995. New beta-glucoside (bgl) genes in Bacillus subtilis: the
bglP gene product has both transport and regulatory functions
similar to those of BglF, its Escherichia coli homolog. J.
Bacteriol. 177:1527-1535); lmrA (Kumano, M., et al. 1997. A 32 kb
nucleotide sequence from the region of the lincomycin-resistance
gene (22 degrees-25 degrees) of the Bacillus subtilis chromosome
and identification of the site of the lin-2 mutation. Microbiology.
143:2775-2782); lrpA gene product from Pyrococcus furiosus
(Brinkman, A. B., et al. 2000. An Lrp-like transcriptional
regulator from the archaeon Pyrococcus furiosus is negatively
autoregulated. J. Biol. Chem. 275:38160-38169); lrpB (Sohenshein,
A. L., J. A. Hoch, and R. Losick (eds.) 2002. Bacillus subtilis and
its closest relatives: from genes to cells. American Society for
Microbiology, Washington D.C.); lrpC (Beloin, C., et al. 1997.
Characterization of an lrp-like OrpC) gene from Bacillus subtilis.
Mol. Gen. Genet. 256:63-71); lytR (Huang, X., and J. D. Helmann.
1998. Identification of target promoters for the Bacillus subtilis
sigma X factor using a consensus-directed search. J. Mol. Biol.
279:165-173); lytT (Sohenshein, A. L., J. A. Hoch, and R. Losick
(eds.) 2002. Bacillus subtilis and its closest relatives: from
genes to cells. American Society for Microbiology, Washington
D.C.); manR gene product from Listeria monocytogenes (Dalet, K., et
al. 2001. A sigma(54)-dependent PTS permease of the mannose family
is responsible for sensitivity of Listeria monocytogenes to
mesentericin Y105. Microbiology. 147:3263-3269); mntR (Que, Q., and
J. D. Helmann. 2000. Manganese homeostasis in Bacillus subtilis is
regulated by MntR, a bifunctional regulator related to the
diphtheria toxin repressor family of proteins. Mol. Microbiol.
35:1454-1468); msmR gene product from Streptococcus mutans
(Russell, R. R., et al. 1992. A binding protein-dependent transport
system in Streptococcus mutans responsible for multiple sugar
metabolism. J. Biol. Chem. 267:4631-4637); mta (Baranova, N. N., et
al. 1999. Mta, a global MerR-type regulator of the Bacillus
subtilis multidrug-efflux transporters. Mol. Microbiol.
31:1549-1559); mtlR (Henstra, S. A., et al. 1999. The Bacillus
stearothermophilus mannitol regulator, MtlR, of the
phosphotransferase system. A DNA-binding protein, regulated by HPr
and iicbmtl-dependent phosphorylation. J. Biol. Chem.
274:4754-4763); mtrB (Gollnick, P., et al. 1990. The mtr locus is a
two-gene operon required for transcription attenuation in the trp
operon of Bacillus subtilis. Proc. Natl. Acad. Sci. 87:8726-8730);
nhaX (Sohenshein, A. L., J. A. Hoch, and R. Losick (eds.) 2002.
Bacillus subtilis and its closest relatives: from genes to cells.
American Society for Microbiology, Washington D.C.); toxR gene
product from Vibrio cholerae (Miller, V. L., and J. J. Mekalanos.
1984. Synthesis of cholera toxin is positively regulated at the
transcriptional level by toxR. Proc. Natl. Acad. Sci.
81:3471-3475); padR gene product from Pediococcus pentosaceus
(Barthelmebs, L., et al. 2000. Inducible metabolism of phenolic
acids in Pediococcus pentosaceus is encoded by an autoregulated
operon which involves a new class of negative transcriptional
regulator. J. Bacteriol. 182:6724-6731); paiA (Sohenshein, A. L.,
J. A. Hoch, and R. Losick (eds.) 2002. Bacillus subtilis and its
closest relatives: from genes to cells. American Society for
Microbiology, Washington D.C.); paiB (Id.); perA (Id.); phoP
(Birkey, S. M., et al. 1994. A pho regulon promoter induced under
sporulation conditions. Gene. 147:95-100); pksA (Sohenshein, A. L.,
J. A. Hoch, and R. Losick (eds.) 2002. Bacillus subtilis and its
closest relatives: from genes to cells. American Society for
Microbiology, Washington D.C.); pucR (Schultz, A. C., et al. 2001.
Functional analysis of 14 genes that constitute the purine
catabolic pathway in Bacillus subtilis and evidence for a novel
regulon controlled by the PucR transcription activator. J.
Bacteriol. 183:3293-3302); purR (Weng, M., et al. 1995.
Identification of the Bacillus subtilis pur operon repressor. Proc.
Natl. Acad. Sci. 92:7455-7459); pyrR (Martinussen, J., et al. 1995.
Two genes encoding uracil phosphoribosyltransferase are present in
Bacillus subtilis. J. Bacteriol. 177:271-274); rbsR (Rodionov, D.
A., et al. 2001. Transcriptional regulation of pentose utilisation
systems in the
Bacillus/Clostridium group of bacteria. FEMS Microbiol. Lett.
205:305-314); resD (Suin, G., et al. 1996. Regulators of aerobic
and anaerobic respiration in Bacillus subtilis. J. Bacteriol.
178:1374-1385); rocR (Gardan, R., et al. 1997. Role of the
transcriptional activator RocR in the arginine-degradation pathway
of Bacillus subtilis. Mol. Microbiol. 24:825-837); rsiX (Tortosa,
P., et al. 2000. Characterization of ylbF, a new gene involved in
competence development and sporulation in Bacillus subtilis. Mol.
Microbiol. 35:1110-1119); sacT (Debarbouille, M., et al. 1990. The
sacT gene regulating the sacPA operon in Bacillus subtilis shares
strong homology with transcriptional antiterminators. J. Bacteriol.
172:3966-3973); sacV (Wong, S. L., et al. 1988. Cloning and
nucleotide sequence of senN, a novel `Bacillus natto` (B. subtilis)
gene that regulates expression of extracellular protein genes. J.
Gen. Microbiol. 134:3269-3276); sacY (Steinmetz, M., et al 1989.
Induction of saccharolytic enzymes by sucrose in Bacillus subtilis:
evidence for two partially interchangeable regulatory pathways. J.
Bacteriol. 171:1519-1523); senS (Wang, L. F., and R. H. Dori. 1990.
Complex character of senS, a novel gene regulating expression of
extracellular-protein genes of Bacillus subtilis. J. Bacteriol.
172:1939-1947); sinR (Bai, U., et al. 1993. SinI modulates the
activity of SinR, a developmental switch protein of Bacillus
subtilis, by protein-protein interaction. Genes Dev. 7:139-148);
slr (Asayama, M., et al. 1998. Translational attenuation of the
Bacillus subtilis spo0B cistron by an RNA structure encompassing
the initiation region. Nucleic Acids Res. 26:824-830); splA
(Fajardo-Cavazos, P., and W. L. Nicholson. 2000. The TRAP-like SplA
protein is a trans-acting negative regulator of spore photoproduct
lyase synthesis during Bacillus subtilis sporulation. J. Bacteriol.
182:555-560); spo0A (Smith, I., et al. 1991. The role of negative
control in sporulation. Res. Microbiol. 142:831-839); spo0F
(Lewandoski, M., et al. 1986. Transcriptional regulation of the
spo0F gene of Bacillus subtilis. J. Bacteriol. 168:870-877);
spoIIID (Kunkel, B., et al. 1989. Temporal and spatial control of
the mother-cell regulatory gene spoIIID of Bacillus subtilis.
Genes. Dev. 3:1735-1744); spoVT (Bagyan, I, et al. 1996. A
compartmentalized regulator of developmental gene expression in
Bacillus subtilis. J. Bacteriol. 178:4500-4507); tenA (Pang, A. S.,
et al. 1991. Cloning and characterization of a pair of novel genes
that regulate production of extracellular enzymes in Bacillus
subtilis. J. Bacteriol. 173:46-54); ten1 (Sohenshein, A. L., J. A.
Hoch, and R. Losick (eds.) 2002. Bacillus subtilis and its closest
relatives: from genes to cells. American Society for Microbiology,
Washington D.C.); tnrA (Wray, L. V., Jr., et al. 1996. TnrA, a
transcription factor required for global nitrogen regulation in
Bacillus subtilis. Proc. Natl. Acad. Sci. 93:8841-8845); treR
(Schock, F., and M. K. Dahl. 1996. Expression of the tre operon of
Bacillus subtilis 168 is regulated by the repressor TreR. J.
Bacteriol. 178:4576-4581); xre (McDonnell, G. E., et al. 1994.
Genetic control of bacterial suicide: regulation of the induction
of PBSX in Bacillus subtilis. J. Bacteriol. 176:5820-5830); xylR
gene product from Bacillus megaterium (Rygus, T., et al. 1991.
Molecular cloning, structure, promoters and regulatory elements for
transcription of the Bacillus megaterium encoded regulon for xylose
utilization. Arch. Microbiol. 155:535:542); yacF (Sohenshein, A.
L., J. A. Hoch, and R. Losick (eds.) 2002. Bacillus subtilis and
its closest relatives: from genes to cells. American Society for
Microbiology, Washington D.C.); and zur (Gaballa, A., and J. D.
Helmann. 1998. Identification of a zinc-specific metalloregulatory
protein, Zur, controlling zinc transport operons in Bacillus
subtilis. J. Bacteriol. 180:5815-5821).
[0318] II.C.5.c. Other Eubacteria
[0319] Regulatory elements, promoters and other expression elements
and factors from prokaryotes other than E. coli and B. subtilis
include without limitation ahyRl gene product from Aeromonas
hydrophila and Aeromonas salmonicida (Swift, S., et al. 1997.
Quorum sensing in Aeromonas hydrophila and Aeromonas salmonicida:
identification of the LuxRI homologs AhyRI and AsaRI and their
cognate N-acylhomoserine lactone signal molecules. J. Bacteriol.
179:5271-5281); angR gene product from Vibrio anguillarum (Salinas,
P. C., et al. 1989. Regulation of the iron uptake system in Vibrio
anguillarum: evidence for a cooperative effect between two
transcriptional activators. Proc. Natl. Acad. Sci. 86:3529-3522);
aphA gene product from Vibrio cholerae (Kovacikova, G., and K.
Skorupski. 2001. Overlapping binding sites for the virulence gene
regulators AphA, AphB and cAMP-CRP at the Vibrio cholerae tcpPH
promoter. Mol. Microbiol. 41:393-407); aphB gene product from
Vibrio cholerae (Kovachikova, G., and K. Skorupski. 2000.
Differential activation of the tcpPH promoter by AphB determines
biotype specificity of virulence gene expression in Vibrio
cholerae. J. Bacteriol. 182:3228-3238); comE gene product from
Streptococcus pneumoniae (Ween, 0., et al. 1999. Identification of
DNA binding sites for ComE, a key regulator of natural competence
in Streptococcus pneumoniae. Mol. Microbiol. 33:817-827); esaI gene
product from Pantoea stewartii subsp. stewartii (von Bodman, S. B.,
et al. 1998. A negative regulator mediates quorum-sensing control
of exopolysaccharide production in Pantoea stewartii subsp.
stewartii. Proc. Natl. Acad. Sci. 95:7687-7692); esaR gene product
from Pantoea stewartii subsp. stewartii (Id.); expl gene product
from Erwinia chrysanthemi (Nasser, W., et al. 1998.
Characterization of the Erwinia chrysanthemi expl-expR locus
directing the synthesis of two N-acyl-homoserine lactone signal
molecules. Mol. Microbiol. 29:1391-1405); expR gene product from
Erwinia chrysanthemi (Id.); gacA gene product from Pseudomonas
aeruginosa (Pessi, G., and D. Haas. 2001. Dual control of hydrogen
cyanide biosynthesis by the global activator GacA in Pseudomonas
aeruginosa PAO1. FEMS Microbiol. Lett. 200:73-78); hapR gene
product from Vibrio cholerae (Jobling, M. G., and R. K. Holmes.
Characterization of hapR, a positive regulator of the Vibrio
cholerae HA/protease gene hap, and its identification as a
functional homologue of the Vibrio harveyi luxR gene. Mol.
Microbiol. 26:1023-1034); hlyR gene product from Vibrio cholerae
(von Mechow, S., et al. 1985. Mapping of a gene that regulates
hemolysin production in Vibrio cholerae. J. Bacteriol.
163:799-802); hupR gene product from Vibrio vulnificus (Litwin, C.
M., and J. Quackenbush. 2001. Characterization of a Vibrio
vulnificus LysR homologue, HupR, which regulates expression of the
haem uptake outer membrane protein, HupA. Microb. Pathog.
31:295-307); lasR gene product from Pseudomonas aerugenosa
(Gambella, M. J., and B. H. Igleweski. 1991. Cloning and
characterization of the Pseudomonas aeruginosa lasR gene, a
transcriptional activator of elastase expression. J. Bacteriol.
173:3000-3009); leuO gene product from Salmonella enterica serovar
Typhimurium (Fang, M., and H. Y. Wu. 1998. A promoter relay
mechanism for sequential gene activation. J. Bacteriol.
180:626-633); luxI gene product from Vibrio cholerae (Engebrecht,
J., and M. Silverman. Nucleotide sequence of the regulatory locus
controlling expression of bacterial genes for bioluminescence.
Nucleic Acids Res. 15:10455-10467); luxO gene product from Vibrio
cholerae (Bassler, B. L., et al. 1994. Sequence and function of
LuxO, a negative regulator of luminescence in Vibrio harveyi. Mol.
Microbiol. 12:403-412); luxR gene product from Vibrio cholerae
(Engebrecht, J., and M. Silverman. Nucleotide sequence of the
regulatory locus controlling expression of bacterial genes for
bioluminescence. Nucleic Acids Res. 15:10455-10467); phzR gene
product from Pseudomonas aureofaciens (Pierson, L. S., et al. 1994.
Phenazine antibiotic biosynthesis in Pseudomonas aureofaciens 30-84
is regulated by PhzR in response to cell density. J. Bacteriol.
176:3966-3974); rhlR gene product from Pseudomonas aeruginosa
(Ochsner, U. A. et al. 1994. Isolation and characterization of a
regulatory gene affecting rhamnolipid biosurfactant synthesis in
Pseudomonas aeruginosa. J. Bacteriol. 176:2044-2054); rsmA gene
product from Erwinia carotovora subsp. carotovora (Cui, Y., et al.
1995. Identification of a global repressor gene, rsmA, of Erwinia
carotovora subsp. carotovora that controls extracellular enzymes,
N-(3-oxohexanoyl)-L-homoserine lactone, and pathogenicity in
soft-rotting Erwinia spp. J. Bacteriol. 177:5108-5115); rsmB gene
product from Erwinia carotovora subsp. carotovora (Cui, Y., et al.
1999. rsmC of the soft-rotting bacterium Erwinia carotovora subsp.
carotovora negatively controls extracellular enzyme and harpin(Ecc)
production and virulence by modulating levels of regulatory RNA
(rsmB) and RNA-binding protein (RsmA). J. Bacteriol.
181:6042-6052); sirA gene product from Salmonella enterica serovar
Typhimurium (Goodier, R. I., and B. M. Ahmer. 2001. SirA orthologs
affects both motility and virulence. J. Bacteriol. 183:2249-2258);
taf gene product from Vibrio cholerae (Salinas, P. C., et al. 1989.
Regulation of the iron uptake system in Vibrio anguillarum:
evidence for a cooperative effect between two transcriptional
activators. Proc. Natl. Acad. Sci. 86:3529-3522); tcpP gene product
from Vibrio cholerae (Hase, C. C., and J. J. Mekalanos. 1998. TcpP
protein is a positive regulator of virulence gene expression in
Vibrio cholerae. Proc. Natl. Acad. Sci. 95:730-734); toxR gene
product from Vibrio cholerae (Miller, V. L., and J. J. Mekalanos.
1984. Synthesis of cholera toxin is positively regulated at the
transcriptional level by toxR. Proc. Natl. Acad. Sci.
81:3471-4375); toxS gene product from Vibrio cholerae (Miller, V.
L., et al. 1989. Identification of toxS, a regulatory gene whose
product enhances toxR-mediated activation of the cholera toxin
promoter. J. Bacteriol. 171:1288-1293); toxT from Vibrio cholerae
(Kaufman, M. R., et al. 1993. Biogenesis and regulation of the
Vibrio cholerae toxin-coregulated pilus: analogies to other
virulence factor secretory systems. Gene. 126:43-49); traM gene
product from Agrobacterium tumefaciens (Faqua, C., et al. 1995.
Activity of the Agrobacterium Ti plasmid conjugal transfer
regulator TraR is inhibited by the product of the traM gene. J.
Bacteriol. 177:1367-1373); traR gene product from Agrobacterium
tumefaciens (Piper, K. R., et al. 1993. Conjugation factor of
Agrobacterium tumefaciens regulates Ti plasmid transfer by
autoinduction. Nature. 362:448-450); vicH gene product from Vibrio
cholerae (Tendeng, C., et al. 2000. Isolation and characterization
of vicH, encoding a new pleiotropic regulator in Vibrio cholerae.
J. Bacteriol. 182:2026-2032); vspR gene product from Vibrio
cholerae (Yildiz, F. H., et al. 2001. VpsR, a Member of the
Response Regulators of the Two-Component Regulatory Systems, Is
Required for Expression of vps Biosynthesis Genes and
EPS(ETr)-Associated Phenotypes in Vibrio cholerae 01 E1 Tor. J.
Bacteriol. 183:1716-1726); gadR gene product from Lactococcus
lactis (Sanders, J. W., et al. 1997. A chloride-inducible gene
expression cassette and its use in induced lysis of Lactococcus
lactis. Appl. Environ. Microbiol. 63:4877-4882); hrpB gene product
from Pseudomonas solanacearum (Van Gijsegem, F., et al. 1995. The
hrp gene locus of Pseudomonas solanacearum, which controls the
production of a type III secretion system, encodes eight proteins
related to components of the bacterial flagellar biogenesis
complex. Mol. Microbiol. 15:1095-1114); carotovora subsp.
carotovora (Cui, Y., et al. 1995. Identification of a global
repressor gene, rsmA, of Erwinia carotovora subsp. carotovora that
controls extracellular enzymes, N-(3-oxohexanoyl)-L-homoserine
lactone, and pathogenicity in soft-rotting Erwinia spp. J.
Bacteriol. 177:5108-5115); rsmB gene product from Erwinia
carotovora subsp. carotovora (Cui, Y., et al. 1999. rsmC of the
soft-rotting bacterium Erwinia carotovora subsp. carotovora
negatively controls extracellular enzyme and harpin(Ecc) production
and virulence by modulating levels of regulatory RNA (rsmB) and
RNA-binding protein (RsmA). J. Bacteriol. 181:6042-6052); sirA gene
product from Salmonella enterica serovar Typhimurium (Goodier, R.
I., and B. M. Ahmer. 2001. SirA orthologs affects both motility and
virulence. J. Bacteriol. 183:2249-2258); taf gene product from
Vibrio cholerae (Salinas, P. C., et al. 1989. Regulation of the
iron uptake system in Vibrio anguillarum: evidence for a
cooperative effect between two transcriptional activators. Proc.
Natl. Acad. Sci. 86:3529-3522); tcpP gene product from Vibrio
cholerae (Hase, C. C., and J. J. Mekalanos. 1998. TcpP protein is a
positive regulator of virulence gene expression in Vibrio cholerae.
Proc. Natl. Acad. Sci. 95:730-734); toxR gene product from Vibrio
cholerae (Miller, V. L., and J. J. Mekalanos. 1984. Synthesis of
cholera toxin is positively regulated at the transcriptional level
by toxR. Proc. Natl. Acad. Sci. 81:3471-4375); toxS gene product
from Vibrio cholerae (Miller, V. L., et al. 1989. Identification of
toxS, a regulatory gene whose product enhances toxR-mediated
activation of the cholera toxin promoter. J. Bacteriol.
171:1288-1293); toxT from Vibrio cholerae (Kaufman, M. R., et al.
1993. Biogenesis and regulation of the Vibrio cholerae
toxin-coregulated pilus: analogies to other virulence factor
secretory systems. Gene. 126:43-49); traM gene product from
Agrobacterium tumefaciens (Faqua, C., et al. 1995. Activity of the
Agrobacterium Ti plasmid conjugal transfer regulator TraR is
inhibited by the product of the traM gene. J. Bacteriol.
177:1367-1373); traR gene product from Agrobacterium tumefaciens
(Piper, K. R., et al. 1993. Conjugation factor of Agrobacterium
tumefaciens regulates Ti plasmid transfer by autoinduction. Nature.
362:448-450); vicH gene product from Vibrio cholerae (Tendeng, C.,
et al. 2000. Isolation and characterization of vicH, encoding a new
pleiotropic regulator in Vibrio cholerae. J. Bacteriol.
182:2026-2032); vspR gene product from Vibrio cholerae (Yildiz, F.
H., et al. 2001. VpsR, a Member of the Response Regulators of the
Two-Component Regulatory Systems, Is Required for Expression of vps
Biosynthesis Genes and EPS(ETr)-Associated Phenotypes in Vibrio
cholerae 01 E1 Tor. J. Bacteriol. 183:1716-1726); lrpA gene product
from Pyrococcus furiosus (Brinkman, A. B., et al. 2000. An Lrp-like
transcriptional regulator from the archaeon Pyrococcus furiosus is
negatively autoregulated. J. Biol. Chem. 275:38160-38169); manR
gene product from Listeria monocytogenes (Dalet, K., et al. 2001. A
sigma(54)-dependent PTS permease of the mannose family is
responsible for sensitivity of Listeria monocytogenes to
mesentericin Y105. Microbiology. 147:3263-3269); msmR gene product
from Streptococcus mutans (Russell, R. R., et al. 1992. A binding
protein-dependent transport system in Streptococcus mutans
responsible for multiple sugar metabolism. toxR gene product from
Vibrio cholerae (Miller, V. L., and J. J. Mekalanos. 1984.
Synthesis of cholera toxin is positively regulated at the
transcriptional level by toxR. Proc. Natl. Acad. Sci.
81:3471-3475); padR gene product from Pediococcus pentosaceus
(Barthelmebs, L., et al. 2000. Inducible metabolism of phenolic
acids in Pediococcus pentosaceus is encoded by an autoregulated
operon which involves a new class of negative transcriptional
regulator. J. Bacteriol. 182:6724-6731); purR (Weng, M., et al.
1995); and xylR gene product from Bacillus megaterium (Rygus, T.,
et al. 1991. Molecular cloning, structure, promoters and regulatory
elements for transcription of the Bacillus megaterium encoded
regulon for xylose utilization. Arch. Microbiol. 155:535:542).
[0320] II.C.5.d. Bacteriophage and Transposable Elements
[0321] Regulatory elements, promoters and other expression elements
from bacteriophage and transposable elements include without
limitation cI gene product from bacteriophage lambda mation and/or
segregated minicells (Reichardt, L. F. 1975. Control of
bacteriophage lambda repressor synthesis: regulation of the
maintenance pathway of the cro and cI products. J. Mol. Biol.
93:289-309); (Love, C. A., et al. 1996. Stable high-copy-number
bacteriophage lambda promoter vectors for overproduction of
proteins in Escherichia coli. Gene. 176:49-53); the c2 gene product
from bacteriophage P22 (Gough, M., and S. Tokuno. 1975. Further
structural and functional analogies between the repressor regions
of phages P22 and lambda. Mol. Gen. Genet. 138:71-79); the cro gene
from bacteriophage lambda (Reichardt, L. F. 1975. Control of
bacteriophage lambda repressor synthesis: regulation of the
maintenance pathway of the cro and cI products. J. Mol. Biol.
93:289-309); the ant gene from bacteriophage P22 (Youderian, P. et
al. 1982. Sequence determinants of promotor activity. Cell.
30:843-853); the mnt gene from bacteriophage P22 (Gough, M. 1970.
Requirement for a functional int product in temperature inductions
of prophage P22 is mnt. J. Virol. 6:320-325; Prell, H. H.1978.
Ant-mediated transactivation of early genes in Salmonella prophage
P22 by superinfecting virulent P22 mutants. Mol. Gen. Genet.
164:331-334); the tetR gene product from the TetR family of
bacterial regulators or homologues of this gene or gene product
found in Tn10 and other members of the bacteriophage, animal virus,
Eubacteria, Eucarya or Archaea may be employed to increase the
efficiency of gene expression and protein production in parent
cells prior to minicell formation and/or segregated minicells
(Moyed, H. S., and K. P. Bertrand. 1983. Mutations in multicopy
Tn10 tet plasmids that confer resistance to inhibitory effects of
inducers of tet gene expression. J. Bacteriol. 155:557-564); the
mnt gene product from bacteriophage SP6 mation and/or segregated
minicells (Mead, D. A., et al. 1985. Single stranded DNA SP6
promoter plasmids for engineering mutant RNAs and proteins:
synthesis of a `stretched` preproparathyroid hormone. Nucleic Acids
Res. 13:1103-1118); and the mnt gene product from bacteriophage T7
mation and/or segregated minicells (Steen, R., et al. 1986. T7 RNA
polymerase directed expression of the Escherichia coli rrnB operon.
EMBO J. 5:1099-1103).
[0322] II.C.5.e. Use of Site-Specific Recombination in Expression
Systems
[0323] Included in the design of the invention are techniques that
increase the efficiency of gene expression and protein production
in minicells. By way of non-limiting example, these techniques may
include modification of endogenous and/or exogenous regulatory
elements responsible for activation and/or repression of proteins
to be expressed from chromosomal and/or plasmid expression vectors.
By way of non-limiting example, this system may be applied to any
of the above regulatory elements/systems. Specifically, each of the
above mentioned regulatory systems may be constructed such that the
promotor regions are oriented in a direction away from the gene to
be expressed, or each of the above mentioned gene(s) to be
expressed may be constructed such that the gene(s) to be expressed
is oriented in a direction away from the regulatory region
promotor. Constructed in this system is a methodology dependent
upon site-specific genetic recombination for inversion and
induction of the gene of interest (Backman, K., et al. 1984. Use of
synchronous site-specific recombination in vivo to regulate gene
expression. Bio/Technology 2:1045-1049; Balakrishnan, R., et al.
1994. A gene cassette for adapting Escherichia coli strains as
hosts for att-Int-mediated rearrangement and pL expression vectors.
Gene 138:101-104; Hasan, N., and W. Szybalaki. 1987. Control of
cloned gene expression by promoter inversion in vivo: construction
of improved vectors with a multiple cloning site and the Ptac
promotor. Gene 56:145-151; Wulfing, C., and A. Pluckthun. 1993. A
versatile and highly repressible Escherichia coli expression system
based on invertible promoters: expression of a gene encoding a
toxic gene product. Gene 136:199-203). These invertible promoters
and/or gene regions will allow tight regulation of potentially
toxic protein products. By way of non-limiting example, these
systems may be derived from bacteriophage lambda, bacteriophage Mu,
and/or bacteriophage P22. In any of these potential systems,
regulation of the recombinase may be regulated by any of the
regulatory systems discussed in section II.C.5 and elsewhere
herein.
[0324] II.C.5.e. Use of Copy Number Control Switches
[0325] A method that can be used to increase the efficiency of gene
expression and protein production in minicells involves the
modification of endogenous and/or introduction of exogenous genetic
expression systems such that the number of copies of a gene
encoding a protein to be expressed can be modulated. Copy number
control systems comprise elements designed to modulate copy number
in a controlled fashion.
[0326] In an exemplary mode, copy number is controlled to decrease
the effects of "leaky" (uninduced) expression of toxic gene
products. This allows one to maintain the integrity of a
potentially toxic gene product during processes such as cloning,
culture maintenance, and periods of growth prior to
minicell-induction. That is, decreasing the copy number of a gene
is expected to decrease the opportunity for mutations affecting
protein expression and/or function to arise. Immediately prior to,
during and/or after minicell formation, the copy number may be
increased to optimize the gene dosage in minicells as desired.
[0327] The replication of eubacterial plasmids is regulated by a
number of factors, some of which are contained within the plasmid,
others of which are located on the chromosome. For reviews, see del
Solar, G., et al. 2000. Plasmid copy number control: an
ever-growing story. Mol Microbiol. 37:492-500; del Solar, G., et
al. 1998. Replication and control of circular bacterial plasmids.
Microbiol. Mol Biol Rev. 62:434-64; and Filutowicz, M., et al.
1987. DNA and protein interactions in the regulation of plasmid
replication. J Cell Sci Suppl. 7:15-31.
[0328] By way of non-limiting example, the pcnB gene product, the
wildtype form of which promotes increased ColE1 plasmid copy number
(Soderbom, F., et al. 1997. Regulation of plasmid R1 replication:
PcnB and RNase E expedite the decay of the antisense RNA, CopA.
Mol. Microbiol. 26:493-504), is modulated; and/or mutant forms of
the pcnB gene are introduced into a cell. In an exemplary cell type
that may be used in the methods of the invention, the wildtype pcnB
chromosomal gene is replaced with a mutant pcnB80 allele (Lopilato,
J., et al. 1986. Mutations in a new chromosomal gene of Escherichia
coli K-12, pcnB, reduce plasmid copy number of pBR322 and its
derivatives. Mol. Gen. Genet. 205:285-290). In such cells the copy
number of a ColE1-derived plasmid is decreased. The cell may
further comprise an expression element comprising an inducible
promoter operably linked to an ORF encoding the wild-type pcnB.
Because the wild-type pcnB gene is dominant to the mutant pcnB80
gene, and because the wild-type pcnB gene product promotes
increased ColE1 plasmid copy number, induction of a wild-type pcnB
in the pcnB80 background will increase the plasmid copy number of
ColE1-derived plasmids. Such copy number control systems may be
expressed from the chromosome and/or plasmid to maintain either low
or high plasmid copy number in the absence of induction. Other
non-limiting examples of gene and/or gene products that may be
employed in copy number control systems for ColE1-based replicons
include genes or homologs of genes encoding RNA I, RNA II, rop,
RNAse H, enzymes involved in the process of polyadenylation, RNAse
E, DNA polymerase I, and DNA polymerase III.
[0329] In the case of IncFII-derived replicons, non-limiting
examples of gene and/or gene products that may be employed in copy
number control systems to control plasmid copy include genes or
homologs of the copA, copB, repA, and repB genes. Copy number
control systems may additionally or alternatively include
manipulation of repC, trfA, dnaA, dnaB, dnaC, seqA, genes protein
Pi, genes encoding HU protein subunits (hupA, hupB) and genes
encoding IHF subunits.
[0330] Other elements may also be included to optimize these
plasmid copy number control systems. Such additional elements may
include the addition or deletion of iteron nucleic acid sequences
(Chattoraj, D. K. 2000. Control of plasmid DNA replication by
iterons: no longer paradoxical. Mol. Microbiol. 37:467-476), and
modification of chaperone proteins involved in plasmid replication
(Konieczny, I., et al. 1997. The replication initiation protein of
the broad-host-range plasmid RK2 is activated by the ClpX
chaperone. Proc Natl Acad Sci USA 94:14378-14382).
[0331] II.C.6. Transportation of Inducible and Inhibitory
Compounds
[0332] Included in the design of the invention are techniques that
increase the efficiency of gene expression and protein production
in minicells. By way of non-limiting example, these techniques may
include utilization and/or modification of factors and systems that
modulate the transport of compounds, including but not limited to
inducers and/or inhibitors of expression elements that control
expression of a gene in a parent cell prior to minicell formation
and/or in segregated minicells. Such manipulations may result in
increased or decreased production, and/or changes in the
intramolecular and intermolecular functions, of a protein in a
minicell or its parent cell. The techniques may be employed to
increase the efficiency of gene expression and protein production
in parent cells prior to minicell formation and/or in segregated
minicells.
[0333] II.C.6.a. Escherichia Coli Genes
[0334] By way of non-limiting example, manipulation of the abpS
gene or gene product from E. coli, or homologs of this gene or gene
product found in other members of the Prokaryotes, Eukaryotes,
Archaebacteria and/or organelles (e.g., mitochondria, chloroplasts,
plastids and the like) may be employed to increase the efficiency
of gene expression and protein production in parent cells prior to
minicell formation and/or in segregated minicells (Celis, R. T.
1982. Mapping of two loci affecting the synthesis and structure of
a periplasmic protein involved in arginine and ornithine transport
in Escherichia coli K-12. J. Bacteriol. 151(3):1314-9).
[0335] In addition to abpS, other exemplary E. coli genes encoding
factors involved in the transport of inducers, inhibitors and other
compounds include, but are not limited to, araE (Khlebnikov, A., et
al. 2001. Homogeneous expression of the P(BAD) promoter in
Escherichia coli by constitutive expression of the low-affinity
high-capacity AraE transporter. Microbiology. 147(Pt 12):3241-7);
araG (Kehres, D. G., and Hogg, R. W. 1992. Escherichia coli K12
arabinose-binding protein mutants with altered transport
properties. Protein Sci. 1(12):1652-60); araH (Id.); argP (Celis,
R. T. 1999. Repression and activation of arginine transport genes
in Escherichia coli K 12 by the ArgP protein. J. Mol. Biol. 17;
294(5):1087-95); aroT (aroR, trpR) (Edwards, R. M., and Yudkin, M.
D. 1982. Location of the gene for the low-affinity
tryptophan-specific permease of Escherichia coli. Biochem. J.
204(2):617-9); artI (Wissenbach, U., et al. 1995. A third
periplasmic transport system for L-arginine in Escherichia coli:
molecular characterization of the artPIQMJ genes, arginine binding
and transport. Mol. Microbiol. 17(4):675-86); artJ (Id.); artM
(Id.); artP (Id.); artQ (Id.); bioP (bir, birB) (Campbell, A., et
al. Biotin regulatory (bir) mutations of Escherichia coli. 1980. J.
Bacteriol. 142(3):1025-8); brnQ (hrbA) (Yamato, I., and Anraku, Y.
1980. Genetic and biochemical studies of transport systems for
branched-chain amino acids in Escherichia coli K-12: isolation and
properties of mutants defective in leucine-repressible transport
activities. J. Bacteriol. 144(1):36-44); brnR (Id.); brnS (Id.);
brnT (Id.); btuC (Friedrich, M. J., et al. 1986. Nucleotide
sequence of the btuCED genes involved in vitamin B12 transport in
Escherichia coli and homology with components of
periplasmic-binding-protein-dependent transport systems. J.
Bacteriol. 167(3):928-34); btuD (Id.) (Friedrich, M. J., et al.
1986. Nucleotide sequence of the btuCED genes involved in vitamin
B12 transport in Escherichia coli and homology with components of
periplasmic-binding-protein-dependent transport systems. J.
Bacteriol. 167(3):928-34); caiT (Eichler, K. 1994. Molecular
characterization of the cai operon necessary for carnitine
metabolism in Escherichia coli. Mol. Microbiol. 13(5):775-86); celA
(Parker, L. L., and Hall, B. G. 1990. Characterization and
nucleotide sequence of the cryptic cel operon of Escherichia coli
K12. Genetics. 124(3):455-71); celB (Id.); celC (Id.); citA (Berlyn
et al., "Linkage Map of Escherichia coli K-12, Edition 9," Chapter
109 in: Escherichia coli and Salmonella typhimurium: Cellular and
Molecular Biology, 2nd Ed., Neidhardt, Frederick C., Editor in
Chief, American Society for Microbiology, Washington, D.C., 1996,
Volume 2, pages 1715-1902, and references cited therein); citB
(Id.); codB (Danielsen, S., et al. 1992. Characterization of the
Escherichia coli codBA operon encoding cytosine permease and
cytosine deaminase. Mol. Microbiol. 6(10):1335-44); cysA
(Karbonowska, H., et al. 1977. Sulphate permease of Escherichia
coli K12. Acta. Biochim. Pol. 24(4):329-34); cysU (cysT) (Sirko,
A., et al. 1995. Sulfate and thiosulfate transport in Escherichia
coli K-12: evidence for a functional overlapping of sulfate- and
thiosulfate-binding proteins. J. Bacteriol. 177(14):4134-6); cysW
(Id.); dctA (Lo, T. C., and Bewick, M. A. 1978. The molecular
mechanisms of dicarboxylic acid transport in Escherichia coli K12.
The role and orientation of the two membrane-bound dicarboxylate
binding proteins. J. Biol. Chem. 10; 253(21):7826-31); dctB (Id.);
dcuA (genA) (Six, S., et al. 1994. Escherichia coli possesses two
homologous anaerobic C4-dicarboxylate membrane transporters (DcuA
and DcuB) distinct from the aerobic dicarboxylate transport system
(Dct). J. Bacteriol. 176(21):6470-8); dcuB (genF) (.); dgoT (Berlyn
et al., "Linkage Map of Escherichia coli K-12, Edition 9," Chapter
109 in: Escherichia coli and Salmonella typhimurium: Cellular and
Molecular Biology, 2nd Ed., Neidhardt, Frederick C., Editor in
Chief, American Society for Microbiology, Washington, D.C., 1996,
Volume 2, pages 1715-1902, and references cited therein); exuT
(Nemoz, G., et al. 1976. Physiological and genetic regulation of
the aldohexuronate transport system in Escherichia coli. J.
Bacteriol. 127(2):706-18); fepD (Ozenberger, B. A., et al. 1987.
Genetic organization of multiple fep genes encoding ferric
enterobactin transport functions in Escherichia coli. J. Bacteriol.
169(8):3638-46); fepG (Chenault, S. S., and Earhart, C. F. 1991.
Organization of genes encoding membrane proteins of the Escherichia
coli ferrienterobactin permease. Mol. Microbiol. 5(6):1405-13);
fucP (prd) (Chen, Y. M. 1987. The organization of the fuc regulon
specifying L-fucose dissimilation in Escherichia coli K12 as
determined by gene cloning. Mol. Gen. Genet. 210(2):331-7); glnP
(Masters, P. S., and Hong, J. S. 1981. Genetics of the glutamine
transport system in Escherichia coli. J. Bacteriol. 147(3):805-19);
glnQ (Nohno, T. 1986. Cloning and complete nucleotide sequence of
the Escherichia coli glutamine permease operon (glnHPQ). Mol. Gen.
Genet. 205(2):260-9); glnR (Masters, P. S., and Hong, J. S. 1981.
Genetics of the glutamine transport system in Escherichia coli. J.
Bacteriol. 147(3):805-19); glpT (Boos, W., et al. 1977.
Purification and properties of a periplasmic protein related to
sn-glycerol-3-phosphate transport in Escherichia coli. Eur. J.
Biochem. 72(3):571-81); gltP (Deguchi, Y., et al. 1989. Molecular
cloning of gltS and gltP, which encode glutamate carriers of
Escherichia coli. B. J. Bacteriol. 171(3):1314-9); gltS (Id.); gntR
(Bachi, B., and Kornberg, H. L. 1975. Genes involved in the uptake
and catabolism of gluconate by Escherichia coli. J. Gen. Microbiol.
90(2):321-35); gntS (Id.); gntT (gntM, usgA) (Id.); gntU (Tong, S.
1996. Cloning and molecular genetic characterization of the
Escherichia coli gntR, gntK, and gntU genes of GntI, the main
system for gluconate metabolism. J. Bacteriol. 178(11):3260-9);
hisM (Berlyn et al., "Linkage Map of Escherichia coli K-12, Edition
9," Chapter 109 in: Escherichia coli and Salmonella typhimurium:
Cellular and Molecular Biology, 2nd Ed., Neidhardt, Frederick C.,
Editor in Chief, American Society for Microbiology, Washington,
D.C., 1996, Volume 2, pages 1715-1902, and references cited
therein); hisP (Id.); hisQ (Id.); livG (hrbB, hrbC, hrbD) (Landick,
R., et al. 1980. Regulation of high-affinity leucine transport in
Escherichia coli. J. Supramol. Struct. 14(4):527-37); livH (hrbB,
hrbC, hrbD) (Id.); livJ (hrbB, hrbC, hrbD) (Id.); livK (hrbB, hrbC,
hrbD) (Id.); livM (Id.); lldP (lctP) (Dong, J. M., et al. 1993.
Three overlapping lct genes involved in L-lactate utilization by
Escherichia coli. J. Bacteriol. 175(20):6671-8); lysP (cadR)
(Steffes, C., et al. 1992. The lysP gene encodes the
lysine-specific permease. J. Bacteriol. 174(10):3242-9); malF
(malB) (Bavoil, P., et al. 1980. Identification of a cytoplasmic
membrane-associated component of the maltose transport system of
Escherichia coli. J. Biol. Chem. 255(18):8366-9); malG (malB)
(Dassa, E., and Hofnung, M. 1985. Sequence of gene malG in E. coli
K12: homologies between integral membrane components from binding
protein-dependent transport systems. EMBO J. 4(9):2287-93); malK
(malB) (Id.); mglC (PMG, mglP) (Harayama, S. 1983. Characterization
of the mgl operon of Escherichia coli by transposon mutagenesis and
molecular cloning. J. Bacteriol. 153(1):408-15); nanT (Vimr, E. R.,
and Troy, F. A. 1985. Identification of an inducible catabolic
system for sialic acids (nan) in Escherichia coli. J. Bacteriol.
164(2):845-53); nupC (cru) (Craig, J. E., et al. 1994. Cloning of
the nupC gene of Escherichia coli encoding a nucleoside transport
system, and identification of an adjacent insertion element, IS
186. Mol. Microbiol. 11(6):1159-68); nupG (Westh Hansen, S. E., et
al. 1987. Studies on the sequence and structure of the Escherichia
coli K-12 nupG gene, encoding a nucleoside-transport system. Eur.
J. Biochem. 168(2):385-91); panF (Vallari, D. S., and Rock, C. O.
1985. Isolation and characterization of Escherichia coli
pantothenate permease (panF) mutants. J. Bacteriol. 164(1):136-42);
potA (Kashiwagi, K., et al. 1993. Functions of potA and potD
proteins in spermidine-preferential uptake system in Escherichia
coli. J. Biol. Chem. 268(26):19358-63); potG (Pistocchi, R., et al.
1993. Characteristics of the operon for a putrescine transport
system that maps at 19 minutes on the Escherichia coli chromosome.
J. Biol. Chem. 268(1):146-52); potH (Id.); potl (Id.); prop (Wood,
J. M., and Zadworny, D. 1980. Amplification of the put genes and
identification of the put gene products in Escherichia coli K12.
Can. J. Biochem. 58(10):787-96); proT (Id.); proV (proU) (Faatz,
E., et al. 1988. Cloned structural genes for the osmotically
regulated binding-protein-dependent glycine betaine transport
system (ProU) of Escherichia coli K-12. Mol. Microbiol.
2(2):265-79); proW (proU) (Id.); proX (proU) (Id.); pstA (R2pho,
phoR2b, phoT) (Amemura, M., et al. 1985. Nucleotide sequence of the
genes involved in phosphate transport and regulation of the
phosphate regulon in Escherichia coli. J. Mol. Biol.
184(2):241-50); pstB (phoT) (Id.); pstC (phoW) (Rao, N,N., and
Torriani, A. 1990. Molecular aspects of phosphate transport in
Escherichia coli. Mol. Microbiol. 4(7):1083-90); pstS (R2pho, nmpA,
phoR2a, phoS) (Makino, K., et al. 1988. Regulation of the phosphate
regulon of Escherichia coli. Activation of pstS transcription by
PhoB protein in vitro. J. Mol. Biol. 203(1):85-95); purP (Burton,
K. 1994. Adenine transport in Escherichia coli. Proc. R. Soc. Lond.
B. Biol. Sci. 255(1343):153-7); putP (Stalmach, M. E., et al. 1983.
Two proline porters in Escherichia coli K-12. J. Bacteriol.
156(2):481-6); rbsA (rbsP, rbsT) (Lida, A., et al. 1984. Molecular
cloning and characterization of genes required for ribose transport
and utilization in Escherichia coli K-12. J. Bacteriol.
158(2):674-82); rbsC (rbsP, rbsT) (Id.); rbsD (rbsP) (Id.); rhaT
(Baldoma, L., et al. 1990. Cloning, mapping and gene product
identification of rhaT from Escherichia coli K12. FEMS Microbiol.
Lett. 60(1-2):103-7); sdaC (Shao, Z., et al. 1994. Sequencing and
characterization of the sdaC gene and identification of the sdaCB
operon in Escherichia coli K12. Eur. J. Biochem. 222(3):901-7);
tnaB (trpP) (Sarsero, J. P., et al. 1991. A new family of integral
membrane proteins involved in transport of aromatic amino acids in
Escherichia coli. J. Bacteriol. 173(10):3231-4); tyrR (Whipp, M.
J., and Pittard, A. J. 1977. Regulation of aromatic amino acid
transport systems in Escherichia coli K-12. J. Bacteriol.
132(2):453-61); ugpC (Schweizer, H., and Boos, W. 1984.
Characterization of the ugp region containing the genes for the
phoB dependent sn-glycerol-3-phosphate transport system of
Escherichia coli. Mol. Gen. Genet. 197(1):161-8); uhpT (Weston, L.
A., and Kadner, R. J. 1987. Identification of uhp polypeptides and
evidence for their role in exogenous induction of the sugar
phosphate transport system of Escherichia coli K-12. J. Bacteriol.
169(8):3546-55); and xylF (xylT) (Sumiya, M., et al. 1995.
Molecular genetics of a receptor protein for D-xylose, encoded by
the gene xylF, in Escherichia coli. Receptors Channels.
3(2):117-28).
[0336] II.C.6.b. Bacillus Subtilis Genes
[0337] By way of non-limiting example, manipulation of the aapA
gene or gene product from B. subtilis, or homologs of this gene or
gene product found in other members of the Prokaryotes, Eukaryotes,
Archaebacteria and/or organelles (e.g., mitochondria, chloroplasts,
plastids and the like) may be employed to increase the efficiency
of gene expression and protein production in parent cells prior to
minicell formation and/or in segregated minicells (Sohenshein, A.
L., J. A. Hoch, and R. Losick (eds.) 2002. Bacillus subtilis and
its closest relatives: from genes to cells. American Society for
Microbiology, Washington D.C.).
[0338] In addition to aapA, other exemplary B. subtilis genes
encoding factors involved in the transport of inducers, inhibitors
and other compounds include, but are not limited to, amyC
(Sekiguchi, J., et al. 1975. Genes affecting the productivity of
alpha-amylase in Bacillus subtilis. J. Bacteriol. 121(2):688-94);
amyD (Id.); araE (Sa-Nogueira, I., and Mota, L. J. 1997. Negative
regulation of L-arabinose metabolism in Bacillus subtilis:
characterization of the araR (araC) gene. J. Bacteriol.
179(5):1598-608); araN (Sa-Nogueira, I., et al. 1997. The Bacillus
subtilis L-arabinose (ara) operon: nucleotide sequence, genetic
organization and expression. Microbiology. 143 (Pt 3):957-69); araP
(Id.); araQ (Id.); csbC (Akbar, S., et al. 1999. Two genes from
Bacillus subtilis under the sole control of the general stress
transcription factor sigmaB. Microbiology. 145 (Pt 5):1069-78);
cysP (Mansilla, M. C., and de Mendoza, D. 2000. The Bacillus
subtilis cysP gene encodes a novel sulphate permease related to the
inorganic phosphate transporter (Pit) family. Microbiology. 146 (Pt
4):815-21); dctB (Sohenshein, A. L., J. A. Hoch, and R. Losick
(eds.) 2002. Bacillus subtilis and its closest relatives: from
genes to cells. American Society for Microbiology, Washington
D.C.); exuT (Rivolta, C., et al. 1998. A 35.7 kb DNA fragment from
the Bacillus subtilis chromosome containing a putative 12.3 kb
operon involved in hexuronate catabolism and a perfectly
symmetrical hypothetical catabolite-responsive element.
Microbiology. 144 (Pt 4):877-84); gabP (Ferson, A. E., et al. 1996.
Expression of the Bacillus subtilis gabP gene is regulated
independently in response to nitrogen and amino acid availability.
Mol. Microbiol. 22(4):693-701); gamP (Sohenshein, A. L., J. A.
Hoch, and R. Losick (eds.) 2002. Bacillus subtilis and its closest
relatives: from genes to cells. American Society for Microbiology,
Washington D.C.); glcP (Paulsen, I. T., et al. 1998.
Characterization of glucose-specific catabolite
repression-resistant mutants of Bacillus subtilis: identification
of a novel hexose:H+ symporter. J. Bacteriol. 180(3):498-504); glcU
(Sohenshein, A. L., J. A. Hoch, and R. Losick (eds.) 2002. Bacillus
subtilis and its closest relatives: from genes to cells. American
Society for Microbiology, Washington D.C.); glnH (Id.); glnM (Id);
glnP (Sohenshein, A. L., J. A. Hoch, and R. Losick (eds.) 2002.
Bacillus subtilis and its closest relatives: from genes to cells.
American Society for Microbiology, Washington D.C.); glnQ (Id.);
glpT (Nilsson, R. P., et al. 1994. The glpT and glpQ genes of the
glycerol regulon in Bacillus subtilis. Microbiology. 140 (Pt
4):723-30); gltP (Tolner, B., et al. 1995. Characterization of the
proton/glutamate symport protein of Bacillus subtilis and its
functional expression in Escherichia coli. J. Bacteriol.
177(10):2863-9); gltT (Tolner, B., et al. 1995. Characterization of
the proton/glutamate symport protein of Bacillus subtilis and its
functional expression in Escherichia coli. J. Bacteriol.
177(10):2863-9); gntP (Reizer, A., et al. Analysis of the gluconate
(gnt) operon of Bacillus subtilis. Mol. Microbiol. 5(5):1081-9);
gutP (Sohenshein, A. L., J. A. Hoch, and R. Losick (eds.) 2002.
Bacillus subtilis and its closest relatives: from genes to cells.
American Society for Microbiology, Washington D.C.); hutM (Oda, M.,
et al. 1988. Cloning and nucleotide sequences of histidase and
regulatory genes in the Bacillus subtilis but operon and positive
regulation of the operon. J. Bacteriol. 170(7):3199-205); iolF
(Sohenshein, A. L., J. A. Hoch, and R. Losick (eds.) 2002. Bacillus
subtilis and its closest relatives: from genes to cells. American
Society for Microbiology, Washington D.C.); kdgT (Pujic, P., et al.
1998. The kdgRKAT operon of Bacillus subtilis: detection of the
transcript and regulation by the kdgR and ccpA genes. Microbiology.
144 (Pt 11):3111-8); lctP (Cruz, Ramos H., et al. 2000.
Fermentative metabolism of Bacillus subtilis: physiology and
regulation of gene expression. J. Bacteriol. 182(11):3072-80); maeN
(Ito, M., et al. 2000. Effects of nonpolar mutations in each of the
seven Bacillus subtilis mrp genes suggest complex interactions
among the gene products in support of Na(+) and alkali but not
cholate resistance. J. Bacteriol. 182(20):5663-70); malP
(Sohenshein, A. L., J. A. Hoch, and R. Losick (eds.) 2002. Bacillus
subtilis and its closest relatives: from genes to cells. American
Society for Microbiology, Washington D.C.); manP (Id.); mleN (Id.);
nasA (Ogawa, K., et al. 1995. The nasB operon and nasA gene are
required for nitrate/nitrite assimilation in Bacillus subtilis. J.
Bacteriol. 177(5):1409-13); nupC (Sohenshein, A. L., J. A. Hoch,
and R. Losick (eds.) 2002. Bacillus subtilis and its closest
relatives: from genes to cells. American Society for Microbiology,
Washington D.C.); opuAB (Kempf, B., et al. 1997. Lipoprotein from
the osmoregulated ABC transport system OpuA of Bacillus subtilis:
purification of the glycine betaine binding protein and
characterization of a functional lipidless mutant. J. Bacteriol.
179(20):6213-20); opuBA (Sohenshein, A. L., J. A. Hoch, and R.
Losick (eds.) 2002. Bacillus subtilis and its closest relatives:
from genes to cells. American Society for Microbiology, Washington
D.C.); pbuG (Saxild, H. H., et al. 2001. Definition of the Bacillus
subtilis PurR operator using genetic and bioinformatic tools and
expansion of the PurR regulon with glyA, guaC, pbuG, xpt-pbuX,
yqhZ-folD, and pbuO. J. Bacteriol. 183(21):6175-83); pbuX (Saxild,
H. H., et al. 2001. Definition of the Bacillus subtilis PurR
operator using genetic and bioinformatic tools and expansion of the
PurR regulon with glyA, guaC, pbuG, xpt-pbuX, yqhZ-folD, and pbuO.
J. Bacteriol. 183(21):6175-83); pstC (Takemaru, K., et al. 1996. A
Bacillus subtilis gene cluster similar to the Escherichia coli
phosphate-specific transport (pst) operon: evidence for a tandemly
arranged pstB gene. Microbiology. 142 (Pt 8):2017-20); pstS (Qi,
Y., et al. 1997. The pst operon of Bacillus subtilis has a
phosphate-regulated promoter and is involved in phosphate transport
but not in regulation of the pho regulon. J. Bacteriol.
179(8):2534-9); pucJ (Schultz, A. C., et al. 2001. Functional
analysis of 14 genes that constitute the purine catabolic pathway
in Bacillus subtilis and evidence for a novel regulon controlled by
the PucR transcription activator. J. Bacteriol. 183(11):3293-302);
pucK (Schultz, A. C., et al. 2001. Functional analysis of 14 genes
that constitute the purine catabolic pathway in Bacillus subtilis
and evidence for a novel regulon controlled by the PucR
transcription activator. J. Bacteriol. 183(11):3293-302); pyrP
(Turner, R. J., et al. 1994. Regulation of the Bacillus subtilis
pyrimidine biosynthetic (pyr) gene cluster by an autogenous
transcriptional attenuation mechanism. J. Bacteriol.
176(12):3708-22); rbsB (Sohenshein, A. L., J. A. Hoch, and R.
Losick (eds.) 2002. Bacillus subtilis and its closest relatives:
from genes to cells. American Society for Microbiology, Washington
D.C.); rbsC (Sohenshein, A. L., J. A. Hoch, and R. Losick (eds.)
2002. Bacillus subtilis and its closest relatives: from genes to
cells. American Society for Microbiology, Washington D.C.); rbsD
(Id.); rocC (Gardan, R., et al. 1995. Expression of the rocDEF
operon involved in arginine catabolism in Bacillus subtilis. J.
Mol. Biol. 23; 249(5):843-56); rocE (Gardan, R., et al. 1995.
Expression of the rocDEF operon involved in arginine catabolism in
Bacillus subtilis. J. Mol. Biol. 23; 249(5):843-56); ssuA (Coppee,
J. Y., et al. 2001. Sulfur-limitation-regulated proteins in
Bacillus subtilis: a two-dimensional gel electrophoresis study.
Microbiology. 147(Pt 6):1631-40); ssuB (van der Ploeg, J. R., et
al. 1998. Bacillus subtilis genes for the utilization of sulfur
from aliphatic sulfonates. Microbiology. 144 (Pt 9):2555-61); ssuC
(van der Ploeg, J. R., et al. 1998. Bacillus subtilis genes for the
utilization of sulfur from aliphatic sulfonates. Microbiology. 144
(Pt 9):2555-61); treP (Yamamoto, H., et al. 1996. Cloning and
sequencing of a 40.6 kb segment in the 73 degrees-76 degrees region
of the Bacillus subtilis chromosome containing genes for trehalose
metabolism and acetoin utilization. Microbiology. 142 (Pt
11):3057-65); xynP (Sohenshein, A. L., J. A. Hoch, and R. Losick
(eds.) 2002. Bacillus subtilis and its closest relatives: from
genes to cells. American Society for Microbiology, Washington
D.C.); ybaR (Id.); ybgF (Id.); ybgH (Id.); ycbE (Id.); ycgO (Id.);
yckl (Id.); yckf (Id.); yckK (Id.); ydgF (Id.); yecA (Borriss, R.,
et al. 1996. The 52 degrees-55 degrees segment of the Bacillus
subtilis chromosome: a region devoted to purine uptake and
metabolism, and containing the genes cotA, gabP and guaA and the
pur gene cluster within a 34960 bp nucleotide sequence.
Microbiology. 142 (Pt 11):3027-31); yesP (Sohenshein, A. L., J. A.
Hoch, and R. Losick (eds.) 2002. Bacillus subtilis and its closest
relatives: from genes to cells. American Society for Microbiology,
Washington D.C.); yesQ (Id.): yfiS (Id.); yhcL (Id.); yhjB (Id.);
yjkB (Id.); ykbA (Id.); yoaB (Id.); yocN (Id.); yodF (Id.); yojA
(Id.); yqiY (Id.); ytlD (Id.); ytlP (Id.); ytmL (Id.); ytmM (Id.);
ytnA (Id.); yurM (Id.); yurN (Id.); yvbW (Id.); yvdH (Id.); yvdl
(Id.); yveA (Pereira, Y., et al. 2001. The yveB gene, Encoding
endolevanase LevB, is part of the sacB-yveB-yveA levansucrase
tricistronic operon in Bacillus subtilis. Microbiology. 147(Pt
12):3413-9); yvfH (Sohenshein, A. L., J. A. Hoch, and R. Losick
(eds.) 2002. Bacillus subtilis and its closest relatives: from
genes to cells. American Society for Microbiology, Washington
D.C.); yvfL (Id.); yvfM (Id.); yvgM (Id.); yvrO (Id.); yvsH (Id.);
ywbF (Id.); ywcJ (Id.); ywoD (Id.); ywoE (Id.); yxeN (Id.); and
yxeR (Id.).
[0339] II.C.7. Catabolite Repression
[0340] Included in the design of the invention are techniques that
increase the efficiency of gene expression and protein production
in minicells. By way of non-limiting example, these techniques may
include utilization and/or modification of factors and systems
involved in the synthesis, degradation or transport of catabolites
that modulate the genetic expression of a preselected protein. Such
manipulations may result in increased or decreased production,
and/or changes in the intramolecular and intermolecular functions,
of a protein in a minicell or its parent cell; in the latter
instance, the protein may be one that is desirably retained in
segregated minicells.
[0341] By way of non-limiting example, it is known in the art to
use promoters from the trp, cst-1, and llp operons of E. coli,
which are induced by, respectively, reduced tryptophan levels,
glucose starvation, and lactose. Manipulation of the catabolites
tryptophan, glucose and lactose, respectively, will influence the
degree of expression of genes operably linked to these promoters.
(Makrides, Savvas C., Strategies for Achieving High-Level
Expression of Genes in Escherichia coli. Microbiological Reviews.
1996. 60:512-538.)
[0342] As another non-limiting example, expression elements from
the E. coli L-arabinose (ara) operon are used in expression
systems. AraC is a protein that acts as a repressor of ara genes in
the absence of arabinose, and also as an activator of ara genes
when arabinose is present. Induction of ara genes also involves
cAMP, which modulates the activity of CRP (cAMP receptor protein),
which in turn is required for full induction of ara genes (Schleif,
Robert, Regulation of the L-arabinose operon of Escherichia coli.
2000. TIG 16:559-564. Thus, maximum expression from an ara-based
expression system is achieved by adding cAMP and arabinose to host
cells, and optimizing the expression of CRP in hostcells.
[0343] As one example, manipulation of the acpS gene or gene
product of E. coli (Pollacco M. L., and J. E. Cronan Jr. 1981. A
mutant of Escherichia coli conditionally defective in the synthesis
of holo-[acyl carrier protein]. J. Biol. Chem. 256:5750-5754); or
homologs of this gene or its gene product found in other
prokaryotes, eukaryotes, archaebacteria or organelles
(mitochondria, chloroplasts, plastids and the like) may be employed
to increase the efficiency of gene expression and protein
production in parent cells prior to minicell formation and/or in
segregated minicells.
[0344] In addition to acpS, other exemplary E. coli genes include
the b2383 gene (Berlyn et al., "Linkage Map of Escherichia coli
K-12, Edition 9," Chapter 109 in: Escherichia coli and Salmonella
typhimurium: Cellular and Molecular Biology, 2nd Ed., Neidhardt,
Frederick C., Editor in Chief, American Society for Microbiology,
Washington, D.C., 1996, Volume 2, pages 1715-1902, and references
cited therein. b2387 gene; the celA gene (Parker L. L., and B. G.
Hall. 1990. Characterization and nucleotide sequence of the cryptic
cel operon of Escherichia coli K12. Genetics. 124:455-471); the
celB gene (Cole S. T., and B. Saint-Joanis, and A. P. Pugsley.
1985. Molecular characterisation of the colicin E2 operon and
identification of its products. Mol Gen Genet. 198:465-472); the
celC gene (Parker L. L., and B. G. Hall. 1990. Characterization and
nucleotide sequence of the cryptic cel operon of Escherichia coli
K12. Genetics. 124:455-471); the cmtB gene (Ezhova N. M., Zaikina,
N. A, Shataeva, L. K., Dubinina, N. I., Ovechkina, T. P. and J. V.
Kopylova. [Sorption properties of carboxyl cation exchangers with a
bacteriostatic effect]. 1980. Prikl Bioikhim Mikrobiol.
16:395-398); the creB gene (Berlyn et al., "Linkage Map of
Escherichia coli K-12, Edition 9," Chapter 109 in: Escherichia coli
and Salmonella typhimurium: Cellular and Molecular Biology, 2nd
Ed., Neidhardt, Frederick C., Editor in Chief, American Society for
Microbiology, Washington, D.C., 1996, Volume 2, pages 1715-1902,
and references cited therein; the creC gene (Wanner B. L. Gene
regulation by phosphate in enteric bacteria. 1993. J Cell Biochem.
51:47-54); the crp gene (Sabourn D., and J. Beckwith. Deletion of
the Escherichia coli crp gene. 1975. J. Bacteriology. 122:338-340);
the crr (gsr, iex, tgs, treD) gene (Jones-Mortimer M. C., and H. L.
Kornberg, and r. Maltby, and P. D. Watts. Role of the crr-gene in
glucose uptake by Escherichia coli. 1977. FEBS Lett. 74:17-19); the
cya gene (Bachi B., and H. L. Kornberg. Utilization of gluconate by
Escherichia coli. A role of adenosine 3':5'-cyclic monophosphate in
the induction of gluconate catabolism. 1975. Biochem J.
150:123-128); the fruA gene (Prior T. I., and H. L. Kornberg.
Nucleotide sequence of fruA, the gene specifying enzyme Iifru of
the phosphoenopyruvate-dependent sugar phosphotranssferase system
in Escherichia coli K12. 1988. J Gen Microbiol. 134:2757-2768); the
fruB gene (Bol'shakova T. N. and R. S. Erlagaeva, and Dobrynina
Oiu, and V. N. Gershanovich. [Mutation fruB in the fructose regulon
affecting beta-galactosidase synthesis and adenylate cyclase
activity of E. coli K12]. 1988. Mol Gen Mikrobiol virusol.
3:33-39); the fruR gene (Jahreis K., and P. W. Postma, and J. W.
Lengeler. Nucleotide sequence of the ilvH-frR gene region of
Escherichia coli K12 and Salmonella typhimurium LT2. 1991. Mol Gen
Genet. 226:332-336); the frvA gene (Berlyn et al., "Linkage Map of
Escherichia coli K-12, Edition 9," Chapter 109 in: Escherichia coli
and Salmonella typhimurium: Cellular and Molecular Biology, 2nd
Ed., Neidhardt, Frederick C., Editor in Chief, American Society for
Microbiology, Washington, D.C., 1996, Volume 2, pages 1715-1902,
and references cited therein); the frwB gene (Id.); the frvD gene
(Id.); the gatB gene (Nobelmann B., and J. W. Lengeler. Molecular
analysis of the gat genes from Escherichia coli and of their roles
in galactitol transport and metabolism. 1996. J. Bacteriol.
178:6790-6795); the gatC gene (Id.); the malX gene (Reidel J., W.
Boos. The malX malY operon of Escherichia coli encodes a novel
enzyme II of the photophotransferase system recognizing glucose and
maltose and an enzyme abolishing the endogenous induction of the
maltose system. 1991. J. Bacteriol. 173:4862-4876); the manX (gptB,
mpt, ptsL, ptsM, ptsX, manIII) gene (Plumbridge J., and A. Kolb.
CAP and Nag repressor binding to the regulatory regions of the
nagE-B and manX genes of Escherichia coli. 1991. J Mol. Biol.
217:661-679); the manY (pel, ptsM, ptsP, manPII) gene (Henderson P.
J., and R. A. Giddens, and M.C. Jones-Mortimer. Transport of
galactose, glucose and their molecular analogues by Escherichia
coli K12. 1977. Biochem J. 162:309-320); the manZ (gptB, mpt, ptsM,
ptsX) gene (Williams N., and D. K. Fox, and C. Shea and S. Roseman.
Pel, the protein that permits lambda DNA penetration of Escherichia
coli, is encoded by a gene in ptsM and is required for mannose
utilization by the phosphotransferase system. 1986. Proc Natl Acad
Sci USA. 83:8934-8938); the mtlA gene (Lengeler J. Mutations
affecting transport of the hexitols D-mannitol, D-glucitol, and
galactitol in Escherichia coli K-12: isolation and mapping. 1975. J
Bacteriol. 124:26-38.); the nagE (pstN) gene (Rogers M. J., and T.
Ohgi, and J. Plumbridge, and D. Soll. Nucleotide sequences of the
Escherichia coli nagE and nagB genes: the structural genes for the
N-acetylglucosamine transport protein of the bacterial
phosphoenolpyruvate: sugar phosphotransferase system and for
glucosamine-6-phosphate deaminase. 1988. Gene. 62:197-207); the
pstA gene (Cox G. B., H. Rosenberg, and J. A. Downie, and S.
Silver. Genetic analysis of mutants affected in the Pst inorganic
phosphate transport system. 1981. J. Bacteriol. 148:1-9); the pstB
(gutB) gene (Id.); the pstG gene (Cox G. B., H. Rosenberg, and J.
A. Downie, and S. Silver. Genetic analysis of mutants affected in
the Pst inorganic phosphate transport system. 1981. J. Bacteriol.
148:1-9); the pstH gene (Id.); the pstI gene (Id.); the pstN gene
(Id.); the pstO gene (Id.); the ptxA (yifU) gene (Berlyn et al.,
"Linkage Map of Escherichia coli K-12, Edition 9," Chapter 109 in:
Escherichia coli and Salmonella typhimurium: Cellular and Molecular
Biology, 2nd Ed., Neidhardt, Frederick C., Editor in Chief,
American Society for Microbiology, Washington, D.C., 1996, Volume
2, pages 1715-1902, and references cited therein); the sgcA (yjhL)
gene (Id.); the sgcC (yjhN) gene (Id.); the treB gene (Boos W., U.
Ehmann, H. Forkl, W. Klein, M. Rimmele, and P. Postma. Trehalose
transport and metabolism in Escherichia coli. 1990. J. Bacteriol.
172:3450-3461); the usg gene (Arps P. J., and M. E. Winkler M E.
Structural analysis of the Escherichia coli K-12 hisT operon by
using a kanamycin resistance cassette. 1987. J. Bacteriol.
169:1061-1070); the wcaD gene (Mao Y., and M. P. Doyle, and J.
Chen. Insertion mutagenisis of wca reduces acide and heat tolerance
of enterohemorrhagic Escherichia coli_O157:H7. 2001. J. Bacteriol.
183:3811-3815); the yadI gene (Berlyn et al., "Linkage Map of
Escherichia coli K-12, Edition 9," Chapter 109 in: Escherichia coli
and Salmonella typhimurium: Cellular and Molecular Biology, 2nd
Ed., Neidhardt, Frederick C., Editor in Chief, American Society for
Microbiology, Washington, D.C., 1996, Volume 2, pages 1715-1902,
and references cited therein); and the ycgC gene (Gutknecht R., and
R. Beutler, and L. F. Garcia-Alles, and U. Baumann, and B. Erni.
The dihydroxyacetone kinase of Escherichia coli utilizes a
phosphoprotein instead of ATP as phosphoryl donor. 2001. EMBO J.
20:2480-2486).
[0345] II.C.8. General Deletions and Modifications
[0346] Included in the design of the invention are techniques that
increase the efficiency of gene expression and protein production
in minicells. By way of non-limiting example, these techniques may
include modification or deletion of endogenous gene(s) from which
their respective gene product decreases the induction and
expression efficiency of a desired protein in the parent cell prior
to minicell formation and/or the segregated minicell. By way of
non-limiting example, these protein components may be the enzymes
that degrade chemical inducers of expression, proteins that have a
dominant negative affect upon a positive regulatory elements,
proteins that have proteolytic activity against the protein to be
expressed, proteins that have a negative affect against a chaperone
that is required for proper activity of the expressed protein,
and/or this protein may have a positive effect upon a protein that
either degrades or prevents the proper function of the expressed
protein. These gene products that require deletion or modification
for optimal protein expression and/or function may also be
antisense nucleic acids that have a negative affect upon gene
expression.
[0347] II.C.9. Cytoplasmic Redox State
[0348] Included in the design of the invention are techniques that
increase the efficiency of gene expression and functional protein
production in minicells. By way of non-limiting example, these
techniques may include modification of endogenous and/or exogenous
protein components that alter the redox state of the parental cell
cytoplasm prior to minicell formation and/or the segregated
minicell cytoplasm. By way of non-limiting example, this protein
component may be the product of the trxA, grx, dsbA, dsbB, and/or
dsbc genes from E. coli or homologs of this gene or gene product
found in other members of the Eubacteria, Eucarya or Archae (Mark
et al., Genetic mapping of trxA, a gene affecting thioredoxin in
Escherichia coli K12, Mol Gen Genet. 155:145-152, 1977; (Russel et
al., Thioredoxin or glutaredoxin in Escherichia coli is essential
for sulfate reduction but not for deoxyribonucleotide synthesis, J.
Bacteriol. 172:1923-1929, 1990); Akiyama et al., In vitro catalysis
of oxidative folding of disulfide-bonded proteins by the
Escherichia coli dsbA (ppfA) gene product, J Biol. Chem.
267:22440-22445, 1992); (Whitney et al., The DsbA-DsbB system
affects the formation of disulfide bonds in periplasmic but not in
intramembraneous protein domains, FEBS Lett. 332:49-51, 1993);
(Shevchik et al., Characterization of DsbC, a periplasmic protein
of Erwinia chrysanthemi and Escherichia coli with disulfide
isomerase activity, EMB J. 13:2007-2012, 1994). These applications
may, but are not limited to increased or decreased production,
increased or decreased intramolecular TrxA activity, increased or
decreased physiological function of the above-mentioned gene
products. By way of non-limiting example, increased production of
gene product (gene expression) may occur through increased gene
dosage (increased copy number of a given gene under the control of
the native or artificial promotor where this gene may be on a
plasmid or in more than one copy on the chromosome), modification
of the native regulatory elements, including, but not limited to
the promotor or operator region(s) of DNA, or ribosomal binding
sites on RNA, relevant repressors/silencers, relevant
activators/inhancers, or relevant antisense nucleic acid or nucleic
acid analog, cloning on a plasmid under the control of the native
or artificial promotor, and increased or decreased production of
native or artificial promotor regulatory elements) controlling
production of the gene. By way of non-limiting example, decreased
gene expression production may occur through modification of the
native regulatory elements, including, but not limited to the
promotor or operator region(s) of DNA, or ribosomal binding sites
on RNA, relevant repressors/silencers, relevant
activators/inhancers, or relevant antisense nucleic acid or nucleic
acid analog, through cloning on a plasmid under the control of the
native regulatory region containing mutations or an artificial
promotor, either or both of which resulting in decrease gene
expression, and through increased or decreased production of native
or artificial promotor regulatory element(s) controlling gene
expression. By definition, intramolecular activity refers to the
enzymatic function, structure-dependent function, e.g. the capacity
off a gene product to interact in a protein-protein,
protein-nucleic acid, or protein-lipid complex, and/or carrier
function, e.g. the capacity to bind, either covalently or
non-covalently small organic or inorganic molecules, protein(s)
carbohydrate(s), fatty acid(s), lipid(s), and nucleic acid(s). By
way of non-limiting example, alteration of intramolecular activity
may be accomplished by mutation of the gene, in vivo or in vitro
chemical modification of the gene product, inhibitor molecules
against the function of the gene product, e.g. competitive,
non-competitive, or uncompetitive enzymatic inhibitors, inhibitors
that prevent protein-protein, protein-nucleic acid, or
protein-lipid interactions, e.g. expression or introduction of
dominant-negative or dominant-positive or other protein
fragment(s), or other carbohydrate(s), fatty acid(s), lipid(s), and
nucleic acid(s) that may act directly or allosterically upon the
gene product, and/or modification of protein, carbohydrate, fatty
acid, lipid, or nucleic acid moieties that modify the gene or gene
product to create the functional protein. By definition,
physiological function refers to the effects resulting from an
intramolecular interaction between the gene product and other
protein, carbohydrate, fatty acid, lipid, nucleic acid, or other
molecule(s) in or on the cell or the action of a product or
products resulting from such an interaction.
[0349] By way of non-limiting example, physiological function may
be the act or result of intermolecular phosphorylation,
biotinylation, methylation, acylation, glycosylation, and/or other
signaling event; this function may be the result of
protein-protein, protein-nucleic acid, or protein-lipid interaction
resulting in a functional moiety; this function may be to interact
with the membrane to recruit other molecules to this compartment of
the cell; this function may be to regulate the transcription and/or
translation of trxA, other protein, or nucleic acid; and this
function may be to stimulate the function of another process that
is not yet described or understood.
[0350] II.C.10. Transcriptional Terminators
[0351] Included in the design of the invention are techniques that
increase the efficiency of gene expression and protein production
in parental cell cytoplasm prior to minicell formation and/or the
segregated minicell cytoplasm. By way of non-limiting example,
these techniques may include modification of terminator regions of
DNA templates or RNA transcripts so that transcription and/or
translation of these nucleic acid regions will terminate at greater
efficiency. By way of non-limiting example, these techniques may
include stem-loop structures, consecutive translational
terminators, polyadenylation sequences, or increasing the
efficiency of rho-dependent termination. Stem loop structures may
include, but are not limited to, inverted repeats containing any
combination of deoxyribonucleic acid or ribonucleic acid molecule,
more than one such inverted repeat, or variable inverted repeats
such that the rate of transcriptional/translational termination may
be moderated dependent on nucleic acid and/or amino acid
concentration, e.g. the mechanism of regulatory attenuation
(Oxdender et al., Attenuation in the Escherichia coli tryptophan
operon: role of RNA secondary structure involving the tryptophan
codon region, Proc. Natl. Acad. Sci. 76:5524-5528, 1979). See also,
Yager and von Hippel, "Transcript Elongation and Termination in e.
Col. And Landick and Yanofsky, "Transcriptional Attenuation,"
Chapters 76 and 77, respectively in: Escherichia Coli and
Salmonella Typhimurium: Cellular and Molecular Biology, Neidhardt,
Frederick C., Editor in Chief, American Society for Microbiology,
Washington, D.C., 1987, Volume 1, pages 1241-1275 and 1276-1301,
respectively, and references cited therein.
[0352] II.C.11. Ribosomal Targeting
[0353] Included in the design of the invention are techniques that
increase the efficiency of gene expression and protein production
in parental cell cytoplasm prior to minicell formation and/or the
segregated minicell cytoplasm. By way of non-limiting example,
these techniques may include modifications of endogenous and/or
exogenous ribosomal components such that ribosomes enter the
minicell segregates with higher efficiency. By way of non-limiting
example, these techniques may include increasing the copy number of
ribosomal binding sites on plasmid or like structure to recruit
more ribosomal components or increase the synthesis of ribosomal
subunits prior to segregation (Mawn et al., Depletion of free 30S
ribosomal subunits in Escherichia coli by expression of RNA
containing Shine-Dalgarno-like sequences, J. Bacteriol.
184:494-502, 2002). This construct may also include the use of
plasmid expressed translation initiation factors to assist
ribosomal segregation (Celano et al., Interaction of Escherichia
coli translation-initiation factor IF-1 with ribosomes, Eur. J.
Biochem. 178:351-355 1988). See also Hoopes and McClure,
"Strategies in Regulation of Transcription Initiation," Chapter 75
in: Escherichia Coli and Salmonella Typhimurium: Cellular and
Molecular Biology, Neidhardt, Frederick C., Editor in Chief,
American Society for Microbiology, Washington, D.C., 1987, Volume
2, pages 1231-1240, and references cited therein.
[0354] II.C.12. Proteases
[0355] Included in the design of the invention are techniques that
increase the efficiency of gene expression and protein production
in minicells. By way of non-limiting example, these techniques may
include utilization and/or modification of endogenous and/or
exogenous proteases. Such manipulations may result in increased or
decreased production, and/or changes in the intramolecular and
intermolecular functions, of a protein in a minicell or its parent
cell; in the latter instance, the protein may be one that is
desirably retained in segregated minicells.
[0356] The production or activity of a desired protein gene product
may be increased by decreasing the level and/or activity of a
protease that acts upon the desired protein. The production or
activity of a desired protein gene product may be increased by
increasing the level and/or activity of a protease that acts upon a
protein that inhibits the production or function of the desired
protein.
[0357] The production or activity of a desired nucleic acid gene
product may be increased be increasing the level and/or activity of
a protease that acts upon a protein that that inhibits the
production or function of the nucleic acid gene product. The
production or activity of a desired nucleic acid gene product may
be increased by decreasing the level and/or activity of a protease
that acts upon a protein that stimulates or enhances the production
or function of the desired nucleic acid gene product.
[0358] As one example, manipulation of the alpA gene or gene
product from E. coli (Kirby J. E., and J. E. Trempy, and S.
Gottesman. Excision of a P4-like cryptic prophage leads to Alp
protease expression in Escherichia coli. 1994. J. Bacteriol.
176:2068-2081), or homologs of this gene or gene product found in
other members of the Prokaryotes, Eukaryotes or Archaebacteria, may
be employed to increase the efficiency of gene expression and
protein production in parent cells prior to minicell formation
and/or segregated minicells postpartum.
[0359] In addition to alpA, other exemplary E. coli genes and gene
products include the clpA gene and gene product from E. coli
(Katayama Y., and S. Gottesman, and J. Pumphrey, and S. Rudikoff,
and W. P. Clark, and M. R. Maurizi. The two-component,
ATP-dependent Clp protease of Escherichia coli. Purification,
cloning, and mutational analysis of the ATP-binding component.
1988, J Biol. Chem. 263-15226-15236); the clpB gene product from E.
coli (Kitagawa M., and C. Wada, and S. Yoshioka, and T. Yura.
Expression of ClpB, an analog of the ATP-dependent proteas
regulatory subunit in Escherichia coli, is controlled by a heat
shock sigma factor (sigma 32). J. Bacteriol. 173:4247-4253); the
clpC gene product from E. coli (Msadek T., and F. Kunst, and G.
Rapoport. MecB of Bacillus subtilis, a member of the ClpC ATPase
family, is a pleiotropic regulator controlling competence gene
expression and growth at high temperature. 1994. Proc Natl Acad Sci
USA 91:5788-5792); the clpP gene product from E. coli (Maurizi M.
R., and W. P. Clark, and Y. Katayama, and S. Rudikoff, and J.
Pumphrey, and B. Bowers, and S. Gottesman. Sequence and structure
of ClpP, the proteolytic component of the ATP-dependent Clp
protease of Escherichia coli. 1990. J biol Chem. 265:12536-12545);
the clpX gene product from E. coli (Gottesman S., and W. P. Clark,
and V. de Crecy-Lagard, and M. R. Maurizi. ClpX, an alternative
subunit for the ATP-dependent Clp protease of Escherichia coli.
Sequence and in vivo activities. 1993. J Biol. Chem.
268:22618-22626); the clpY gene product from E. coli (Missiakas D.,
and F. Schwager, J. M. Betton, and C. Georgopoulos, S. Raina.
Identification and characterization of HsIV HsIU (ClpQ ClpY)
proteins involved in overall proteolysis of misfolded proteins in
Escherichia coli. 1996. EMBO J. 15:6899-6909); the dcp gene product
from E. coli (Becker S., and Plapp R. Location of the dcp gene on
the physical map of Escherichia coli. 1992. J. Bacteriol.
174:1698-1699); the degP (htrA) gene product from E. coli (Lipinska
B., and M. Zylicz, and C. Georgopoulos. The HtrA (DegP) protein,
essential for Escherichia coli survival at high temperatures, is an
endopeptidase. 1990. J. Bacteriol. 172:1791-1797); the ggt gene
product from E. coli (Finidori J., and Y. Laperche, and R.
Haguenauer-Tsapis, and R. Barouki, and G. Guellaen, and J. Hanoune.
In vitro biosynthesis and membrane insertion of gamma-glutamyl
transpeptidase. 1984. J Biol. Chem. 259:4687-4690); the hfl gene
product from E. coli (Cheng H. H., and H. Echols. A class of
Escherichia coli proteins controlled by the hflA locus. 1987. J
Mol. Biol. 196:737-740); the hflB gene product from E. coli
(Banuett F., and M. A. Hoyt, and L. McFarlane, and H. Echols, and
I. Herskowitz. HflB, a new Escherichia coli locus regulating
lysogeny and the level of bacteriophage lambda c11 protein. 1986. J
Mol. Biol. 187:213-224); the hflC gene product from E. coli (Noble
J. A., and M. A. Innis, and E. V. Koonin, and K. E. Rudd, and F.
Banuett, and I. Herskowitz, The Escherichia coli hflA locus encodes
a putative GTP-binding protein and two membrane proteins, one of
which contains a protease-like domain. 1993. Proc Natl Acad Sci
USA. 90:10866-10870); the hflK gene product from E. coli (Id.); the
hftX gene product from E. coli (Noble J. A., and M. A. Innis, and
E. V. Koonin, and K. E. Rudd, and F. Banuett, and I. Hertzskowitz.
The Escherichia coli hflA locus encodes a putative GTP-binding
protein and two membrane proteins, one of which contains a
protease-like domain. 1993. Proc Natl Acad Sci USA.
90:10866-10870); the hopD gene product from E. coli (Whitchurch C.
B., and J. S. Mattick Escherichia coli contains a set of genes
homologous to those involved in protein secretion, DNA uptake and
the assembly of type-4 fimbriae in other bacteria. 1994. Gene.
150:9-15); the htrA gene product from E. coli (Lipinska B., and S.
Sharma, and C. Georgopoulos. Sequence analysis and regulation of
the htrA gene of Escherichia coli: a sigma 32-independent mechanism
of heat-inducible transcription. 1988. Nucleic Acids Res.
16:10053-10067); the hycI gene product from E. coli (Rossmann R.,
and T. Maier, and F. Lottspeich, and A. Bock. Characterisation of a
proteas from Escherichia coli involved in hydrogenase maturation.
1995. Eur J. Biochem. 227:545-550); the iap gene product from E.
coli (Nakata A., and M. Yamaguchi, and K. Isutani, and M. Amemura.
Escherichia coli mutants deficient in the production of alkaline
phosphatase isoszymes. 1978. J. Bacteriol. 134:287-294); the lep
gene product from E. coli (Silver P., and W. Wickner. Genetic
mapping of the Escherichia coli leader (signal) peptidase gene
(lep): a new approach for determining the map position of a cloned
gene. 1983. J. Bacteriol. 54:659-572); the lon gene product from E.
coli (Donch J., and J. Greenberg. Genetic analysis of lon mutants
of strain K-12 of Escherichia coli. 1968. Mol Gen Genet.
103:105-115); the lsp gene product from E. coli (Regue M., and J.
Remenick, and M. tokunaga, and G. A. Mackie, and H. C. Wu. Mapping
of the lipoprotein signal peptidase gene (lsp). 1984. J. Bacteriol.
1984 158:632-635); the ompT gene product from E. coli (Akiyama Y.,
and K. SecY protein, a membrane-embedded secretion factor of E.
coli, is cleaved by the ompT proteas in vitro. 1990. Biochem
Biophys Res Commun. 167:711-715); the opdA gene product from E.
coli (Conllin C. A., and C. G. Miller. Location of the prlC (opdA)
gene on the physical map of Escherichia coli. 1993. J. Bacteriiol.
175:5731-5732); the orfX gene product from E. coli (Berlyn, M. K.
B., et al. 1996. Linkage map of Escherichia coli K-12, Edition 9.
In F. C. Neidhardt, R. Curtiss, J. L. Ingraham, E. C. C. Lin, K. B.
Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H.
E. Umbarger (eds.). Escherichia coli and Salmonella typhimurium:
Cellular and Molecular Biology, 2.sup.nd ed. American Society for
Microbiology, Washington D.C.); the pepA gene product from E. coli
(Stirling C. J., and S. D. Colloms, and J. F. Collins, and G.
Szatmari, and D. J. Sherratt. XerB, an Escherichia coli gene
required for plasmid ColE1 site-specific recombination, is
identical to pepA, encoding aminopeptidaseA, a protein with
substantial similarity to bovine lens leucine aminopeptidase. 1989.
EMBO J. 8:1623-1627); the pepD gene product from E. coli (Henrich
B., and U. Schroeder, and R. W. Frank, and R. Plapp. Accurate
mapping of the Escherichia coli pepD gene by sequence analysis of
its 5' flanking region. 1989. Mol Gen Genet. 215:369-373); the pepE
gene product from E. coli (Conlin C. A., and T. M. Knox, and C. G.
Miller. Cloning and physical map position of an alpha-aspartyl
depeptidase gene, pepE, from Escherichia coli. 1994. J. Bacteriol.
176:1552-1553); the pepN gene product from E. coli (Miller C. G.,
and G. Schwartz. Peptidase-deficient mutants of Escherichia coli.
1978. J. Bacteriiol. 135:603-611); the pepP gene product from E.
coli (Id.); the pepQ gene product from E. coli (Id.); the pepT gene
product from E. coli (Miller G. G., and G. Schwartz.
Peptidase-deficient mutants of Escherichia coli. 1978. J.
Bacteriiol. 135:603-611); the pilD gene product from E. coli
(Francetic 0., and S. Lory, and A. P. Pugsley. A second prepilin
peptidase gene in Escherichia coli K-12. 1998, Mol. Microbiol.
27:763-775); the pinA gene product from E. coli (Hilliard J. J.,
and L. D. Simon, and L. Van Melderen, and M. R. Maurizi. PinA
inhibits ATP hydrolysis and energy-dependent protein degradation by
Lon protease. 1998. J Biol. Chem. 273:524-527); the prc(tsp) gene
product from E. coli (Nagasawa H., and Y. Sakagami, and A. Suzuki,
and H. Suzuki, and H. Hara, and Y. Hirota. Determination of the
cleavage site involved in C-terminal processing of
penicillin-binding proein 3 of Escherichia coli. 1989. J.
Bacteriol. 171:5890-5893); the prlC gene product from E. coli
(Jiang X., and M. Zhang, and Y. Ding, and J. Yao, and H. Chen, and
D. Zhu, and M. Muramatu. Escherichia coli prlC gene encodes a
trypsin-like proteinase regulating the cell cycle. 1998. J Biochem
(Tokyo) 128:980-985); the protease V gene product from E. coli
(Berlyn, M. K. B. et al. 1996. Linkage map of Escherichia coli
K-12, Edition 9, In F. C. Neidhardt, R. Curtiss, J. L. Ingraham, E.
C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M.
Schaechter, and H. E. Umbarger (eds.). Escherichia coli and
Salmonella typhimurium: Cellular and Molecular Biology, 2.sup.nd
ed. American Society for Microbiology, Washington, D.C.); the
protease VI gene product from E. coli (Id.); the protease In gene
product from E. coli (Id.); the protease Fa gene product from E.
coli or homologues (Id.); the protease Mi gene product from E. coli
(Id.); the protease So gene product from E. coli (Id.); the ptrA
gene product from E. coli (Id.); the ptrB gene product from E. coli
(Id.); the sypB gene product from E. coli (Barends S., and A. W.
Karzai, and R. T. Sauer, and J. Wower, and B. Kraal. Simultaneous
an functional binding of SmpB and EF-Tu-TP to the analyl acceptor
arm of tmRNA. 2001. J Mol. Biol. 314:9-21); the sohB gene product
from E. coli (Baird L., and B. Lipinska, and S. Raina, and C.
Georgopoulos. Identification of the Escherichia coli sohB gene, a
multicopy suppressor of the HtrA (DegP) null phenotype. 1991. J.
Bacteriol. 173-5763-5770); the sspA gene product from E. coli
(Ichihara S., and T. Suzuki, and M. Suzuki, and C. Mizushima.
Molecular cloning and sequencing of the sppA gene and
characterization of the encoded proteas IV, a signal peptide
peptidase of Escherichia coli. 1986. J Biol. Chem. 261; 9405-9411);
the tesA gene product from E. coli (Cho H., and J. E. Cronan Jr.
Escherichia coli thioesterase I, molecular cloning and sequencing
of the structural gene and identification as a periplasmic enzyme.
1993 J Biol. Chem. 268:9238-9245); the tufA gene product from E.
coli (Ang., and J. S. Lee, and J. D. Friesen. Evidence for an
internal promoter preceding tufA in the str operon of Escherichia
coli. J. Bacteriol. 149:548-553); the tufB gene product from E.
coli (Mihajima A., and M. Shibuya, and Y. Kaziro. Construction and
characterization of the two hybrid Col1E1 plasmids carrying
Escherichia coli tufB gene. 1979. FEBS Lett. 102:207-210); the ybaU
gene product from E. coli (Berlyn, M. K. B., et al. 1996. Linkage
map of Escherichia coli K-12, Edition 9. In F.C. Neidhardt, R.
Curtiss, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W.
S. Resnikoff, M. Riley, M. Schaechter, and H. E. Umbarger (eds.).
Escherichia coli and Salmonella typhimurium: Cellular and Molecular
Biology, 2.sup.nd ed. American Society for Microbiology,
Washington, D.C.); the ssrA gene (tmRNA, 10sA RNA) product from E.
coli (Oh B. K., and A. K. Chauhan, and K. Isono, and D. Apirion.
Location of a gene (ssrA) for a small, stable RNA 910Sa RNA) in the
Escherichia coli chromosome. 1990. J. Bacteriol. 172:4708-4709);
and the ssrB gene from E. coli (Berlyn, M. K. B., et al. 1996.
Linkage map of Escherichia coli K-12, Edition 9. In F.C. Neidhardt,
R. Curtiss, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik,
W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Ummbarger 9
eds.). Escherichia coli and Salmonella typhimurium: Cellular and
Molecular Biology, 2.sup.nd ed. American Society for Microbiology,
Washington, D.C.).
[0360] II.C.13. Chaperones
[0361] Included in the design of the invention are techniques that
increase the efficiency of gene expression and functional protein
production in minicells. By way of non-limiting example, these
techniques may include modification of chaperones and chaperonins,
i.e., endogenous and/or exogenous protein components that monitor
the unfolded state of translated proteins allowing proper folding
and/or secretion, membrane insertion, or soluble multimeric
assembly of expressed proteins in the parental cell prior to
minicell formation and/or the segregated minicell cytoplasm,
membrane, periplasm, and/or extracellular environment. See
Gottesman et al., Protein folding and unfolding by Escherichia coli
chaperones and chaperonins, Current Op. Microbiol. 3:197-202, 2000;
and Mayhew et al., "Molecular Chaperone Proteins," Chapter 61 in:
Escherichia coli and Salmonella typhimurium: Cellular and Molecular
Biology, 2nd Ed., Neidhardt, Frederick C., Editor in Chief,
American Society for Microbiology, Washington, D.C., 1996, Volume
1, pages 922-937, and references cited therein.
[0362] These applications may, but are not limited to increased or
decreased chaperone production, increased or decreased
intramolecular activity of a chaperone, increased or decreased
physiological function of a chaperone, or deletion, substitution,
inversion, translocation or insertion into, or mutation of, a gene
encoding a chaperone. By way of non-limiting example, increased
production of a chaperone may occur through increased chaperone
gene dosage (increased copy number of a given gene under the
control of the native or artificial promotor where this gene may be
on a plasmid or in more than one copy on the chromosome),
modification of the native regulatory elements, including, but not
limited to the promotor or operator region(s) of DNA, or ribosomal
binding sites on RNA, relevant repressors/silencers, relevant
activators/enhancers, or relevant antisense nucleic acid or nucleic
acid analog, cloning on a plasmid under the control of the native
or artificial promotor, and increased or decreased production of
native or artificial promotor regulatory element(s) controlling
production of the chaperone gene or gene product. By way of
non-limiting example, decreased production of a chaperone may occur
through modification of the native regulatory elements, including,
but not limited to the promotor or operator region(s) of DNA, or
ribosomal binding sites on RNA, relevant repressors/silencers,
relevant activators/enhancers, or relevant antisense nucleic acid
or nucleic acid analog, through cloning on a plasmid under the
control of the native regulatory region containing mutations or an
artificial promotor, either or both of which resulting in decreased
chaperone production, and through increased or decreased production
of native or artificial promotor regulatory element(s) controlling
production of the chaperone gene or gene product. By definition,
intramolecular activity refers to the enzymatic function,
structure-dependent function, e.g. the capacity of chaperone to
interact in a protein-protein, protein-nucleic acid, or
protein-lipid complex, and/or carrier function, e.g. the capacity
to bind, either covalently or non-covalently small organic or
inorganic molecules, protein(s), carbohydrate(s), fatty acid(s),
lipid(s), and nucleic acid(s). By way of non-limiting example,
alteration of intramolecular activity may be accomplished by
mutation of the chaperone gene, in vivo or in vitro chemical
modification of Chaperone, inhibitor molecules against the function
of chaperone, e.g. competitive, non-competitive, or uncompetitive
enzymatic inhibitors, inhibitors that prevent protein-protein,
protein-nucleic acid, or protein-lipid interactions, e.g.
expression or introduction of dominant-negative or
dominant-positive chaperone or other protein fragment(s), or other
carbohydrate(s), fatty acid(s), lipid(s), and nucleic acid(s) that
may act directly or allosterically upon Chaperone, and/or
modification of protein, carbohydrate, fatty acid, lipid, or
nucleic acid moieties that modify the chaperone gene or gene
product to create the functional protein. By definition,
physiological function refers to the effects resulting from an
intramolecular interaction between Chaperone and other protein,
carbohydrate, fatty acid, lipid, nucleic acid, or other molecule(s)
in or on the cell or the action of a product or products resulting
from such an interaction. By way of non-limiting example,
physiological function may be the act or result of intermolecular
phosphorylation, biotinylation, methylation, acylation,
glycosylation, and/or other signaling event; this function may be
the result of a protein-protein, protein-nucleic acid, or
protein-lipid interaction resulting in a functional moiety; this
function may be to interact with the membrane to recruit other
molecules to this compartment of the cell; this function may be to
regulate the transcription and/or translation of chaperone, other
protein, or nucleic acid; and this function may be to stimulate the
function of another process that is not yet described or
understood.
[0363] By way of non-limiting example, chaperone genes may be any
of the E. coli genes listed below, as well as any homologs thereof
from prokaryotes, exukariutes, arcahebacteria, or organelles
(mitochondria, chloroplasts, plastids, etc.). Exemplary E. coli
genes encoding chaperones include, by way of non-limiting example,
the cbpA gene (Shiozawa T., and C. Ueguchi, and T. Mizuno. The rpoD
gene functions as a multicopy suppressor for mutations in the
chaperones, CbpA, DnaJ and DnaK, in Escherichia coli. 1996 FEMS
Microbiol Lett. 138:245-250): the clpB gene (Squires C. L., and S.
Pedersen, and B. M. Ross, and C. Squires. ClpB is the Escherichia
coli heat shock protein F84.1. 1991. J. Bacteriol. 173:4254-4262);
the dnaK gene (Kroczynska B., and S. Y. Blond. Cloning and
characterization of a new soluble murine J-domain protein that
stimulates BiP, Hsc70 and DnaK ATPase activity with different
efficiencies. 2001. Gene. 273:267-274); the dnaJ gene (Kedzierska
S., and E. Matuszewska. The effect of co-overproduction of
DnaK/DnaJ/GrpE and ClpB proteins on the removal of heat-aggregated
proteins from Escherichia coli Delta clpB mutant cells--new insight
into the role of Hsp70 in a functional cooperation with Hsp100.
2001. FEMS Microbiol Lett. 204:355-360); the ecpD gene (Raina S.,
and D. Missiakas, and L. Baird, and S. Kumar, and C. Georgopoulos.
Identification and transcriptional analysis of the Escherichia coli
htrE operon which is homologous to pap and related pilin operons.
1993. J. Bacteriol. 175:5009-5021); the ffh gene (Muller, M., et
al. 1002. Protein traffic in bacteria: multiple routes from the
ribosome to and across the membrane. Prog. Nucleic Acid Res. Mol.
Biol. 66:107-157); 4.5S RNA (Muller, M., et al. 1002. Protein
traffic in bacteria: multiple routes from the ribosome to and
across the membrane. Prog. Nucleic Acid Res. Mol. Biol.
66:107-157); the FtsY gene (Muller, M., et al. 1002. Protein
traffic in bacteria: multiple routes from the ribosome to and
across the membrane. Prog. Nucleic Acid Res. Mol. Biol.
66:107-157); the fimC gene (Klemm P., and B. J. Jorgensen, and I.
van Die, and H. de Ree, and H. Bergmans. The fim genes responsible
for synthesis of type 1 fimbriae in Escherichia coli, cloning and
genetic organization. 1985. Mol Gen Genet. 199:410-414); the groE
gene (Burton Z. F., and D. Eisenberg. A procedure for rapid
isolation of both groE protein and glutamine synthetase from E
coli. 1980. Arch Biochem Biophys. 205:478-488); the groL gene
(Berlyn, M. K. B., et al. 1996. Linkage map of Escherichia coli
K-12, Edition 9. In F. C. Neidhardt, R. Curtiss, J. L. Ingraham, E.
C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M.
Schaechter, and H. E. Umbarger (eds.). Escherichia coli and
Salmonella typhimurium: cellular and molecular biology, 2nd ed.
American Society for Microbiology, Washington D.C.); the groS gene
(Berlyn, M. K. B., et al. 1996. Linkage map of Escherichia coli
K-12, Edition 9. In F. C. Neidhardt, R. Curtiss, J. L. Ingraham, E.
C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M.
Schaechter, and H. E. Umbarger (eds.). Escherichia coli and
Salmonella typhimurium: cellular and molecular biology, 2nd ed.
American Society for Microbiology, Washington D.C.); the hptG gene
(Berlyn, M. K. B., et al. 1996. Linkage map of Escherichia coli
K-12, Edition 9. In F. C. Neidhardt, R. Curtiss, J. L. Ingraham, E.
C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M.
Schaechter, and H. E. Umbarger (eds.). Escherichia coli and
Salmonella typhimurium: cellular and molecular biology, 2nd ed.
American Society for Microbiology, Washington D.C.); the hscA gene
(Takahashi Y., and M. Nakamura. Functional assignment of the
ORF2-iscS-iscU-iscA-hscB-hscA-fdx-ORF3 gene cluster involved in the
assembly of Fe--S clusters in Escherichia coli.1999. J Biochem
(Tokyo). 126:917-926); the ibpA gene (Lund P. A. Microbial
molecular chaperones. 2001. Adv Microb Physiol. 44:93-140); the
papJ gene (Tennent, J. M., et al. 1990. Integrity of Escherichia
coli P pili during biogenesis: properties and role of PapJ. Mol.
Microbiol. 4:747-758); the secB gene (Lecker, S., et al. 1989.
Three pure chaperone proteins of Escherichia coli--SecB, trigger
factor and GroEL--form soluble complexes with precursor proteins in
vitro. EMBO J. 8:2703-2709); and the tig gene (Lecker, S., et al.
1989. Three pure chaperone proteins of Escherichia coli--SecB,
trigger factor and GroEL--form soluble complexes with precursor
proteins in vitro. EMBO J. 8:2703-2709); the secE gene (Muller, M.,
et al. 1002. Protein traffic in bacteria: multiple routes from the
ribosome to and across the membrane. Prog. Nucleic Acid Res. Mol.
Biol. 66:107-157); and the secY gene (Muller, M., et al. 1002.
Protein traffic in bacteria: multiple routes from the ribosome to
and across the membrane. Prog. Nucleic Acid Res. Mol. Biol.
66:107-157).
[0364] II.C.14. Export Apparatus and Membrane Targeting
[0365] Included in the design of the invention are techniques that
increase the efficiency of gene expression and protein production
in parental cells prior to minicell formation and/or in the
segregated minicells. By way of non-limiting example, these
techniques may include construction of chimeric proteins including,
but not limited to, coupling the expressed protein of interest with
native Eubacterial, Eukaryotic, Archeabacterial or organellar
leader sequences to drive membrane insertion or secretion of the
protein of interest to the periplasm or extracellular environment.
In addition to using native leader sequences, these minicell
expression constructs may also include proteolytic cleavage sites
to remove the leader sequence following insertion into the membrane
or secretion. These proteolytic cleavage sites may be native to the
organism from which the minicell is derived or non-native. In the
latter example, also included in the system are the non-native
protease that recognizes the non-native proteolytic cleavage
site.
[0366] Non-limiting examples of these leader sequences may be the
leader from the STII protein (Voss, T., et al. 1994. Periplasmic
expression of human interferon-alpha 2c in Escherichia coli results
in a correctly folded molecule. Biochem. J. 298:719-725), maltose
binding protein (malE) (Ito, K. 1982. Purification of the precursor
form of maltose-binding protein, a periplasmic protein of
Escherichia coli. J. Biol. Chem. 257:9895-9897), phoA (Jobling, M.
G., et al. 1997. Construction and characterization of versatile
cloning vectors for efficient delivery of native foreign proteins
to the periplasm of Escherichia coli. Plasmid. 38:158-173), lamB
(Wong, E. Y., et al. 1988. Expression of secreted insulin-like
growth factor-1 in Escherichia coli. Gene. 68:193-203), ompA (Loo,
T., et al. 2002. Using secretion to solve a solubility problem:
high-yield expression in Escherichia coli and purification of the
bacterial glycoamidase PNGase F. Protein Expr. Purif. 24:90-98), or
pelB (Molloy, P. E., et al. 1998. Production of soluble
single-chain T-cell receptor fragments in Escherichia coli trxB
mutants. Mol. Immunol. 35:73-81).
[0367] In addition to these leader sequences, mutations in the
cellular export machinery may be employed to increase the
promiscuity of export to display or export sequences with
non-optimized leader sequences. Non-limiting examples of genes that
may be altered to increase export promiscuity are mutations in secY
(prlA4) (Derman, A. I., et al. 1993. A signal sequence is not
required for protein export in prlA mutants of Escherichia coli.
EMBO J. 12:879-888), and secE (Harris, C. R., and T. J. Silhavy.
1999. Mapping an interface of SecY (PrlA) and SecE (PrlG) by using
synthetic phenotypes and in vivo cross-linking. J. Bacteriol.
181:3438-3444).
[0368] II.C.15. Increasing Stability and Solubility
[0369] Included in the design of the invention are techniques that
increase the efficiency of gene expression and protein production
in parental cells prior to minicell formation and/or in the
segregated minicells. By way of non-limiting example, these
techniques may include construction of chimeric/fusion proteins
including, but not limited to, coupling the expressed protein of
interest with native Eubacterial, Eukaryotic, Archeabacterial or
organellar solublizing sequences. As used herein, "solublizing
sequences" are complete or truncated amino acid sequences that
increase the solubility of the expressed membrane protein of
interest. This increased solubility may be used to increase the
lifetime of the soluble state until proper membrane insertion may
take place. By way of non-limiting example, these soluble chimeric
fusion proteins may be ubiquitin (Power, R. F., et al. 1990. High
level expression of a truncated chicken progesterone receptor in
Escherichia coli. J. Biol. Chem. 265:1419-1424), thioredoxin
(LaVallie, E. R., et al. 1993. A thioredoxin gene fusion expression
system that circumvents inclusion body formation in the E. coli
cytoplasm. Biotechnology (N.Y.) 11:187-193; Kapust, R. B., and D.
S. Waugh. 1999. Escherichia coli maltose-binding protein is
uncommonly effective at promoting the solubility of polypeptides to
which it is fused. Protein Sci. 8:1668-1674), the dsbA gene product
(Winter, J., et al. 2001. Increased production of human proinsulin
in the periplasmic space of Escherichia coli by fusion to DsbA. J.
Biotechnol. 84:175-185), the SPG protein (Murphy, J. P., et al.
1992. Amplified expression and large-scale purification of protein
G'. Bioseparation 3:63-71), the malE gene product (maltose-binding
protein) (Hampe, W., et al. 2000. Engineering of a proteolytically
stable human beta 2-adrenergic receptor/maltose-binding protein
fusion and production of the chimeric protein in Escherichia coli
and baculovirus-infected insect cells. J. Biotechnol. 77:219-234;
Kapust et al., Escherichia coli maltose-binding protein is
uncommonly effective at promoting the solubility of polypeptides to
which it is fused, Protein Sci. 8:1668-1674, 1999),
glutathione-s-transferase (GST); and/or nuclease A (Meeker et al.,
A fusion protein between serum amyloid A and staphylococcal
nuclease--synthesis, purification, and structural studies, Proteins
30:381-387, 1998). In addition to these proteins, Staphylococcal
protein A, beta-galactosidase, S-peptide, myosin heavy chain,
dihydrofolate reductase, T4 p55, growth hormone N terminus, E. coli
Hemolysin A, bacteriophage lambda cIl protein, TrpE, and TrpLE
proteins may also be used as fusion proteins to increase protein
expression and/or solubility (Makrides, Strategies for Achieving
High-Level Expression of Genes in Escherichia coli, Microbiol. Rev.
60:512-538).
[0370] III. Preparation of Minicells
[0371] III.A. Parent Cell Mutations
[0372] Although it has been reported that relatively few molecules
of endogenous RNA polymerase segregate into minicells (Shepherd et
al., Cytoplasmic RNA Polymerase in Escherichia coli, J Bacteriol
183:2527-34, 2001), other reports and results indicate that many
RNA Polymerase molecules follow plasmids into minicells (Funnell
and Gagnier, Partition of P1 plasmids in Escherichia coli mukB
chromosomal partition mutants, J Bacteriol 177:2381-6, 1995). In
any event, applicants have discovered that the introduction of an
exogenous RNA polymerase to minicell-producing cells enhances
expression of episomal elements in minicells. Such enhanced
expression may allow for the successful expression of proteins in
minicells, wherein such proteins are expressed poorly or not at all
in unmodified minicells. In order to maximize the amount of RNA
transcription from episomal elements in minicells,
minicell-producing cell lines that express an RNA polymerase
specific for certain episomal expression elements may be used. An
example of an E. coli strain of this type, designated MC-T7, was
created and used as is described in the Examples. Those skilled in
the art will be able to make and use equivalent strains based on
the present disclosure and their knowledge of the art.
[0373] Minicell-producing cells may comprise mutations that augment
preparative steps. For example, lipopolysaccharide (LPS) synthesis
in E. coli includes the lipid A biosynthetic pathway. Four of the
genes in this pathway have now been identified and sequenced, and
three of them are located in a complex operon that also contains
genes involved in DNA and phospholipid synthesis. The rfa gene
cluster, which contains many of the genes for LPS core synthesis,
includes at least 17 genes. The rfb gene cluster encodes protein
involved in O-antigen synthesis, and rfb genes have been sequenced
from a number of serotypes and exhibit the genetic polymorphism
anticipated on the basis of the chemical complexity of the O
antigens. See Schnaitman and Klena, Genetics of lipopolysaccharide
biosynthesis in enteric bacteria, Microbiol. Rev. 57:655-82, 1993.
When present, alone, or in combination, the rfb and oms mutations
cause alterations in the eubacterial membrane that make it more
sensitive to lysozyme and other agents used to process minicells.
Similarly, the rfa (Chen, L., and W. G. Coleman Jr. 1993. Cloning
and characterization of the Escherichia coli K-12 rfa-2 (rfaC)
gene, a gene required for lipopolysaccharide inner core synthesis.
J. Bacteriol. 175:2534-2540), lpcA (Brooke, J. S., and M. A.
Valvano. 1996. Biosynthesis of inner core lipopolysaccharide in
enteric bacteria identification and characterization of a conserved
phosphoheptose isomerase. J. Biol. Chem. 271:3608-3614), and 1 pcB
(Kadrman, J. L., et al. 1998. Cloning and overexpression of
glycosyltransferases that generate the lipopolysaccharide core of
Rhizobium leguminosarum. J. Biol. Chem. 273:26432-26440) mutations,
when present alone or in combination, cause alterations in
lipopolysaccharides in the outer membrane causing cells to be more
sensitive to lysozyme and agents used to process minicells. In
addition, such mutations can be used to reduce the potential
antigenicity and/or toxicity of minicells.
[0374] III.B. Culturing Conditions
[0375] Included in the design of the invention are the conditions
to grow parental cells from which minicells will be produced.
Non-limiting examples herein are drawn to conditions for growing E.
coli parental cells to produce minicells derived from E. coli
parental cells. Non-limiting examples for growth media may include
rich media, e.g. Luria broth (LB), defined minimal media, e.g. M63
salts with defined carbon, nitrogen, phosphate, magnesium, and
sulfate levels, and complex minimal media, e.g. defined minimal
media with casamino acid supplement. This growth may be performed
in culture tubes, shake flasks (using a standard air incubator, or
modified bioreactor shake flask attachment), or bioreactor. Growth
of parental cells may include supplemented additions to assist
regulation of expression constructs listed in the sections above.
These supplements may include, but are not limited to dextrose,
phosphate, inorganic salts, ribonucleic acids, deoxyribonucleic
acids, buffering agents, thiamine, or other chemical that
stimulates growth, stabilizes growth, serves as an osmo-protectant,
regulates gene expression, and/or applies selective pressure to
mutation, and/or marker selection. These mutations may include an
amino acid or nucleotide auxotrophy, while the selectable marker
may include transposable elements, plasmids, bacteriophage, and/or
auxotrophic or antibiotic resistance marker. Growth conditions may
also require temperature adjustments, carbon alternations, and/or
oxygen-level modifications to stimulate temperature sensitive
mutations found in designed gene products for a given desired
phenotype and optimize culture conditions.
[0376] By way of non-limiting example, production of minicells and
protein production may occur by using either of two general
approaches or any combination of each. First, minicells may be
formed, purified, and then contained expression elements may be
stimulated to produce their encoded gene products. Second, parental
cells, from which the minicells are derived, may be stimulated to
express the protein of interest and segregate minicells
simultaneously. Finally, any timing variable of minicell formation
and protein production may be used to optimize protein and minicell
production to best serve the desired application. The two general
approaches are shown in the sections below.
[0377] III.C. Manipulation of Genetic Expression in Minicell
Production
[0378] Included in the design of the invention are methods that
increase the efficiency, rate and/or level of gene expression and
protein production in parent cells and/or minicells. Such methods
include, but are not limited to, the following.
[0379] By way of non-limiting example, parental cells are grown
overnight in the appropriate media. From this culture, the cells
are subcultured into the same media and monitored for growth. At
the appropriate cell density, the cultures are induced for minicell
production using any of the switching mechanisms discussed in
section II.B. regulating any construct discussed in section II.A.
Non-limiting examples of this minicell-inducing switching mechanism
may be the ileR gene product regulating the production of the has
minicell-inducing gene product or the melR gene product regulating
the production of the minB minicell-inducing gene product.
Following minicell induction, the culture is allowed to continue
growth until the desired concentration of minicells is obtained. At
this point, the minicells are separated from the parental cells as
described in section II.E. Purified minicells are induced for
protein production by triggering the genetic switching mechanism
that segregated into the minicell upon separation from the parental
cell. By way of non-limiting example, this genetic switching
mechanism may be any of those discussed in section I.B. regulating
the production of any protein of interest. Furthermore, at this
point or during the production of minicells the peripheral gene
expression, production, and assembly machinery discussed in section
II.C. may be triggered to assist in this process. By way of
non-limiting example, the groEL complex may be triggered using the
temperature sensitive lambda cI inducible system from a
co-segregant plasmid to assist in the proper assembly of the
expressed protein of interest.
[0380] III.D. Separation of Minicells From Parent Cells
[0381] A variety of methods are used to separate minicells from
parent cells (i.e., the cells from which the minicells are
produced) in a mixture of parent cells and minicells. In general,
such methods are physical, biochemical and genetic, and can be used
in combination.
[0382] III.D.1. Physical Separation of Minicells from Parent
Cells
[0383] By way of non-limiting example, minicells are separated from
parent cells glass-fiber filtration (Christen et al., Gene
23:195-198, 1983), and differential and zonal centrifugation
(Barker et al., J. Gen. Microbiol. 111:387-396, 1979),
size-exclusion chromatography, e.g. gel-filtration, differential
sonication (Reeve, J. N., and N. H. Mendelson. 1973. Pronase
digestion of amino acid binding components on the surface of
Bacillus subtilis cells and minicells. Biochem. Biophys. Res.
Commun. 53:1325-1330), and UV-irradiation (Tankersley, W. G., and
J. M. Woodward. 1973. Induction and isoloation of non-replicative
minicells of Salmonella typhimuium and their use as immunogens in
mice. Bacteriol. Proc. 97).
[0384] Some techniques involve different centrifugation techniques,
e.g., differential and zonal centrifugation. By way of non-limiting
example, minicells may be purified by the double sucrose gradient
purification technique described by Frazer and Curtiss, Curr.
Topics Microbiol. Immunol. 69:1-84, 1975. The first centrifugation
involves differential centrifugation, which separates parent cells
from minicells based on differences in size and/or density. The
percent of sucrose in the gradient (graduated from about 5 to about
20%), Ficol or glycerol is designed to allow only parent cells to
pass through the gradient.
[0385] The supernatant, which is enriched for minicells, is then
separated from the pellet and is spun at a much higher rate (e.g.,
.gtoreq.11,000 g). This pellets the minicells and any parent cells
that did not pellet out in the first spin. The pellet is then
resuspended and layered on a sucrose gradient.
[0386] The band containing minicells is collected, pelleted by
centrifugation, and loaded on another gradient. This procedure is
repeated until the minicell preparation is essentially depleted of
parent cells, or has a concentration of parent cells that is low
enough so as to not interfere with a chosen minicell application or
activity. By way of non-limiting example, buffers and media used in
these gradient and resuspension steps may be LB, defined minimal
media, e.g. M63 salts with defined carbon, nitrogen, phosphate,
magnesium, and sulfate levels, complex minimal media, e.g. defined
minimal media with casamino acid supplement, and/or other buffer or
media that serves as an osmo-protectant, stabilizing agent, and/or
energy source, or may contain agents that limit the growth of
contaminating parental cells, e.g. azide, antibiotic, or lack an
auxotrophic supplemental requirement, e.g. thiamine.
[0387] Other physical methods may also be used to remove parent
cells from minicell preparations. By way of non-limiting example,
mixtures of parent cells and minicells are frozen to -20.degree. C.
and then thawed slowly (Frazer and Curtiss, Curr. Topics Microbiol.
Immunol. 69:1-84, 1975).
[0388] III.D.2. Biochemical Separation of Minicells From Parent
Cells
[0389] Contaminating parental cells may be eliminated from minicell
preparations by incubation in the presence of an agent, or under a
set of conditions, that selectively kills dividing cells. Because
minicells can neither grow nor divide, they are resistant to such
treatments.
[0390] Examples of biochemical conditions that prevent or kill
dividing parental cells is treatment with a antibacterial agent,
such as penicillin or derivatives of penicillin. Penicillin has two
potential affects. First, penicillin prevent cell wall formation
and leads to lysis of dividing cells. Second, prior to lysis
dividing cells form filaments that may assist in the physical
separation steps described in section III.E.1. In addition to
penicillin and its derivatives, other agents may be used to prevent
division of parental cells. Such agents may include azide. Azide is
a reversible inhibitor of electron transport, and thus prevents
cell division. As another example, D-cycloserine or phage MS2 lysis
protein may also serve as a biochemical approach to eliminate or
inhibit dividing parental cells. (Markiewicz et al., FEMS
Microbiol. Lett. 70:119-123, 1992). Khachatourians (U.S. Pat. No.
4,311,797) states that it may be desirable to incubate
minicell/parent cell mixtures in brain heart infusion broth at
36.degree. C. to 38.degree. C. prior to the addition of penicillin
G and further incubations.
[0391] III.D.3. Genetic Separation of Minicells From Parent
Cells
[0392] Alternatively or additionally, various techniques may be
used to selectively kill, preferably lyse, parent cells. For
example, although minicells can internally retain M13 phage in the
plasmid stage of the M13 life cycle, they are refractory to
infection and lysis by M13 phage (Staudenbauer et al., Mol. Gen.
Genet. 138:203-212, 1975). In contrast, parent cells are infected
and lysed by M13 and are thus are selectively removed from a
mixture comprising parent cells and minicells. A mixture comprising
parent cells and minicells is treated with M13 phage at an M.O.I.=5
(phage:cells). The infection is allowed to continue to a point
where .gtoreq.50% of the parent cells are lysed, preferably
.gtoreq.75%, more preferably .gtoreq.95% most preferably
.gtoreq.99%; and .ltoreq.25% of the minicells are lysed or killed,
preferably .ltoreq.15%, most preferably .ltoreq.1%.
[0393] As another non-limiting example of a method by which parent
cells can be selectively killed, and preferably lysed, a chromosome
of a parent cell may include a conditionally lethal gene. The
induction of the chromosomal lethal gene will result in the
destruction of parent cells, but will not affect minicells as they
lack the chromosome harboring the conditionally lethal gene. As one
example, a parent cell may contain a chromosomal integrated
bacteriophage comprising a conditionally lethal gene. One example
of such a bacteriophage is an integrated lambda phage that has a
temperature sensitive repressor gene (e.g., lambda cI857).
Induction of this phage, which results in the destruction of the
parent cells but not of the achromosomal minicells, is achieved by
simply raising the temperature of the growth media. A preferred
bacteriophage to be used in this method is one that kills and/or
lyses the parent cells but does not produce infective particles.
One non-limiting example of this type of phage is one that lyses a
cell but which has been engineered to as to not produce capsid
proteins that are surround and protect phage DNA in infective
particles. That is, capsid proteins are required for the production
of infective particles.
[0394] As another non-limiting example of a method by which parent
cells can be selectively killed or lysed, toxic proteins may be
expressed that lead to parental cell lysis. By way of non-limiting
example, these inducible constructs may employ a system described
in section II.B. to control the expression of a phage holing gene.
Holin genes fall with in at least 35 different families with no
detectable orthologous relationships (Grundling, A., et al. 2001.
Holins kill without warning. Proc. Natl. Acad. Sci. 98:9348-9352)
of which each and any may be used to lyse parental cells to improve
the purity of minicell preparations.
[0395] Gram negative eubacterial cells and minicells are bounded by
an inner membrane, which is surrounded by a cell wall, wherein the
cell wall is itself enclosed within an outer membrane. That is,
proceeding from the external environment to the cytoplasm of a
minicell, a molecule first encounters the outer membrane (OM), then
the cell wall and finally, the inner membrane (IM). In different
aspects of the invention, it is preferred to disrupt or degrade the
OM, cell wall or IM of a eubacterial minicell. Such treatments are
used, by way of non-limiting example, in order to increase or
decrease the immunogenicity, and/or to alter the permeability
characteristics, of a minicell.
[0396] Eubacterial cells and minicells with altered membranes
and/or cell walls are called "Poroplasts.TM." "spheroplasts," and
"protoplasts." Herein, the terms "spheroplast" and "protoplast"
refer to spheroplasts and protoplasts prepared from minicells. In
contrast, "cellular spheroplasts" and "cellular protoplasts" refer
to spheroplasts and protoplasts prepared from cells. Also, as used
herein, the term "minicell" encompasses not only minicells per se
but also encompasses Poroplasts.TM., spheroplasts and
protoplasts.
[0397] In a poroplast, the eubacterial outer membrane (OM) and LPS
have been removed. In a spheroplast, portions of a disrupted
eubacterial OM and/or disrupted cell wall either may remain
associated with the inner membrane of the minicell, but the
membrane and cell wall is nonetheless porous because the
permeability of the disrupted OM and cell wall has been increased.
A membrane is said to be "disrupted" when the membrane's structure
has been treated with an agent, or incubated under conditions, that
leads to the partial degradation of the membrane, thereby
increasing the permeability thereof. In contrast, a membrane that
has been "degraded" is essentially, for the applicable intents and
purposes, removed. In preferred embodiments, irrespective of the
condition of the OM and cell wall, the eubacterial inner membrane
is not disrupted, and membrane proteins displayed on the inner
membrane are accessible to compounds that are brought into contact
with the minicell, poroplast, spheroplast, protoplast or cellular
poroplast, as the case may be.
[0398] III.E.2. Poroplasts.TM.
[0399] For various applications poroplasted minicells are capable
of preserving the cytoplasmic integrity while producing increased
stability over that of naked protoplasts. Maintenance of the cell
wall in poroplasted minicells increases the osmotic resistance,
mechanical resistance and storage capacity over protoplasts while
permitting passage of small and medium size proteins and molecules
through the porous cell wall. A poroplast is a Gram negative
bacterium that has its outer membrane only removed. The production
of poroplasts involves a modification of the procedure to make
protoplasts to remove the outer membrane (Birdsell et al.,
Production and ultrastructure of lysozyme and
ethylenediaminetetraacetate-Lysozyme Spheroplasts of Escherichia
coli, J. Bacteriology 93: 427-437, 1967; Weiss, Protoplast
formation in Escherichia coli. J. Bacteriol. 128:668-670, 1976).
Like protoplasts, measuring the total LPS that remains in the
poroplast preparation may be used to monitor the removal of the
outer membrane. Endotoxin kits and antibodies reactive against LPS
may be used to measure LPS in solution; increasing amounts of
soluble LPS indicates decreased retention of LPS by protoplants.
This assay thus makes it possible to quantify the percent removal
of total outer membrane from the poroplasted minicells.
[0400] Several chemical and physical techniques have been employed
to remove the outer membrane of gram negative bacteria. Chemical
techniques include the use of EDTA in Tris to make cells
susceptible to hydrophobic agents such as actinomycin C. Leive L.
The barrier function of the gram-negative envelope. Ann N Y Acad.
Sci. 1974 May 10; 235(0):109-29; Voll MJ, Leive L. Actinomycin
resistance and actinomycin excretion in a mutant of Escherichia
coli. J. Bacteriol. 1970 May; 102(2):600-2; Lactic Acid disruption
of the outer membrane as measured by the uptake of hydrophobic
fluorophores; Alakomi H L, Skytta E, Saarela M, Mattila-Sandholm T,
Latva-Kala K, Helander I M. Lactic acid permeabilizes gram-negative
bacteria by disrupting the outer membrane. Appl Environ Microbiol.
2000 May; 66(5):2001-5; and Polymyxin B disruption as measured by
periplasmic constituent release (Teuber M, Cerny G. Release of the
periplasmic ribonuclease I into the medium from Escherichia coli
treated with the membrane-active polypeptide antibiotic polymyxin
B. FEBS Lett. 1970 May 11; 8(1):49-51). Physical techniques include
the use of osmodifferentiation to facilitate the disruption of the
OM. Neu H C, Heppel L A. The release of enzymes from Escherichia
coli by osmotic shock and during the formation of spheroplasts. J
Biol. Chem. 1965 September; 240(9):3685-92. See also Voll MJ, Leive
L. Actinomycin resistance and actinomycin excretion in a mutant of
Escherichia coli. J Bacteriol. 1970 May; 102(2):600-2; Fiil A,
Branton D. Changes in the plasma membrane of Escherichia coli
during magnesium starvation. J. Bacteriol. 1969 June; 98(3):1320-7;
and Matsuyama S, Fujita Y, Mizushima S. SecD is involved in the
release of translocated secretory proteins from the cytoplasmic
membrane of Escherichia coli. EMBO J. 1993 January;
12(1):265-70.
[0401] III.E.3. Spheroplasts
[0402] A spheroplast is a bacterial minicell that has a disrupted
cell wall and/or a disrupted OM. Unlike eubacterial minicells and
poroplasts, which have a cell well and can thus retain their shape
despite changes in osmotic conditions, the absence of an intact
cell wall in spheroplasts means that these minicells do not have a
rigid form.
[0403] III.E.4. Protoplasts
[0404] A protoplast is a bacterium that has its outer membrane and
cell wall removed. The production of protoplasts involves the use
of lysozyme and high salt buffers to remove the outer membrane and
cell wall (Birdsell et al., Production and ultrastructure of
lysozyme and ethylenediaminetetraacetate-Lysozyme Spheroplasts of
Escherichia coli, J. Bacteriology 93: 427-437, 1967; Weiss,
Protoplast formation in Escherichia coli. J. Bacteriol.
128:668-670, 1976). Various commercially available lysozymes can be
used in such protocols. Measuring the total LPS that remains in the
protoplast preparation is used to monitor the removal of the outer
membrane. Endotoxin kits assays can be used to measure LPS in
solution; increasing amounts of soluble LPS indicates decreased
retention of LPS by protoplasts. This assay thus makes it possible
to quantify the percent removal of total outer membrane from the
minicells. Endotoxin assays are commerically available from, e.g.,
BioWhittaker Molecular Applications (Rockland, Me.)
[0405] For minicell applications that utilize bacterial-derived
minicells, it may be necessary to remove the outer membrane of
Gram-negative cells and/or the cell wall of any bacterial-derived
minicell. For Gram-positive bacterial cells, removal of the cell
wall may be easily accomplished using lysozyme. This enzyme
degrades the cell wall allowing easy removal of now soluble cell
wall components from the pelletable protoplasted minicells. In a
more complex system, the cell wall and outer membrane of
Gram-negative cells may be removed by combination treatment with
EDTA and lysozyme using a step-wise approach in the presence of an
osmoprotecting agent (Birdsell, et al. 1967. Production and
ultrastructure of lysozyme and ethylenediaminetetraacetate-lysozyme
spheroplasts of E. coli, J. Bacteriol. 93:427-437; Weiss, 1976.
Protoplast formation in E. coli. J. Bacteriol. 128:668-670). By
non-limiting By way of non-limiting example, this osmoprotectant
may be sucrose and/or glycerol. It has been found that the
concentration of the osmoprotectant sucrose, the cell wall
digesting enzyme lysozyme, and chelator EDTA can be optimized to
increase the quality of the protoplasts produced. Separation of
either prepared Gram-negative spheroplasts prepared in either
fashion from removed remaining LPS may occur through exposure of
the spheroplast mixture to an anti-LPS antibody. By non-limiting By
way of non-limiting example, the anti-LPS antibody may be
covalently or non-covalently attached to magnetic, agarose,
sepharose, sepheracyl, polyacrylamide, and/or sephadex beads.
Following incubation, LPS is removed from the mixture using a
magnet or slow centrifugation resulting in a protoplast-enriched
supernatant.
[0406] Monitoring loss of LPS may occur through dot-blot analysis
of protoplast mixtures or various commercially available endotoxin
kit assays can be used to measure LPS in solution; increasing
amounts of soluble LPS indicates decreased retention of LPS by
protoplasts. This immuno assay may comprise a step of comparing the
signal to a standard curve in order to quantify the percent removal
of total outer membrane from the minicells. Other endotoxin assays,
such as the LAL Systems from BioWhittaker, are commercially
available. LPS removal has been measured by gas chromatography of
fatty acid methyl esters. Alakomi H L, Skytta E, Saarela M,
Mattila-Sandholm T, Latva-Kala K, Helander I M. Lactic acid
permeabilizes gram-negative bacteria by disrupting the outer
membrane. Appl Environ Microbiol. 2000 May; 66(5):2001-5.
[0407] In order to reduce, preferably eliminate, in vivo antigenic
potential of minicells or minicell protoplasts, minicell
protoplasts may be treated to remove undesirable surface
components. Minicell protoplasts so treated are referred to as
"denuded minicells" a term that encompasses both spheroplasts and
protoplasts. Denuding minicells or minicell protoplasts is
accomplished by treatment with one or more enzymes or conditions
that selectively or preferentially removes or make less antigenic
externally displayed proteins. As one non-limiting example, the
protease trypsin is used to digest exposed proteins on the surface
of these particles. In this example, the proteolytic activity of
trypsin may be modulated or terminated by the additional of a
soybean trypsin inhibitor. Non-limiting examples of other proteases
that additionally or alternatively may be used include
chymotrypsin, papain, elastase, proteinase K and pepsin. For some
such proteases, it may be necessary to limit the extent of
proteolysis by, e.g., using a suboptimal concentration of protease
or by allowing the reaction to proceed for a suboptimal period of
time. By the term "suboptimal," it is meant that complete digestion
is not achieved under such conditions, even though the reactions
could proceed to completion under other (i.e., optimal)
conditions.
[0408] It is sometimes preferred to use molecular genetic
techniques to create mutant derivatives of exogenous proteins that
(1) are resistant to the proteases or other enzymes used to prepare
minicells and (2) retain the desired biological activity of the
receptor that is desired to be retained, i.e., the ability to bind
one or more ligands of interest.
[0409] It is within the scope of the invention to have two or more
exogenous proteins expressed within and preferentially displayed by
minicells in order to achieve combined, preferable synergistic,
therapeutic compositions. Similarly, two or more therapeutic
minicell compositions are formulated into the same composition, or
are administered during the same therapeutic minicell compositions
(i.e., "cocktail" therapies). In other types of "cocktail" therapy,
one or more therapeutic minicell compositions are combined or
co-administered with one or more other therapeutic agents that are
not minicell compositions such as, e.g., organic compounds,
therapeutic proteins, gene therapy constructs, and the like.
[0410] III.F. Minicells from L-form Eubacteria
[0411] L-form bacterial strains may be used to prepare minicells
and are preferred in some embodiments of the invention. L-form
bacterial strains are mutant or variant strains, or eubacteria that
have been subject to certain conditions, that lack an outer
membrane, a cell wall, a periplasmic space and extracellular
proteases. Thus, in L-form Eubacteria, the cytoplasmic membrane is
the only barrier between the cytoplasm and its surrounding
environment. For reviews, see Grichko, V. P., et al. 1999. The
Potential of L-Form Bacteria in Biotechnology, Can. J. Chem.
Engineering 77:973-977; and Gumpert J., et al. 1998 Use of cell
wall-less bacteria (L-forms) for efficient expression and secretion
of heterologous gene products. Curr Opin Biotechnol. 9:506-9.
[0412] Segregation of minicells from L-form eubacterial parent
cells allows for the generation of minicells that are at least
partially deficient in barriers that lie outside of the cytoplasmic
membrane, thus providing direct access to components displayed on
the minicell membrane. Thus, depending on the strains and methods
of preparation used, minicells prepared from L-form eubacterial
parent cells will be similar if not identical to various forms of
poroplasts, spheroplasts and/or protoplasts. Displayed components
that are accessible in L-form minicells include, but are not
limited to, lipids, small molecules, proteins, sugars, nucleic
acids and/or moieties that are covalently or non-covalently
associated with the cytoplasmic membrane or any component
thereof.
[0413] By way of non-limiting example, L-form Eubacteria that can
be used in the methods of the invention include species of
Escherichia, Streptomyces, Proteus, Bacillus, Clostridium,
Pseudomonas, Yersinia, Salmonella, Enterococcus and Erwinia. See
Onoda, T., et al. 1987. Morphology, growth and reversion in a
stable L-form of Escherichia coli K12. J. Gen. Microbiol.
133:527-534; Inanova, E. H., et al. 1997. Effect of Escherichia
coli L-form cytoplasmic membranes on the interaction between
macrophages and Lewis lung carcinoma cells: scanning electron
microscopy. FEMS Immunol. Med. Microbiol. 17:27-36; Onoda, T., et
al. 2000. Effects of calcium and calcium chelators on growth and
morphology of Escherichia coli L-form NC-7. J. Bacteriol.
182:1419-1422; Innes, C. M., et al. 2001. Induction, growth and
antibiotic production of Streptomyces viridifaciens L-form
bacteria. J Appl Microbiol. 90:301-308; Ferguson, C. M., et al.
2000. An ELISA for the detection of Bacillus subtilis L-form
bacteria confirms their symbiosis in strawberry. Lett Appl
Microbiol. 31:390-394; Waterhouse R. N., et al. 1994. An
investigation of enumeration and DNA partitioning in Bacillus
subtilis L-form bacteria. J Appl Bacteriol. 77:497-503; Hoischen,
C., et al. 2002. Novel bacterial membrane surface display system
using cell wall-less L-forms of Proteus mirabilis and Escherichia
coli. Appl. Environ. Microbiol. 68:525-531; Rippmann, J. F., et al.
1998. Procaryotic expression of single-chain variable-fragment
(scFv) antibodies: secretion in L-form cells of Proteus mirabilis
leads to active product and overcomes the limitations of
periplasmic expression in Escherichia coli. Appl. Environ.
Microbiol. 64:4862-4869; Mahony, D. E., et al. 1988. Transformation
of Clostridium perfringens L forms with shuttle plasmid DNA. Appl.
Environ. Microbiol. 54:264-267); Kurona, M., et al. 1983.
Intergenus cell fusions between L-form cells of Pseudomonas
aeruginosa and Escherichia coli. Biken. J. 26:103-111; Ivanova, E.,
et al. 2000. Studies of the interactions of immunostimulated
macrophages and Yersinia enterocolitica 0:8. Can. J. Microbiol.
46:218-228; Allan, E. J., et al. 1993. Growth and physiological
characteristics of Bacillus subtilis L-forms. J. Appl. Bacteriol.
74:588-594; Allan, E. J. 1991. Induction and cultivation of a
stable L-form of Bacillus subtilis. J. Appl. Bacteriol. 70:339-343;
Nishikawa, F., et al. 1994. Protective capacity of L-form
Salmonella typhimurium against murine typhoid in C3H/HeJ mice.
Microbiol. Immunol. 38:129-137; Kita, E., et al. 1993. Isolation of
a cytotoxin from L-form Salmonella typhimurium. FEMS Microbiol.
Lett. 109:179-184; Jass, J., et al. Growth and adhesion of
Enterococcus faecium L-forms. FEMS Microbiol. Lett. 115:157-162;
and U.S. Pat. No. 6,376,245.
[0414] IV. Assaying Minicells
[0415] IV.A. Efficiency of Minicell Production
[0416] The level of minicell production will vary and may be
evaluated using methods described herein. Relatively high levels of
minicell production are generally preferred. Conditions in which
about 40% of cells are achromosomal have been reported (see, e.g.,
Hassan et al., Suppression of initiation defects of chromosome
replication in Bacillus subtilis dnaA and oriC-deleted mutants by
integration of a plasmid replicon into the chromosomes, J Bacteriol
179:2494-502, 1997). Procedures for identifying strains that give
high yields of minicells are known in the art; see, e.g.,
Clark-Curtiss and Curtiss III, Analysis of Recombinant DNA Using
Escherichia coli Minicells, Meth. Enzol. 101:347-362, 1983.
[0417] Minicell production can be assessed by microscopic
examination of late log-phase cultures. The ratio of minicells to
normal cells and the frequency of cells actively producing
minicells are parameters that increase with increasing minicell
production.
[0418] IV.B. Detecting Protein Synthesis in Minicells
[0419] Methods for detecting and assaying protein production are
known in the art. See, e.g., Clark-Curtiss and Curtiss III, Meth
Enzol 101:347-362, 1983. As an exemplary procedure, transformed E.
coli minicell-producing cells are grown in LB broth with the
appropriate antibiotic overnight. The following day the overnight
cultures are diluted 1:50 in fresh media, and grown at 37.degree.
C. to mid-log phase. If it is desired to eliminate whole cells, an
antibiotic that kills growing (whole) cells but not quiescent cells
(minicells) may be used. For example, in the case of cells that are
not ampicillin resistant, ampicillin (100 mg per ml is added), and
incubation is allowed to continue for about 2 more hrs. Cultures
are then centrifuged twice at low speed to pellet most of the large
cells. Minicells are pelleted by spinning 10 min at 10,000 rpm, and
are then resuspended in M63 minimal media supplemented with 0.5%
casamino acids, and 0.5 mM cAMP, or M9 minimal medium supplemented
with 1 mM MgSO.sub.4, 0.1 mM CaCl.sub.2, 0.05% NaCl, 0.2% glucose,
and 1 ng per ml thiamine. Labeled (.sup.35S) methonine is added to
the minicells for about 15 to about 90 minutes, and minicells are
immediately collected afterwards by centrifugation for 10 min at
4.degree. C. and 14,000 rpm. Cells are resuspended in 50 to 100
.mu.g Laemmeli-buffer, and disrupted by boiling and vortexing (2
min for each step). Incorporation of .sup.35S-methionine was
determined by measuring the amount of radioactivity contained in 1
.mu.l of the lysate after precipitation of proteins with
trichloroacetic acid (TCA). Minicell lysates (50,000 to 100,000 cpm
per lane) are subjected to PAGE on, e.g., 10% polyacrylamide gels
in which proteins of known size are also run as molecular weight
standards. Gels are fixed and images there of are generated by,
e.g., autoradiography or any other suitable detection systems.
[0420] IV.C. Evaluating the Therapeutic Potential of Minicells
[0421] Various methods are used at various stages of development of
a therapeutic minicell composition to estimate their therapeutic
potential. As a non-limiting example, the therapeutic potential of
minicells displaying a receptor is examined as follows.
[0422] IV.C.1. Receptors
[0423] The specificity of, rate of association of, rate of
dissociation of, and/or stability of complexes resulting from,
receptor binding to its ligand can be measured in vitro using
methods known in the art.
[0424] In the case of a sphingolipid binding receptor, such as an
S1P receptor, the ligand (S1P) is detectably labeled so that the
specificity of, rate of formation of, and degree of stability of
complexes resulting from the ligand-receptor binding can be
examined by measuring the degree and rate at which the labeled
ligand is removed from solution due to its binding to minicells
displaying the receptor. In order to avoid extraneous factors from
influencing these experiments, they are carried out in buffered
solutions that are as free of contaminating substances as possible.
However, as is understood in the art, stabilizing agents such as
BSA (bovine serum albumin) or protease inhibitors may be desirably
included in these experiments. In a preferred environment, a
sphingolipid binding receptor is the rat EDG-1, rat EDG-3, rat
SCaMPER and human SCaMPER, the sequences of which are set forth
herein.
[0425] Minicell compositions that bind sphingolipids with the
desired specificity are identified from the preceding experiments.
Typically, studies of ligand-receptor binding then proceed to
studies in which the binding capacity of promising minicell
compositions is tested under in vitro conditions that are
increasingly more representative of in vivo conditions. For
example, binding experiments are carried out in the presence of
sera or whole blood in order to determine the therapeutic potential
of minicell compositions in the presence of compounds that are
present within the circulatory system of an animal.
[0426] IV.C.2. Molecular Sponge
[0427] Minicell compositions can also be tested for their ability
to bind and/or interanlize toxic compounds. The therapeutic
potential of such capacity is evaluated using experiments in which
detectably labeled derivatives of a toxic compound are present in
the bloodstream of an anesthetized animal, which may a human. The
blood of the animal is shunted out of the body and past a device
that incorporates a minicell composition before being returned to
the body. The device is constructed so that the blood contacts a
semipermeable membrane that is in contact with the minicell
composition. By "semipermeable" it is meant that certain agents can
be freely exchanged across the membrane, whereas others are
retained on one side of the membrane or the other. For example, the
toxic compound of interest is able to cross the semipermeable
membrane, whereas minicells and blood cells are separately retained
in their respective compartments. Detectably labeled derivatives of
the toxic compound are present in the bloodstream of the animal.
The capacity of the minicells to take up the toxic compound
corresponds with a reduction of the levels of detectably labeled
material in the blood and an increase in detectably labeled
material in the minicell composition.
[0428] The above types of minicell-comprising compositions,
devices, and procedures may be incorporated into ex vivo modalities
such as ex vivo gene therapy and dialysis machines. An "ex vivo
modality" is one in which a biological sample, such as a blood
sample, is temporarily removed from an animal, altered through in
vitro manipulation, and then returned to the body. In "ex vivo gene
therapy," cells in the sample from the animal are transformed with
DNA in vitro and then returned to the body. A "dialysis machine" is
a device in which a fluid such as blood of an animal is temporarily
removed therefrom and processed through one or more physical,
chemical, biochemical, binding or other processes designed to
remove undesirable substances including but are not limited to
toxins, venoms, overexpressed or overactive endogenous agents, and
pathogens or molecules derived therefrom.
[0429] Intraminicellular co-expression of a second molecule that is
displayed on the surface of minicells, and which is a ligand for a
binding moiety that is immobilized, can optionally be used in order
to remove minicells from the sample before it is returned to the
body. In the latter aspect, minicells that bind undesirable
substances are preferably removed with the undesirable compound
remaining bound to the minicells. Minicells that have been used for
ex vivo gene therapy, but which have failed to deliver a nucleic
acid to any cells in the sample, can be removed in a similar
manner.
[0430] IV.C.3. Minicell-Solubilized Receptors
[0431] It is known in the art to use recombinant DNA technology to
prepare soluble (hydrophilic) receptor fragments from receptors
that bind a bioactive ligand. Unlike the native, membrane-bound
receptor, which is relatively insoluble in water (hydrophobic),
soluble receptor fragments can be formulated for therapeutic
delivery using techniques that are known to have been used to
formulate soluble agents.
[0432] Typically, soluble receptor fragments are used to
competitively inhibit the binding of the receptor to its ligand.
That is, the soluble receptor fragments bind the ligand at the
expense of the membrane-bound receptor. Because less of the ligand
is bound to its receptor, the cellular response to the ligand is
attenuated. Common cellular responses that are desirably attenuated
include but are not limited to the uptake of an undesirable agent
(e.g., a toxin, a pathogen, etc.) and activation of a signaling
pathway having undesirable consequences (e.g., inflammation,
apoptosis, unregulated growth, etc.).
[0433] Preparing a soluble fragment derived from a receptor is not
trivial. Typically, the three dimensional structure of the receptor
is not known, and must be predicted based on homology with other
receptors or by using software that predicts the tertiary structure
of a polypeptide based on its amino acid sequence. Using the
hypothetical structure of the receptor, a series of polypeptides
are prepared that comprise amino acid sequences from the receptor
but lack regions thereof that are thought to be membrane-anchoring
or transmembrane domain(s) of the receptor. Some of the
polypeptides prepared this way may be soluble, some may retain the
binding activity of the receptor, and a few may have both
characteristics. Members of the latter class of polypeptides are
soluble receptor fragments, some of which may be amenable to
development as a therapeutic or diagnostic agent.
[0434] For any given receptor, there is always the possibility that
none of the soluble fragments derived from the receptor will
specifically bind its ligand with sufficient affinity as to be
therapeutically effective. Thus, in some instances, it may not be
possible to prepare a receptor fragment that is both soluble and
sufficiently biologically active.
[0435] The minicells of the invention provide a "universal carrier"
for receptors that allows the hydrophobic receptors to be
solubilized in the sense that, although they remain associated with
a membrane, the minicell is a small, soluble particle. That is, as
an alternative to preparing a set of polypeptides to see which, if
any of them, are water soluble receptor fragments, one may, using
the teachings of the disclosure, prepare soluble minicells that
display the receptor.
[0436] IV.C.4. Reducing Toxicity
[0437] For in vivo use of minicells for the purposes of eliciting
an immune response or for therapeutic and diagnostic applications
involving delivery of minicells to a human or to an anima, it may
be useful to minimize minicell toxicity by using
endotoxin-deficient mutants of parent cells. Without being limited
to the following example, lipopolysaccharide (LPS) deficient E.
coli strains could be conjugated with minicell producing cells to
make parent cells lacking the endotoxin. LPS synthesis in E. coli
includes the lipid A biosynthetic pathway. Four of the genes in
this pathway have now been identified and sequenced, and three of
them are located in a complex operon which also contains genes
involved in DNA and phospholipid synthesis. The rfa gene cluster,
which contains many of the genes for LPS core synthesis, includes
at least 17 genes. The rfb gene cluster encodes protein involved in
O-antigen synthesis, and rfb genes have been sequenced from a
number of serotypes and exhibit the genetic polymorphism
anticipated on the basis of the chemical complexity of the O
antigens (Schnaitman and Klena. 1993. Genetics of
lipopolysaccharide biosynthesis in enteric bacteria. Microbiol.
Rev. 57:655-82). When present alone or in combination the rfb and
oms mutations cause alterations in the eubacterial membrane that
make it more sensitive to lysozyme and other agents used to process
minicells. Similarly, the rfa (Chen, L., and W. G. Coleman Jr.
1993. Cloning and characterization of the Escherichia coli K-12
rfa-2 (rfaC) gene, a gene required for lipopolysaccharide inner
core synthesis. J. Bacteriol. 175:2534-2540), lpcA (Brooke, J. S.,
and M. A. Valvano. 1996. Biosynthesis of inner core
lipopolysaccharide in enteric bacteria identification and
characterization of a conserved phosphoheptose isomerase. J. Biol.
Chem. 271:3608-3614), and 1 pcB (Kadrman, J. L., et al. 1998.
Cloning and overexpression of glycosyltransferases that generate
the lipopolysaccharide core of Rhizobium leguminosarum. J. Biol.
Chem. 273:26432-26440) mutations, when present alone or in
combination, cause alterations in lipopolysaccharides in the outer
membrane causing cells to be more sensitive to lysozyme and agents
used to process minicells. In addition, such mutations can be used
to reduce the potential antigenicity and/or toxicity of
minicells.
[0438] Minicell-producing cells may comprise mutations that augment
preparative steps. For example, lipopolysaccharide (LPS) synthesis
in E. coli includes the lipid A biosynthetic pathway. Four of the
genes in this pathway have now been identified and sequenced, and
three of them are located in a complex operon that also contains
genes involved in DNA and phospholipid synthesis. The rfa gene
cluster, which contains many of the genes for LPS core synthesis,
includes at least 17 genes. The rfb gene cluster encodes protein
involved in O-antigen synthesis, and rfb genes have been sequenced
from a number of serotypes and exhibit the genetic polymorphism
anticipated on the basis of the chemical complexity of the O
antigens. See Schnaitman and Klena, Genetics of lipopolysaccharide
biosynthesis in enteric bacteria, Microbiol. Rev. 57:655-82, 1993.
When present, alone, or in combination, the rfb and oms mutations
cause alterations in the eubacterial membrane that make it more
sensitive to lysozyme and other agents used to process minicells.
Similarly, the rfa (Chen, L., and W. G. Coleman Jr. 1993. Cloning
and characterization of the Escherichia coli K-12 rfa-2 (rfaC)
gene, a gene required for lipopolysaccharide inner core synthesis.
J. Bacteriol. 175:2534-2540), lpcA (Brooke, J. S., and M. A.
Valvano. 1996. Biosynthesis of inner core lipopolysaccharide in
enteric bacteria identification and characterization of a conserved
phosphoheptose isomerase. J. Biol. Chem. 271:3608-3614), and 1 pcB
(Kadrman, J. L., et al. 1998. Cloning and overexpression of
glycosyltransferases that generate the lipopolysaccharide core of
Rhizobium leguminosarum. J. Biol. Chem. 273:26432-26440) mutations,
when present alone or in combination, cause alterations in
lipopolysaccharides in the outer membrane causing cells to be more
sensitive to lysozyme and agents used to process minicells. In
addition, such mutations can be used to reduce the potential
antigenicity and/or toxicity of minicells.
[0439] V. Genetic Expression in Minicells
[0440] Various minicells of the invention use recombinant DNA
expression systems to produce a non-eubacterial protein, which may
be a membrane protein that is preferably "displayed" on the surface
of minicells, a membrane protein that projects portions not
associated with a membrane towards the interior of a minicell, or a
soluble protein present in the exterior of the minicells. By
"displayed" it is meant that a protein is present on the surface of
a cell (or minicell) and is thus in contact with the external
environment of the cell. Non-limiting examples of displayed
exogenous proteins of the invention include mammalian receptors and
fusion proteins comprising one or more transmembrane domains. In
other aspects of the invention, minicells use expression elements
to produce bioactive nucleic acids from templates therefor.
[0441] V.A. Expression Systems
[0442] In vivo and in vitro protein expression systems provide a
variety of techniques that allow scientists to transcribe and
translate amino acid polypeptides proteins from recombinant DNA
templates (Kaufman, Overview of vector design for mammalian gene
expression. Mol Biotechnol, 2001. 16: 151-160; and Kozak,
Initiation of translation in prokaryotes and eukaryotes. Gene,
1999. 234: 187-208).
[0443] Although minicells are virtually depleted of chromosomal DNA
(Tudor et al., Presence of nuclear bodies in some minicells of
Escherichia coli. J Bacteriol, 1969. 98: 298-299), it has been
reported that minicells have all the elements required to express
nucleotide sequences that are present in episomal expression
elements therein (Levy, Very stable prokaryote messenger RNA in
chromosomeless Escherichia coli minicells. Proc Natl Acad Sci USA,
1975. 72: 2900-2904; Hollenberg et al., Synthesis of high molecular
weight polypeptides in Escherichia coli minicells directed by
cloned Saccharomyces cerevisiae 2-micron DNA. Gene, 1976. 1: 33-47;
Crooks et al., Transcription of plasmid DNA in Escherichia coli
minicells. Plasmid, 1983. 10: 66-72; Clark-Curtiss, Analysis of
recombinant DNA using Escherichia coli minicells. Methods Enzymol,
1983. 101: 347-362).
[0444] Preferred expression vectors and constructs according to the
invention are episomal genetic elements. By "episomal" it is meant
that the expression construct is not always linked to a cell's
chromosome but may instead be retained or maintained in host cells
as a distinct molecule entity. Minicells can retain, maintain and
express episomal expression constructs such as, e.g., plasmids,
bacteriophage, viruses and the like (Crooks et al., Plasmin
10:66-72, 1983; Clark-Curtiss, Methods Enzymology 101:347-62, 1983;
Witkiewicz et al., Acta. Microbiol. Pol. A 7:21-24, 1975; Ponta et
al., Nature 269:440-2, 1977). By "retained" it is meant that the
episomal expression construct is at least temporarily present and
expressed in a host parent cell and/or minicell; by "maintained" it
is meant that the episomal expression construct is capable of
autonomous replication within a host parent cell and/or minicell.
In the context of episomal elements, the term "contained"
encompasses both "retained" and "maintained." A preferred type of
an episomal element according to the invention is one that is
always an extrachromocomal element, or which is part of a
chromosome but becomes an extrachromosomal element before or during
minicell production.
[0445] The fact that minicells do not contain chromosomal DNA but
do contain episomal expression elements, such as plasmids, that can
be used as templates for RNA synthesis means that the only proteins
that are actively produced in minicells are those that are encoded
by the expression elements that they contain. Minicell-producing E.
coli cells can be made competent and transformed with expression
elements that direct the expression of proteins encoded by the
expression elements. An expression element segregates into
minicells as they are produced. In isolated minicells that contain
expression elements, there is a single DNA template RNA for
transcription. Therefore, the only nucleic acids and proteins that
are actively produced (expressed) by minicells are those that are
encoded by sequences on the expression vector. In the context of
the invention, sequences that encode amino acid sequences are
designated "open reading frames" or "ORFs." One feature of minicell
expression systems of interest as regards the present invention is
that endogenous (i.e., chromosomally located) genes are not present
and are thus not expressed, whereas genes present on the episomal
element are expressed (preferably over-expressed) in the minicells.
As a result, the amount of endogenous proteins, including membrane
proteins, decreases as the minicells continue to express genes
located on episomal expression constructs.
[0446] The minicell system can reduce or eliminate undesirable
features associated with the transcription and translation of
endogenous proteins from the E. coli chromosome. For example,
expression of proteins in minicell systems results in low
background signal ("noise") when radiolabeled proteins produced
using recombinant DNA technology (Jannatipour et al., Translocation
of Vibrio Harveyi N,N'-DIacetylchitobiase to the outer membrane of
Escherichia coli. J. Bacteriol, 1987. 169: 3785-3791). A high
background signal can make it difficult to detect a protein of
interest. In whole cell E. coli systems, endogenous proteins
(encoded by the bacterial chromosome) are labeled as well as the
protein(s) encoded by the expression element; whereas, in minicell
systems, only the proteins encoded by the expression element in the
minicells are labeled.
[0447] There are a variety of proteins, both eubacterial and
eukaryotic, that have been expressed from plasmid DNA in minicells
(Clark-Curtiss, Methods Enzymal, 101:347-362, 1983). Some examples
of proteins and nucleic acids that have been expressed in minicells
include the Kdp-ATPase of E. coli (Altendorf et al., Structure and
function of the Kdp-ATPase of Escherichia coli. Acta Physiol Scand,
643: 137-146, 1998); penicillin binding proteins alpha and gamma
(Davies et al., Prediction of signal sequence-dependent protein
translocation in bacteria: Assessment of the Escherichia coli
minicell system. Biochem Biophys Res Commun, 150: 371-375, 1988);
cell surface antigens of Polyromaonas gingivalas (Rigg et al., The
molecular cloning, nucleotide sequence and expression of an
antigenic determinant from Porphyromonas gingivalis. Arch Oral
Biol, 45:41-52, 2000); trkG integral membrane protein of E. coli
(Schlosser et al., Subcloning, Nucleotide sequence, and expression
of trkG, a gene that encodes an integral membrane protein involved
in potassium uptake via the Trk system of Escherichia coli. J.
Bacteriol, 173:3170-3176, 1991); the 34 kDa antigen of Treponema
pallidum (Swancutt et al., Molecular characterization of the
pathogen-specific, 34-kilodalton membrane immunogen of Treponema
pallidum. Infect Immun, 57:3314-23, 1989); late proteins of
bacteriophage MB78 (Colla et al., IUBMB Life 48:493-497, 1999);
uncharacterized DNA from Xenopus laevis (Cohen and Boyer, U.S. Pat.
No. 4,237,224, which issued Dec. 2, 1980); the onc gene v-fos
(MacConnell and Verman, Expression of FBJ-MSV oncogene (fos)
product in bacteria, 131(2) Virology 367 1983); interferon (Edge et
al., Chemical synthesis of a human inteferon-alpha 2 gene and its
expression in Escherichia coli, Nucleic Acids Res. 11:6419, 1983);
bovine growth hormone (Rosner et al., Expression of a cloned bovine
growth hormone gene in Escherichia coli minicells, Can. J. Biochem.
60:521-4, 1982); gastrointestinal hormone (Suzuki et al.,
Production in Escherichia coli of biologically active secretin, a
gastroinstestinal hormone, Proc. Natl. Acad. Sci. USA 79:2475,
1982); and archeabacterial proteins (Lienard and Gottschalk,
Cloning, sequencing and expression of the genes encoding the sodium
translocating N-methyltetrahydromethanopterin: coenzyme M
methyltransferase of the methylotrohic archaeon Methanosarcina
mazei Gol, 425 FEBS Letters 204, 1998; and Lemker et al.,
Overproduction of a functional A1 ATPase from the archaeon
Methanosarcina mazei G1 in Escherichia coli, European Journal of
Biochemistry 268:3744, 2001).
[0448] V.B. Modulating Genetic Expression in Minicells
[0449] Gene expression in minicells, and/or in minicell-producing
(parent) cells, involves the coordinated activity of a variety of
expression factors, regulatory elements and expression sequences.
Any of these may be modified to alter the extent, timing or
regulation of expression of a gene of interest in minicells and/or
their parent cells. Often, the goal of the manipulations is to
increase the efficiency of protein production in minicells.
However, increased expression may, in some instances, desirably
include increased or "tight" negative regulation. This may reduce
or eliminate selective pressure created by toxic gene products, and
allow for functional expression in a controlled fashion by removing
the negative regulation and/or inducing expression of the gene
product at a preselected time. By way of non-limiting example,
these techniques may include modification or deletion of endogenous
gene(s) from which their respective gene product decreases the
induction and expression efficiency of a desired protein in the
parent cell prior to minicell formation and/or the segregated
minicell. By way of non-limiting example, these protein components
may be the enzymes that degrade chemical inducers of expression,
proteins that have a dominant negative affect upon a positive
regulatory elements, proteins that have proteolytic activity
against the protein to be expressed, proteins that have a negative
affect against a chaperone that is required for proper activity of
the expressed protein, and/or this protein may have a positive
effect upon a protein that either degrades or prevents the proper
function of the expressed protein. These gene products that require
deletion or modification for optimal protein expression and/or
function may also be antisense nucleic acids that have a negative
affect upon gene expression.
[0450] VI. Fusion(Chimeric) Proteins
[0451] In certain aspects of the invention, a fusion protein is
expressed and displayed by minicells. One class of fusion proteins
of particular interest are those that are displayed on the surface
of minicells, e.g., fusion proteins comprising one or more
transmembrane domains. Types of displayed fusion proteins of
particular interest are, by way of non-limiting example, those that
have an extracellular domain that is a binding moiety or an
enzymatic moiety. By way of non-limiting example, the fusion
protein ToxR-PhoA has been expressed in and displayed on the
surface of minicells. The ToxR-PhoA fusion protein comprises a
polypeptide corresponding to the normally soluble enzyme, alkaline
phosphatase, anchored to the minicell membrane by the single
transmembrane domain of ToxR (see the Examples). The fusion protein
retains the activity of the enzyme in the context of the minicell
membrane in which it is bound. Nearly all of the fusion protein is
oriented so that the enzyme's catalytic domain is displayed on the
outer surface of the minicell.
[0452] VI.A. Generation of Fusion Proteins
[0453] Polypeptides, which are polymers of amino acids, are encoded
by another class of molecules, known as nucleic acids, which are
polymers of structural units known as nucleotides. In particular,
proteins are encoded by nucleic acids known as DNA and RNA
(deoxyribonucleic acid and ribonucleic acid, respectively).
[0454] The nucleotide sequence of a nucleic acid contains the
"blueprints" for a protein. Nucleic acids are polymers of
nucleotides, four types of which are present in a given nucleic
acid. The nucleotides in DNA are adenine, cytosine and guanine and
thymine, (represented by A, C, G, and T respectively); in RNA,
thymine (T) is replaced by uracil (U). The structures of nucleic
acids are represented by the sequence of its nucleotides arranged
in a 5' ("5 prime") to 3' ("3 prime") direction, e.g.,
TABLE-US-00004 (SEQ ID NO. 361)
5'-A-T-G-C-C-T-A-A-A-G-C-C-G-C-T-C-C-C-T-C-A-3'
[0455] In biological systems, proteins are typically produced in
the following manner. A DNA molecule that has a nucleotide sequence
that encodes the amino acid sequence of a protein is used as a
template to guide the production of a messenger RNA (mRNA) that
also encodes the protein; this process is known as transcription.
In a subsequent process called translation, the mRNA is "read" and
directs the synthesis of a protein having a particular amino acid
sequence.
[0456] Each amino acid in a protein is encoded by a series of three
contiguous nucleotides, each of which is known as a codon. In the
"genetic code," some amino acids are encoded by several codons,
each codon having a different sequence; whereas other amino acids
are encoded by only one codon sequence. An entire protein (i.e., a
complete amino acid sequence) is encoded by a nucleic acid sequence
called a reading frame. A reading frame is a continuous nucleotide
sequence that encodes the amino acid sequence of a protein; the
boundaries of a reading frame are defined by its initiation (start)
and termination (stop) codons.
[0457] The process by which a protein is produced from a nucleic
acid can be diagrammed as follows:
##STR00001##
[0458] A chimeric reading frame encoding a fusion protein is
prepared as follows. A "chimeric reading frame" is a genetically
engineered reading frame that results from the fusion of two or
more normally distinct reading frames, or fragments thereof, each
of which normally encodes a separate polypeptide. Using recombinant
DNA techniques, a first reading frame that encodes a first amino
acid sequence is linked to a second reading frame that encodes a
second amino acid sequence in order to generate a chimeric reading
frame. Chimeric reading frames may also include nucleotide
sequences that encode optional fusion protein elements (see
below).
[0459] A hypothetical example of a chimeric reading frame created
from two normally separate reading frames is depicted in the
following flowchart.
First Open Reading Frame and "Protein-1":
##STR00002##
[0460] Second Open Reading Frame and "Protein-2":
##STR00003##
[0461] Chimeric Reading Frame that Encodes a Fusion Protein Having
Sequences from Protein-1 and Protein-2:
##STR00004##
[0462] In order for a chimeric reading frame to be functional, each
normally distinct reading frame therein must be fused to all of the
other normally distinct reading frames in a manner such that all of
the reading frames are in frame with each other. By "in frame with
each other" it is meant that, in a chimeric reading frame, a first
nucleic acid having a first reading frame is covalently linked to a
second nucleic acid having a second reading frame in such a manner
that the two reading frames are "read" (translated) in register
with each other. As a result, the chimeric reading frame encodes
one extended amino acid sequence that includes the amino acid
sequences encoded by each of the normally separate reading frames.
A fusion protein is thus encoded by a chimeric reading frame.
[0463] The fusion proteins of the invention are used to display
polypeptides on minicells. The fusion proteins comprise (1) at
least one polypeptide that is desired to be displayed by minicells
(a "displayed polypeptide") and (2) at least one membrane
polypeptide, e.g., a transmembrane or a membrane anchoring domain.
For various aspects of the invention, optional fusion protein
elements, as defined herein, may also be included if required or
desired.
[0464] VI.B. Optional Fusion Protein Elements
[0465] The fusion proteins of the invention may optionally comprise
one or more non-biologically active amino acid sequences, i.e.,
optional fusion protein elements. Such elements include, but are
not limited to, the following optional fusion protein elements. It
is understood that a chimeric reading frame will include nucleotide
sequences that encode such optional fusion protein elements, and
that these nucleotide sequences will be positioned so as to be in
frame with the reading frame encoding the fusion protein. Optional
fusion protein elements may be inserted between the displayed
polypeptide and the membrane polypeptide, upstream or downstream
(amino proximal and carboxy proximal, respectively) of these and
other elements, or within the displayed polypeptide and the
membrane polypeptide. A person skilled in the art will be able to
determine which optional element(s) should be included in a fusion
protein of the invention, and in what order, based on the desired
method of production or intended use of the fusion protein.
[0466] Detectable polypeptides are optional fusion protein elements
that either generate a detectable signal or are specifically
recognized by a detectably labeled agent. An example of the former
class of detectable polypeptide is green fluorescent protein (GFP).
Examples of the latter class include epitopes such as a "His tag"
(6 contiguous His residues, a.k.a. 6.times.His), the "FLAG tag" and
the c-myc epitope. These and other epitopes can be detected using
labeled antibodies that are specific for the epitope. Several such
antibodies are commercially available.
[0467] Attachment (support-binding) elements are optionally
included in fusion proteins and can be used to attach minicells
displaying a fusion protein to a preselected surface or support.
Examples of such elements include a "His tag," which binds to
surfaces that have been coated with nickel; streptavidin or avidin,
which bind to surfaces that have been coated with biotin or
"biotinylated" (see U.S. Pat. No. 4,839,293 and Airenne et al.,
Protein Expr. Purif. 17:139-145, 1999); and
glutathione-s-transferase (GST), which binds to surfaces coated
with glutathione (Kaplan et al., Protein Sci. 6:399-406, 1997; U.S.
Pat. No. 5,654,176). Polypeptides that bind to lead ions have also
been described (U.S. Pat. No. 6,111,079).
[0468] Spacers (a.k.a. linkers) are amino acid sequences that are
optionally included in a fusion protein in between other portions
of a fusion protein (e.g., between the membrane polypeptide and the
displayed polypeptide, or between an optional fusion protein
element and the remainder of the fusion protein). Spacers can be
included for a variety of reasons. For example, a spacer can
provide some physical separation between two parts of a protein
that might otherwise interfere with each other via, e.g., steric
hindrance. The ability to manipulate the distance between the
membrane polypeptide and the displayed polypeptide allows one to
extend the displayed polypeptide to various distances from the
surface of minicells.
[0469] VI.C. Interactions with Recipient Cells
[0470] Many Gram-negative pathogens use a type III secretion
machine to translocate protein toxins across the bacterial cell
envelope (for a review, see Cheng L W, Schneewind O. Type III
machines of Gram-negative bacteria: delivering the goods. Trends
Microbiol 2000 May; 8(5):214-20). For example, pathogenic Yersinia
spp. export over a dozen Yop proteins via a type III mechanism,
which recognizes secretion substrates by signals encoded in yop
mRNA or chaperones bound to unfolded Yop proteins. A 70-kb
virulence plasmid found in pathogenic Yersinia spp. to survive and
multiply in the lymphoid tissues of the host. The virulence plasmid
encodes the Yop virulon, an integrated system allowing
extracellular bacteria to inject bacterial proteins into cells. The
Yop virulon comprises a variety of Yop proteins and a dedicated
type III secretion apparatus, called Ysc (for a review, see
Cornelis G R, Boland A, Boyd A P, Geuijen C, Iriarte M, Neyt C,
Sory M P, Stainier I. The virulence plasmid of Yersinia, an
antihost genome. Microbiol. Mol Biol Rev 1998 62(4):1315-52).
[0471] VII. Minicell Display
[0472] Included in the design of the invention is the use of
minicells to express and display soluble or membrane-bound protein
libraries to identify a soluble or membrane-bound protein that
binds a known ligand or to identify proteins (e.g. orphan
receptors) for which the known ligand or substrate is not known but
for which a reporter could be engineered into the minicell that
would signal the presence of the encoded protein. In the preferred
embodiment of the invention, this `minicell display` technique is
analogous to phage display for the purpose of identifying genes
that encode receptor-like or antibody-like proteins against known
ligand. This approach will allow identification of an unknown
receptor protein for which a known ligand has affinity. These known
ligands may have been identified as having a pharmacological,
biological, or other effect without knowledge of the site of
effect. In these cases the knowledge of receptor will allow basic
research to understand the molecular and/or physiological response
and permit directed modification of the ligand for better
pharmacological or biological response or modification of the
receptor for employment in ligand-binding applications. In another
non-limiting embodiment of the invention, the ligand need not be
known but some general characteristic of the protein would be.
[0473] For purposes of this application, soluble or membrane-bound
protein libraries may be constructed by random cloning of DNA
fragments or directed cloning using reverse transcriptase
polymerase chain reaction (RT-PCR). In either method, DNA fragments
may be placed under the regulation of any regulatory element listed
in section II.B. on any plasmid or chromosomal construct. In the
case of soluble protein receptors, they will be fused to form a
chimeric protein with a known transmembrane domain (TMD), e.g. the
TMD from the toxR gene product. Upon induction of the soluble or
membrane-bound protein library, minicells, minicell protoplasts, or
minicell poroplasts (as the experiment requires) will be mixed with
the known ligand. Without being limited to the following example,
screening could be accomplished by first labeling the known ligand
with a molecular fluorophore, e.g. TAMRA, FTC, or in some cases a
fluorescent protein, e.g. GFP. A positive interaction between the
minicells displaying the receptor for the labeled ligand will be
identified and separated from the library population by
fluorescent-activated cell sorting (FACS). Isolated, positive
receptor-ligand interactions will be identified by PCR
amplification, subcloned into a clean background, and sequenced
using plasmid-specific oligonucleotides. Subcloned proteins will be
re-screened for interaction with the labeled ligand, and their
binding patterns characterized.
[0474] Positive interacting receptor proteins may be employed in
mutagenesis or other directed evolutionary process to improve or
decrease the binding affinity to the ligand. In another
application, the receptor-ligand pair may be further employed in a
screening process to identify new compounds that may interfere with
the interaction. Thus, using a known substance to identify the
receptor and the identified receptor-ligand pair to identify other
interfering compounds. Chimeric-soluble or membrane-bound protein
libraries may be screened versus a protein-array chip that presents
a variety of known protein compounds or peptide variations. In this
application, the minicell, minicell protoplast, or minicell
poroplast will also contain a label, signaling component, and/or
antigen recognizable by an antibody for identification of a
positive interaction on the protein chip array. Other approaches
for identification may include packaged fluorescent molecules or
proteins that are constitutively produced, induced by the positive
interaction with the ligand, or regulated by a regulatory element
described in section II.B.
[0475] In a preferred embodiment of the invention, cDNA libraries
could be constructed from isolated B-cells, activated B-cell or
T-cells for the purpose of identifying receptors or antibodies that
are encoded by these cells of the immune system. In a non-limiting
example, a small molecule could be used to immunize an experimental
animal (e.g., rat, mouse, rabbit), the spleen could be removed, or
blood could be drawn and used as a source of mRNA. Reverse
transcription reactions could then be used to construct a cDNA
library that would eventually be transformed into the minicell
parent bacteria, as described above. The minicells would then be
isolated, induced and subjected to FACS analysis with subsequent
amplification and sequencing of the cDNA fragment of interest (see
above). The PCR-amplified plasmid-containing cDNA fragment encoding
the "receptor" or "antibody" of interest would be ready for
transformation and expression in the minicell context for
diagnostic, therapeutic research or screening applications of the
invention.
[0476] In a related, non-limiting embodiment of the invention,
minicells expressing a particular antigen (e.g., protein,
carbohydrate, small molecule, lipid) on their surfaces (described
elsewhere in this application) are used to generate an immunogenic
response. The advantages of presenting an antigen on the surfaces
of minicells are that the minicells themselves may be an adjuvant
that stimulates the immune response, particularly if administered
subcutaneously (SC) or intramuscularly (IM). Moreover, the
minicells are not readily eliminated by the renal system and are
present in the circulatory system of an immunized animal for a
longer time. In addition, small molecules could be tethered to the
minicell in a way that presents the desired moiety of the molecule.
Animals are presented with minicell-based immunogens, and the
antibodies produced in the animals are prepared and used in
therapeutic, diagnostic, research and screening applications.
Although this aspect of the invention may be used to make
antibodies to any molecule displayed on their surface, the
extracellular domains of membrane proteins are of particular
interest.
[0477] Minicell display could be used to identify orphan receptors
or other proteins for which a ligand or substrate is not known. As
a non-limiting example, orphan G protein coupled receptors (GPCRs)
or novel RNA and DNA polymerases could be identified from organisms
living in extreme environments. A cDNA library could be is
constructed from an organism and expressed in minicells that
co-express a reporter system that indicates the presence of the
novel protein. In a non-limiting example of GPCRs, the minicells
used for minicell display are engineered to express a G-protein in
a manner that would signal an interaction with the orphan GPCR.
[0478] VIII. Aptamers
[0479] Traditionally, techniques for detecting and purifying target
molecules have used polypeptides, such as antibodies, that
specifically bind such targets. While nucleic acids have long been
known to specifically bind other nucleic acids (e.g., ones having
complementary sequences), aptamers (i.e., nucleic acids that bind
non-nucleic target molecules) have been disclosed. See, e.g.,
Blackwell et al., Science (1990) 250:1104-1110; Blackwell et al.,
Science 250:1149-1152; Tuerk et al., Science (1990) 249:505-510;
Joyce, Gene (1989) 82:83-87; and U.S. Pat. No. 5,840,867 entitled
"Aptamer analogs specific for biomolecules".
[0480] As applied to aptamers, the term "binding" specifically
excludes the "Watson-Crick"-type binding interactions (i.e., A:T
and G:C base-pairing) traditionally associated with the DNA double
helix. The term "aptamer" thus refers to a nucleic acid or a
nucleic acid derivative that specifically binds to a target
molecule, wherein the target molecule is either (i) not a nucleic
acid, or (ii) a nucleic acid or structural element thereof that is
bound through mechanisms other than duplex- or triplex-type base
pairing. Such a molecule is called a "non-nucleic molecule"
herein.
[0481] VIII.A. Structures of Nucleic Acids
[0482] "Nucleic acids," as used herein, refers to nucleic acids
that are isolated a natural source; prepared in vitro, using
techniques such as PCR amplification or chemical synthesis;
prepared in vivo, e.g., via recombinant DNA technology; or by any
appropriate method. Nucleic acids may be of any shape (linear,
circular, etc.) or topology (single-stranded, double-stranded,
supercoiled, etc.). The term "nucleic acids" also includes without
limitation nucleic acid derivatives such as peptide nucleic acids
(PNA's) and polypeptide-nucleic acid conjugates; nucleic acids
having at least one chemically modified sugar residue, backbone,
internucleotide linkage, base, nucleoside, or nucleotide analog; as
well as nucleic acids having chemically modified 5' or 3' ends; and
nucleic acids having two or more of such modifications. Not all
linkages in a nucleic acid need to be identical.
[0483] Nucleic acids that are aptamers are often, but need not be,
prepared as oligonucleotides. Oligonucleotides include without
limitation RNA, DNA and mixed RNA-DNA molecules having sequences of
lengths that have minimum lengths of 2, 4, 6, 8, 10, 11, 12, 13,
14, 15, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides, and
maximum lengths of about 100, 75, 50, 40, 25, or 15 or more
nucleotides, irrespectively. In general, a minimum of 6
nucleotides, preferably 10 nucleotides, more preferably 14 to 20
nucleotides, is necessary to effect specific binding.
[0484] In general, the oligonucleotides may be single-stranded (ss)
or double-stranded (ds) DNA or RNA, or conjugates (e.g., RNA
molecules having 5' and 3' DNA "clamps") or hybrids (e.g., RNA:DNA
paired molecules), or derivatives (chemically modified forms
thereof). However, single-stranded DNA is preferred, as DNA is
often less labile than RNA. Similarly, chemical modifications that
enhance an aptamer's specificity or stability are preferred.
[0485] VIII.B.Chemical Modifications of Nucleic Acids
[0486] Chemical modifications that may be incorporated into
aptamers and other nucleic acids include, with neither limitation
nor exclusivity, base modifications, sugar modifications, and
backbone modifications.
[0487] Base modifications: The base residues in aptamers may be
other than naturally occurring bases (e.g., A, G, C, T, U, 5MC, and
the like). Derivatives of purines and pyrimidines are known in the
art; an exemplary but not exhaustive list includes
aziridinylcytosine, 4-acetylcytosine, 5-fluorouracil,
5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethylaminomethyluracil, inosine, N6-isopentenyladenine,
1-methyladenine, 1-methylpseudouracil, 1-methylguanine,
1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,
2-methylguanine, 3-methylcytosine, 5-methylcytosine (5MC),
N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil,
5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,
5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine,
uracil-5-oxyacetic acid methylester, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid, and 2,6-diaminopurine. In
addition to nucleic acids that incorporate one or more of such base
derivatives, nucleic acids having nucleotide residues that are
devoid of a purine or a pyrimidine base may also be included in
aptamers.
[0488] Sugar modifications: The sugar residues in aptamers may be
other than conventional ribose and deoxyribose residues. By way of
non-limiting example, substitution at the 2'-position of the
furanose residue enhances nuclease stability. An exemplary, but not
exhaustive list, of modified sugar residues includes 2' substituted
sugars such as 2'-O-methyl-, 2'-O-alkyl, 2'-O -allyl, 2'-S-alkyl,
2'-S-allyl, 2'-fluoro-, 2'-halo, or 2'-azido-ribose, carbocyclic
sugar analogs, alpha-anomeric sugars, epimeric sugars such as
arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,
sedoheptuloses, acyclic analogs and abasic nucleoside analogs such
as methyl riboside, ethyl riboside or propylriboside.
[0489] Backbone modifications: Chemically modified backbones
include, by way of non-limiting example, phosphorothioates, chiral
phosphorothioates, phosphorodithioates, phosphotriesters,
aminoalkylphosphotriesters, methyl and other alkyl phosphonates
including 3'-alkylene phosphonates and chiral phosphonates,
phosphinates, phosphoramidates including 3'-amino phosphoramidate
and aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, and
boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs
of these, and those having inverted polarity wherein the adjacent
pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to
5'-2'. Chemically modified backbones that do not contain a
phosphorus atom have backbones that are formed by short chain alkyl
or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl
or cycloalkyl internucleoside linkages, or one or more short chain
heteroatomic or heterocyclic internucleoside linkages, including
without limitation morpholino linkages; siloxane backbones;
sulfide, sulfoxide and sulfone backbones; formacetyl and
thioformacetyl backbones; methylene formacetyl and thioformacetyl
backbones; alkene containing backbones; sulfamate backbones;
methyleneimino and methylenehydrazino backbones; sulfonate and
sulfonamide backbones; and amide backbones.
[0490] VIII.C. Preparation and Identification of Aptamers
[0491] In general, techniques for identifying aptamers involve
incubating a preselected non-nucleic target molecule with mixtures
(2 to 50 members), pools (50 to 5,000 members) or libraries (50 or
more members) of different nucleic acids that are potential
aptamers under conditions that allow complexes of target molecules
and aptamers to form. By "different nucleic acids" it is meant that
the nucleotide sequence of each potential aptamer may be different
from that of any other member, that is, the sequences of the
potential aptamers are random with respect to each other.
Randomness can be introduced in a variety of manners such as, e.g.,
mutagenesis, which can be carried out in vivo by exposing cells
harboring a nucleic acid with mutagenic agents, in vitro by
chemical treatment of a nucleic acid, or in vitro by biochemical
replication (e.g., PCR) that is deliberately allowed to proceed
under conditions that reduce fidelity of replication process;
randomized chemical synthesis, i.e., by synthesizing a plurality of
nucleic acids having a preselected sequence that, with regards to
at least one position in the sequence, is random. By "random at a
position in a preselected sequence" it is meant that a position in
a sequence that is normally synthesized as, e.g., as close to 100%
A as possible (e.g., 5'-C-T-T-A-G-T-3') is allowed to be randomly
synthesized at that position (C-T-T-N-G-T, wherein N indicates a
randomized position where, for example, the synthesizing reaction
contains 25% each of A,T,C and G; or x % A, w % T, y % C and z % G,
wherein x+w+y+z=100. In later stages of the process, the sequences
are increasingly less randomized and consensus sequences may
appear; in any event, it is preferred to ultimately obtain an
aptamer having a unique nucleotide sequence.
[0492] Aptamers and pools of aptamers are prepared, identified,
characterized and/or purified by any appropriate technique,
including those utilizing in vitro synthesis, recombinant DNA
techniques, PCR amplification, and the like. After their formation,
target:aptamer complexes are then separated from the uncomplexed
members of the nucleic acid mixture, and the nucleic acids that can
be prepared from the complexes are candidate aptamers (at early
stages of the technique, the aptamers generally being a population
of a multiplicity of nucleotide sequences having varying degrees of
specificity for the target). The resulting aptamer (mixture or
pool) is then substituted for the starting apatamer (library or
pool) in repeated iterations of this series of steps. When a
limited number (e.g., a pool or mixture, preferably a mixture with
less than 100 members, more preferably less than 10 members, most
preferably 1, of nucleic acids having satisfactory specificity is
obtained, the aptamer is sequenced and characterized. Pure
preparations of a given aptamer are generated by any appropriate
technique (e.g., PCR amplification, in vitro chemical synthesis,
and the like).
[0493] For example, Tuerk and Gold (Science (1990) 249:505-510)
disclose the use of a procedure termed "systematic evolution of
ligands by exponential enrichment" (SELEX). In this method, pools
of nucleic acid molecules that are randomized at specific positions
are subjected to selection for binding to a nucleic acid-binding
protein (see, e.g., PCT International Publication No. WO 91/19813
and U.S. Pat. No. 5,270,163). The oligonucleotides so obtained are
sequenced and otherwise characterization. Kinzler, K. W., et al.
(Nucleic Acids Res. (1989) 17:3645-3653) used a similar technique
to identify synthetic double-stranded DNA molecules that are
specifically bound by DNA-binding polypeptides. Ellington, A. D.,
et al. (Nature (1990) 346: 818-822) disclose the production of a
large number of random sequence RNA molecules and the selection and
identification of those that bind specifically to specific dyes
such as Cibacron blue.
[0494] Another technique for identifying nucleic acids that bind
non-nucleic target molecules is the oligonucleotide combinatorial
technique disclosed by Ecker, D. J. et al. (Nuc. Acids Res. 21,
1853 (1993)) known as "synthetic unrandomization of randomized
fragments" (SURF), which is based on repetitive synthesis and
screening of increasingly simplified sets of oligonucleotide
analogue libraries, pools and mixtures (Tuerk et al., Science
249:505, 1990). The starting library consists of oligonucleotide
analogues of defined length with one position in each pool
containing a known analogue and the remaining positions containing
equimolar mixtures of all other analogues. With each round of
synthesis and selection, the identity of at least one position of
the oligomer is determined until the sequences of optimized nucleic
acid ligand aptamers are discovered.
[0495] Once a particular candidate aptamer has been identified
through a SURF, SELEX or any other technique, its nucleotide
sequence can be determined (as is known in the art), and its
three-dimensional molecular structure can be examined by nuclear
magnetic resonance (NMR). These techniques are explained in
relation to the determination of the three-dimensional structure of
a nucleic acid ligand that binds thrombin in Padmanabhan et al., J.
Biol. Chem. 24, 17651 (1993); Wang et al., Biochemistry 32, 1899
(1993); and Macaya et al., Proc. Nat'l. Acad. Sci. USA 90, 3745
(1993). Selected aptamers may be resynthesized using one or more
modified bases, sugars or backbone linkages. Aptamers consist
essentially of the minimum sequence of nucleic acid needed to
confer binding specificity, but may be extended on the 5' end, the
3' end, or both, or may be otherwise derivatized or conjugated.
[0496] IX. Polypeptidic Binding Moieties
[0497] A variety of binding moities can be attached to a minicell
of the invention for a variety of purposes. In a preferred
embodiment, the binding moiety is directed to a ligand that is
displayed by a cell into which it is desired to deliver the
therapeutic content of a minicell.
[0498] IX.A. Antibodies and Antibody Derivatives
[0499] The term "antibody" is meant to encompass an immunoglobulin
molecule obtained by in vitro or in vivo generation of an
immunogenic response, and includes polyclonal, monospecific and
monoclonal antibodies, as well as antibody derivatives, e.g.
single-chain antibody fragments (scFv). An "immunogenic response"
is one that results in the production of antibodies directed to one
or more proteins after the appropriate cells have been contacted
with such proteins, or polypeptide derivatives thereof, in a manner
such that one or more portions of the protein function as epitopes.
An epitope is a single antigenic determinant in a molecule. In
proteins, particularly denatured proteins, an epitope is typically
defined and represented by a contiguous amino acid sequence.
However, in the case of nondenatured proteins, epitopes also
include structures, such as active sites, that are formed by the
three-dimensional folding of a protein in a manner such that amino
acids from separate portions of the amino acid sequence of the
protein are brought into close physical contact with each
other.
[0500] Wildtype antibodies have four polypeptide chains, two
identical heavy chains and two identical light chains. Both types
of polypeptide chains have constant regions, which do not vary or
vary minimally among antibodies of the same class (i.e., IgA, IgM,
etc.), and variable regions. Variable regions are unique to a
particular antibody and comprise an "antigen binding domain" that
recognizes a specific epitope. Thus, an antibody's specificity is
determined by the variable regions located in the amino terminal
regions of the light and heavy chains.
[0501] As used herein, the term "antibody" encompasses derivatives
of antibodies such as antibody fragments that retain the ability to
specifically bind to antigens. Such antibody fragments include Fab
fragments (i.e., an antibody fragment that contains the
antigen-binding domain and comprises a light chain and part of a
heavy chain bridged by a disulfide bond); Fab' (an antibody
fragment containing a single anti-binding domain comprising an Fab
and an additional portion of the heavy chain through the hinge
region); F(ab')2 (two Fab' molecules joined by interchain disulfide
bonds in the hinge regions of the heavy chains; the Fab' molecules
may be directed toward the same or different epitopes); a
bispecific Fab (an Fab molecule having two antigen binding domains,
each of which may be directed to a different epitope); a single
chain Fab chain comprising a variable region, a.k.a., a sFv (the
variable, antigen-binding determinative region of a single light
and heavy chain of an antibody linked together by a chain of about
10 to about 25 amino acids).
[0502] The term "antibody" includes antibodies and antibody
derivatives that are produced by recombinant DNA techniques and
"humanized" antibodies. Humanized antibodies have been modified, by
genetic manipulation and/or in vitro treatment to be more human, in
terms of amino acid sequence, glycosylation pattern, etc., in order
to reduce the antigenicity of the antibody or antibody fragment in
an animal to which the antibody is intended to be administered (Gus
sow et al., Methods Enz. 203:99-121, 1991).
[0503] A single-chain antibody (scFv) is a non-limiting example of
a binding moiety that may be displayed on minicells. Single-chain
antibodies are produced by recombinant DNA technology and may be
incorporated into fusion proteins. The term "single chain" denotes
the fact that scFv's are found in a single polypeptide. In
contrast, wildtype antibodies have four polypeptide chains, two
identical heavy chains and two identical light chains. Both types
of polypeptide chains have constant regions, which do not vary or
vary minimally among antibodies of the same class (i.e., IgA, IgM,
etc.), and variable regions. An antibody's specificity is
determined by the variable regions located in the amino terminal
regions of the light and heavy chains. The variable regions of a
light chain and associated heavy chain form an "antigen binding
domain" that recognizes a specific epitope. In a single chain
antibody, the amino acid sequences of the variable light and
variable heavy regions of an antibody are present in one comtiguous
polypeptide. Methods of producing single chain antibodies are known
in the art. See, for example, U.S. Pat. Nos. 4,946,778; 5,260,203;
5,455,030; 5,518,889; 5,534,621; 5,869,620; 6,025,165; 6,027,725
and 6,121,424.
[0504] Antibody derivatives and other polypeptides that are binding
moieties can be isolated from protein display libraries, in which a
library of candidate binding agents is displayed on a phage or
other agent that comprises a nucleic acid encoding the protein it
displays. Thus, an agent that binds to the target compound can be
isolated, and nucleic acid prepared therefrom, providing for the
rapid isolation of binding moieties and nucleic acids that can be
used to produce them. For reviews, see Benhar I. Biotechnological
applications of phage and cell display. Biotechnology Adv. 2001
(19):1-33; FitzGerald K. In vitro display technologies--new tools
for drug discovery. Drug Discov Today. 2000 5(6):253-258; and
Hoogenboom HR, Chames P. Natural and designer binding sites made by
phage display technology. Immunol Today. 2000 August;
21(8):371-8.
[0505] A variety of protein display systems are known in the art
and include various phage display systems such as those described
in Jung S, Arndt K, Muller K, Pluckthyn A. Selectively infective
phage (SIP) technology: scope and limitations. J Immunol Methods.
1999 (231):93-104; Katz B. Structural and mechanistic determinants
of affinity and specificity of ligands discovered or engineered by
phage display. Annu Rev Biophys Biomol Struct. 1997 (26):27-45;
Forrer P, Jung S, Pluckthun A. Beyond binding: using phage display
to select for structure, folding and enzymatic activity in
proteins. Curr Opin Struct Biol. 1999 August; 9(4):514-20; Rondot
S, Koch J, Breitling F, Dubel S. A helper phage to improve
single-chain antibody presentation in phage display. Nat.
Biotechnol. 2001 January; 19(1):75-8. Giebel L B, Cass R T,
Milligan D L, Young D C, Arze R, Johnson C R. Screening of cyclic
peptide phage libraries identifies ligands that bind streptavidin
with high affinities. Biochemistry. 1995 Nov. 28; 34(47):15430-5;
de Kruif J, Logtenberg T. Leucine zipper dimerized bivalent and
bispecific scFv antibodies from a semi-synthetic antibody phage
display library. J Biol. Chem. 1996 Mar. 29; 271(13):7630-4;
Hoogenboom H R, Henderikx P, de Haard H. Creating and engineering
human antibodies for immunotherapy. Adv Drug Deliv Rev. 1998 Apr.
6; 31(1-2):5-31; Helfrich W, Haisma HJ, Magdolen V, Luther T, Bom
VJ, Westra J, van der Hoeven R, Kroesen BJ, Molema G, de Leij L. A
rapid and versatile method for harnessing scFv antibody fragments
with various biological effector functions. J Immunol Methods. 2000
Apr. 3; 237(1-2):131-45; Hoess R H. Bacteriophage lambda as a
vehicle for peptide and protein display. Curr Pharm Biotechnol 2002
March; 3(1):23-8; Baek H, Suk K H, Kim YH, Cha S. An improved
helper phage system for efficient isolation of specific antibody
molecules in phage display. Nucleic Acids Res. 2002 Mar. 1;
30(5):e18; and Rondot S, Koch J, Breitling F, Dubel S. A helper
phage to improve single-chain antibody presentation in phage
display. Nat. Biotechnol. 2001 January; 19(1):75-8.
[0506] Other display systems include without limitation "Yeast
Display" (Curr Opin Biotechnol 1999 October; 10(5):422-7.
Applications of yeast in biotechnology: protein production and
genetic analysis. Cereghino G P, Cregg J M.); "Baculovirus Display"
(Kost T A, Condreay J P. Recombinant baculoviruses as expression
vectors for insect and mammalian cells. Curr Opin Biotechnol. 1999
October; 10(5):428-33; and Liang M, Dubel S, Li D, Queitsch I, Li
W, Bautz E K. Baculovirus expression cassette vectors for rapid
production of complete human IgG from phage display selected
antibody fragments. J Immunol Methods. 2001 Jan. 1;
247(1-2):119-30); "Ribosome Display" (Hanes J, Schaffitzel C,
Knappik A, Pluckthun A. Picomolar affinity antibodies from a fully
synthetic naive library selected and evolved by ribosome display.
Nat. Biotechnol. 2000 December; 18(12):1287-92; Hanes J, Jermutus
L, Pluckthun A. Selecting and evolving functional proteins in vitro
by ribosome display. Methods Enzymol. 2000; 328:404-30; Schaffitzel
C, Hanes J, Jermutus L, Pluckthun A. Ribosome display: an in vitro
method for selection and evolution of antibodies from libraries. J
Immunol Methods. 1999 Dec. 10; 231(1-2):119-35; Hanes J, Jermutus
L, Weber-Bornhauser S, Bosshard H R, Pluckthun A. Ribosome display
efficiently selects and evolves high-affinity antibodies in vitro
from immune libraries. Proc Natl Acad Sci USA. 1998 Nov. 24;
95(24):14130-5; Hanes J, Pluckthun A. In vitro selection and
evolution of functional proteins by using ribosome display. Proc
Natl Acad Sci USA. 1997 May 13; 94(10):4937-42; Coia G, Pontes-Braz
L, Nuttall S D, Hudson P J, Irving R A Panning and selection of
proteins using ribosome display. J Immunol Methods. 2001 Aug. 1;
254(1-2):191-7; Irving R A, Coia G, Roberts A, Nuttall S D, Hudson
P J. Ribosome display and affinity maturation: from antibodies to
single V-domains and steps towards cancer therapeutics. J Immunol
Methods. 2001 Feb. 1; 248(1-2):31-45); and "Bacterial Display"
(Hoischen C, Fritsche C, Gumpert J, Westermann M, Gura K, Fahnert
B. Novel bacterial membrane surface display system using cell
wall-less L-forms of Proteus mirabilis and Escherichia coli. Appl
Environ Microbiol. 2002 February; 68(2):525-31; Etz H, Minh D B,
Schellack C, Nagy E, Meinke A. Bacterial phage receptors, versatile
tools for display of polypeptides on the cell surface. J.
Bacteriol. 2001 December; 183(23):6924-35; Patel D, Vitovski S,
Senior HJ, Edge M D, Hockney R C, Dempsey M J, Sayers J R.
Continuous affinity-based selection: rapid screening and
simultaneous amplification of bacterial surface-display libraries.
Biochem J. 2001 Aug. 1; 357(Pt 3):779-85; Lang H. Outer membrane
proteins as surface display systems. Int J Med. Microbiol. 2000
December; 290(7):579-85; Earhart CF. Use of an Lpp-OmpA fusion
vehicle for bacterial surface display. Methods Enzymol. 2000;
326:506-16; Benhar I, Azriel R, Nahary L, Shaky S, Berdichevsky Y,
Tamarkin A, Wels W. Highly efficient selection of phage antibodies
mediated by display of antigen as Lpp-OmpA' fusions on live
bacteria. J Mol. Biol. 2000 Aug. 25; 301(4):893-904; Xu Z, Lee SY.
Display of polyhistidine peptides on the Escherichia coli cell
surface by using outer membrane protein C as an anchoring motif.
Appl Environ Microbiol. 1999 November; 65(11):5142-7; Daugherty P
S, Olsen M J, Iverson B L, Georgiou G. Development of an optimized
expression system for the screening of antibody libraries displayed
on the Escherichia coli surface. Protein Eng. 1999 July;
12(7):613-21; Chang HJ, Sheu S Y, Lo S J. Expression of foreign
antigens on the surface of Escherichia coli by fusion to the outer
membrane protein traT. J Biomed Sci. 1999 January; 6(1):64-70;
Maurer J, Jose J, Meyer T F. Autodisplay: one-component system for
efficient surface display and release of soluble recombinant
proteins from Escherichia coli. J. Bacteriol. 1997 February;
179(3):794-804.
[0507] Antibodies, particularly single-chain antibodies, directed
to surface antigens specific for a particular cell type may also be
used as cell- or tissue-specific targeting elements. Single-chain
antibody amino acid sequences have been incorporated into a variety
of fusion proteins, including those with transmembrane domains
and/or membrane-anchoring domains. See, for example, Kuroki et al.,
"Specific Targeting Strategies of Cancer Gene Therapy Using a
Single-Chain Variable Fragment (scFv) with a High Affinity for
CEA," Anticancer Res., pp. 4067-71, 2000; U.S. Pat. No. 6,146,885,
to Dornburg, entitled "Cell-Type Specific Gene Transfer Using
Retroviral Vectors Containing Antibody-Envelope Fusion Proteins";
Jiang et al., "In Vivo Cell Type-Specific Gene Delivery With
Retroviral Vectors That Display Single Chain Antibodies," Gene
Ther. 1999, 6:1982-7; Engelstadter et al., "Targeting Human T Cells
By Retroviral Vectors Displaying Antibody Domains Selected From A
Phage Display Library," Hum. Gene Ther. 2000, 11:293-303; Jiang et
al., "Cell-Type-Specific Gene Transfer Into Human Cells With
Retroviral Vectors That Display Single-Chain Antibodies," J. Virol
1998,72:10148-56; Chu et al., "Toward Highly Efficient
Cell-Type-Specific Gene Transfer With Retroviral Vectors Displaying
Single-Chain Antibodies," J. Virol 1997, 71:720-5; Chu et al.,
"Retroviral Vector Particles Displaying The Antigen-Binding Site Of
An Antibody Enable Cell-Type-Specific Gene Transfer," J. Virol
1995, 69:2659-63; Chu et al., "Cell Targeting With Retroviral
Vector Particles Containing Antibody-Envelope Fusion Proteins,"
Gene Ther. 1994, 1:292-9; Esshar et al., "Specific activation and
targeting of cytotoxic lymphocytes through chimeric single chains
consisting of antibody-binding domains and the .quadrature. or
.quadrature. subunits of the immunoglobulin and T-cell receptors,"
Proc. Natl. Acad. Sci. USA, 1993, Vol. 90:720-724; Einfeld et al.,
"Construction of a Pseudoreceptor That Mediates Transduction by
Adenoviruses Expressing a Ligand in Fiber or Penton Base," J.
Virol. 1999, 73:9130-9136; Marin et al., "Targeted Infection of
Human Cells via Major Histocompatibility Complex Class I Molecules
by Moloney Murine Leukemia Virus-Derived Viruses Displaying
Single-Chain Antibody Fragment-Envelope Fusion Proteins," J.
Virol., 1996, 70:2957-2962; Somia et al., "Generation of targeted
retroviral vectors by using single-chain variable fragment: An
approach to in vivo gene delivery," Proc. Natl. Acad. Sci. USA,
1995, 92:7570-7574; Liu et al., "Treatment of B-Cell Lymphoma With
Chimeric IgG and Single-Chain Fv Antibody-Interleukin-2 Fusion
Proteins," Blood, 1998, 92:2103-2112; Martin et al., "Retrovirus
Targeting by Tropism Restriction to Melanoma Cells," J. Virol.,
1999, 73:6923-6929; Ramjiawan et al., "Noninvasive Localization of
Tumors by Immunofluorescence Imaging Using a Single Chain Fv
Fragment of a Human Monoclonal Antibody with Broad Cancer
Specificity," Amer. Cancer Society, 2000, 89:1134-1144; Snitkovsky
et al., "A TVA-Single-Chain Antibody Fusion Protein Mediates
Specific Targeting of a Subgroup A Avian Leukosis Virus Vector to
Cells Expressing a Tumor-Specific Form of Epidermal Growth Factor
Receptor," J. Virol., 2000, 74:9540-9545; Chu et al., "Toward
Highly Efficient Cell-Type-Specific Gene Transfer with Retroviral
Vectors Displaying Single-Chain Antibodies," J. Virol., 1997,
71:720-725; Kulkarni et al., Programmed cell death signaling via
cell-surface expression of a single-chain antibody transgene,
Transplantation 2000 Mar. 27; 69(6):1209-17.
[0508] IX.B. Non-Catalytic Derivatives of Active Sites of
Enzymes
[0509] Enzymes bind their substrates, at least transiently, in
regions known as "active sites." It is known in the art that
non-catalytic derivatives of enzymes, which bind but do not
chemically alter their substrates may be prepared. Non-catalytic
enzymes, particularly the mutant active sites thereof, are used to
bind substrate molecules.
[0510] As a non-limiting example, enzymes from which biologically
inactive (non-catalytic) sphingolipid-binding derivatives are
obtained. Such derivatives of these enzymes bind their substrate
sphingolipid. Sphingosine-1-phosphate (S1P) is bound by
non-catalytic derivatives of enzymes having S1P as a substrate,
e.g., S1P lyase and S1P phosphatase. Sphingosine (SPH) is bound by
non-catalytic derivatives of enzymes having SPH as a substrate,
e.g., SPH kinase and ceramide synthase. Ceramide (CER) is bound by
non-catalytic derivatives of enzymes having CER as a substrate,
such as, by way of non-limiting example, ceramidase, sphingomyelin
synthase, ceramide kinase, and glucosylceramide synthase.
Sphingomyelin is bound by non-catalytic derivatives of
sphingomyelinase, an enzyme having sphingomyelin as a
substrate.
[0511] IX.C. Nucleic Acid Binding Domains
[0512] Nucleic acid binding polypeptide domains may bind nucleic
acids in a sequence-dependent or sequence-independent fashion
and/or in a manner that is specific for various nucleic acids
having different chemical structures (e.g., single- or
double-stranded DNA or RNA, RNA:DNA hybrid molecules, etc.).
Non-limiting examples of membrane-based transcription factors and
DNA-binding protein include Smad proteins (Miyazono et al.,
TGF-beta signaling by Smad proteins (Review), Adv Immunol
75:115-57, 2000); SREBPs (sterol regulatory element binding
proteins) (Ye et al., Asparagine-proline sequence within
membrane-spanning segment of SREBP triggers intramembrane cleavage
by site-2 protease, Proc Natl Acad Sci USA 97:5123-8, 2000;
Shimomura et al., Cholesterol feeding reduces nuclear forms of
sterol regulatory element binding proteins in hamster liver, Proc
Natl Acad Sci USA 94:12354-9, 1997; Brown and Goldstein, The SREBP
pathway: regulation of cholesterol metabolism by proteolysis of a
membrane-bound transcription factor (Review), Cell 89:331-40, 1997;
Scheek et al., Sphingomyelin depletion in cultured cells blocks
proteolysis of sterol regulatory element binding proteins at site
1, Proc Natl Acad Sci USA 94:11179-83, 1997); mitochondrial
DNA-binding membrane proteins, e.g., Abf2p and YhmZp (Cho et al., A
novel DNA-binding protein bound to the mitochondrial inner membrane
restores the null mutation of mitochondrial histone Abf2p in
Saccharomyces cerevisiae, Mol Cell Biol 18:5712-23, 1998); and
bacterial DNA-binding membrane proteins (Smith et al.,
Transformation in Bacillus subtilis: purification and partial
characterization of a membrane-bound DNA-binding protein., J
Bacteriol 156:101-8, 1983).
[0513] IX.D. Attaching Binding Moities, or Other Compounds, to
Minicells
[0514] Binding compounds or moieties can be chemically attached
(conjugated) to minicells via membrane proteins that are displayed
on the minicells. The compound to be conjugated to minicells (the
"attachable compound") may of any chemical composition, i.e., a
small molecule, a nucleic acid, a radioisotope, a lipid or a
polypeptide. One type of attachable compound that can be covalently
attached to minicells is a binding moiety, e.g., an antibody or
antibody derivative. Another non-limiting example of attachable
compounds is polyethylene glycol ("PEG"), which lowers the uptake
in vivo of minicells by the reticuloendothelial system (RES).
Another non-limiting example of creating stealth minicells to avoid
the RES is to express proteins or other molecules on the surfaces
of minicells whose lipid compositions have been modified, such as
anionic lipid-rich minicells.
[0515] By way of non-limiting example, it is possible to prepare
minicells that express transmembrane proteins with cysteine
moieties on extracellular domains. Linkage of the membrane protein
may be achieved through surface cysteinyl groups by, e.g.,
reduction with cysteinyl residues on other compounds to form
disulfide bridges (S.dbd.S). If appropriate cysteinyl residues are
not present on the membrane protein they may be introduced by
genetic manipulation. The substitution of cysteine for another
amino acid may be achieved by methods well-known to those skilled
in the art, for example, by using methods described in Maniatis,
Sambrook, and Fritsch (Molecular Cloning: A Laboratory Manual, Cold
Spring Harbor Laboratory Press, 1989). As a non-limiting example,
bioactive lysosphingolipids (e.g., sphingosine,
sphingosine-1-phosphate, sphingosylphosphoryl choline) are
covalently linked to proteins expressed on the surfaces of
minicells such that these bioactive lipids are on the surface of
the minicells and accessible for therapeutic or diagnostic uses in
vivo or in vitro.
[0516] When the attachable moiety and the membrane protein both
have a reduced sulfhydryl group, a homobifunctional cross-linker
that contains maleimide, pyridyl disulfide, or
beta-alpha-haloacetyl groups may be used for cross-linking.
Examples of such cross-linking reagents include, but are not
limited to, bismaleimidohexane (BMH) or
1,4-Di-[3'-(2'-pyridyldithio)propionamido]butane (DPDPB).
Alternatively, a heterobifunctional cross-linker that contains a
combination of maleimide, pyridyl disulfide, or
beta-alpha-haloacetyl groups can be used for cross-linking.
[0517] As another non-limiting example, attachable moieties may be
chemically conjugated using primary amines. In these instances, a
homobifunctional cross-linker that contains succiminide ester,
imidoester, acylazide, or isocyanate groups may be used for
cross-linking. Examples of such cross-linking reagents include, but
are not limited to: Bis [2-(succinimidooxycarbonyloxy)ethyl]sulfone
(BSOCOES); Bis [2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfone
(sulfo-BSCOCOES); Disuccinimidyl suberate (DSS);
Bis-(Sulfosuccinimidyl) Suberate (BS3); Disuccinimidyl glutarate
(DSG); Dithiobis (succinimidylpropionate) (DSP);
Dithiobois(sulfosuccinimidylpropionate) (DTSSP);
Disulfosuccinimidyl tartrate (sulfo-DST);
Dithio-bis-maleimidoethane (DTME); Disuccinimidyl tartrate (DST);
Ethylene glycolbis(sulfosuccinimidylsuccinate) (sulfo-EGS);
Dimethyl malonimidate.2HCl (DMM); Ethylene
glycolbis(succinimidylsuccinate) (EGS); Dimethyl succinimidate.2HCl
(DMSC); Dimethyl adipimidate.2HCl (DMA); Dimethyl pimelimidate.2
HCl (DMP); and Dimethyl suberimidate.2.HCl (DMS), and Dimethyl
3,3'-dithiobispropionimidate.2HCl (DTBP). Heterobifunctional
cross-linkers that contains a combination of imidoester or
succinimide ester groups may also be used for cross-linking.
[0518] As another non-limiting example, attachable moieties may be
chemically conjugated using sulfhydryl and primary amine groups. In
these instances, heterobifunctional cross-linking reagents are
preferable used. Examples of such cross-linking reagents include,
but are not limited to: N-succinimidyl
3-(2-pyridyldithio)propionate (DPDP); N-succinimidyl
6-[3'-(2-pyridyldithio)-propionamido]hexanoate (sulfo-LC-SPDP);
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS);
m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS);
succinimidyl 4-[P-maleimidophenyl]butyrate (SMPB);
sulfosuccinimidyl 4-[p-maleimidophenyl]butyrate (sulfo-SMPB);
N-[-Maleimidobutyryloxy]succinimide ester (GMBS),
N-[-maleimidobutyryloxy]sulfosuccinimide ester (sulfo-GMBS);
N-[-maleimidocaproyloxy]succinimide ester (EMCS);
N-[-maleimidocaproyloxy]sulfosuccinimide ester (sulfo-EMCS);
N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB);
sulfosuccinimidyl(4-iodacetyl)aminobenzo ate (sulfo-SIAB);
succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC);
sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate
(sulfo-SMCC);
succiminidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amido-caproate-
) (LC-SMCC); 4-succinimidyloxycarbonyl-methyl-(2-pyridyldithio)
toluene (SMPT); and sulfo-LC-SMPT.
[0519] As an exemplary protocol, a minicell suspension is made 5 mM
EDTA/PBS, and a reducing solution of 2-mercaptoethylamine in 5 mM
EDTA/PBS is added to the minicells. The mixture is incubated for 90
minutes at 37.degree. C. The minicells are washed with EDTA/PBS to
remove excess 2-mercaptoethylamine. The attachable moiety is
dissolved in PBS, pH 7.2. A maleimide crosslinker is added to the
solution, which is then incubated for 1 hour at room temperature.
Excess maleimide is removed by column chromatography.
[0520] The minicells with reduced sulfhydryl groups are mixed with
the derivatized compounds having an attachable moiety. The mixture
is allowed to incubate at 4.degree. C. for 2 hours or overnight to
allow maximum coupling. The conjugated minicells are washed to
remove unreacted (unattached) compounds having the attachable
moiety. Similar protocols are used for expressed membrane proteins
with other reactive groups (e.g., carboxyl, amine) that can be
conjugated to an attachable moiety.
[0521] IX.E. Non-Genetic Methods for Directing Compounds to
Membranes
[0522] Included within the scope of the invention are compounds
that can be inserted into the membrane of segregated minicells.
Such compounds include attachable moieties that are chemically
conjugated to the surface of a minicell, and compounds that
associate with and/or insert into a membrane "spontaneously," i.e.,
by virtue of their chemical nature. By way of non-limiting example,
proteins that "spontaneously" insert into membranes include but are
not limited to Thykaloid membrane proteins (Woolhead et al., J.
Biol. Chem. 276:14607-14613, 2001), the mitochondrial adenine
nucleotide translocator (Jacotot et al., J. Exp. Med. 193:509-519,
2001), and polypeptides obtained using the methods of Hunt et al.
(Spontaneous, pH-dependent membrane insertion of a transbilayer
alpha-helix, Biochem 36:15177-15192, 1997). Lipids, gangliosides,
sphingomyelins, plasmalogens glycosyl diacylglycerols, and sterols
can also be incorporated into the membranes of segregated
minicells.
[0523] X. Membrane Proteins
[0524] In certain aspects of the invention, membrane proteins from
non-eubacterial organisms are expressed and displayed by minicells.
The cellular membrane (a.k.a. the "plasma membrane") is a lipid
bilayer that forms the boundary between the interior of a cell and
its external environment. The term "membrane proteins" refers to
proteins that are found in membranes including without limitation
cellular and organellar membranes.
[0525] X.A. Types of Membrane Proteins
[0526] X.A.1. In General
[0527] Membrane proteins consist, in general, of two types,
peripheral membrane proteins and integral membrane proteins.
[0528] Integral membrane proteins can span both layers (or
"leaflets") of a lipid bilayer. Thus, such proteins may have
extracellular, transmembrane, and intracellular domains.
Extracellular domains are exposed to the external environment of
the cell, whereas intracellular domains face the cytosol of the
cell. The portion of an integral membrane protein that traverses
the membrane is the "transmembrane domain." Transmembrane domains
traverse the cell membrane often by one or more regions comprising
15 to 25 hydrophobic amino acids which are predicted to adopt an
alpha-helical conformation.
[0529] Integral membrane proteins are classified as bitopic or
polytopic (Singer, (1990) Annu. Rev. Cell Biol. 6:247-96). Bitopic
proteins span the membrane once while polytopic proteins contain
multiple membrane-spanning segments.
[0530] A peripheral membrane protein is a membrane protein that is
bound to the surface of the membrane and is not integrated into the
hydrophobic layer of a membrane region. Peripheral membrane
proteins do not span the membrane but instead are bound to the
surface of a membrane, one layer of the lipid bilayer that forms a
membrane, or the extracellular domain of an integral membrane
protein.
[0531] X.A.2. In General
[0532] The invention can be applied to any membrane protein,
including but not limited to the following exemplary receptors and
membrane proteins. The proteins include but are not limited to are
receptors (e.g., GPCRs, sphingolipid receptors, neurotransmitter
receptors, sensory receptors, growth factor receptors, hormone
receptors, chemokine receptors, cytokine receptors, immunological
receptors, and compliment receptors, FC receptors), channels (e.g.,
potassium channels, sodium channels, calcium channels), pores
(e.g., nuclear pore proteins, water channels), ion and other pumps
(e.g., calcium pumps, proton pumps), exchangers (e.g.,
sodium/potassium exchangers, sodium/hydrogen exchangers,
potassium/hydrogen exchangers), electron transport proteins (e.g.,
cytochrome oxidase), enzymes and kinases (e.g., protein kinases,
ATPases, GTPases, phosphatases, proteases), structural/linker
proteins (e.g., Caveolins, clathrin), adapter proteins (e.g., TRAD,
TRAP, FAN), chemotactic/adhesion proteins (e.g., ICAM11, selectins,
CD34, VCAM-1, LFA-1,VLA-1), and phospholipases such as PI-specific
PLC and other phospholipiases.
[0533] X.A.3. Receptors
[0534] Within the scope of the invention are any receptor,
including without limitation:
[0535] The nuclear receptors, e.g. the nuclear export receptor;
[0536] The peripheral (mitochondrial) benzodiazephine receptor
(Gavish et al., "Enigma of the Peripheral Benzodiazephine
Receptor," Pharmacological Reviews, Vol. 51, No. 4);
[0537] Adrenergic and muscarinic receptors (Brodde et al.,
"Adrenergic and Muscarinic Receptors in the Human Heart",
Pharmacological Review, Vol. 51, No. 4);
[0538] Gamma-aminobutyric acid.sub.A receptors (Barnard et al.,
"International Union of Pharmacology. IV. Subtypes of -Aminobutyric
Acid.sub.A Receptors: Classification on the Basis of Submit
Structure and Receptor Function," Pharmacological Reviews, Vol. 50,
No. 2);
[0539] Kinin B.sub.1 receptors (Marceau et al., "The B.sub.1
Receptors for Kinins," Pharmacological Reviews, Vol. 50, No.
3);
[0540] Chemokine receptors (Murphy et al., "International Union of
Pharmacology. XXII. Nomenclature for Chemokine Receptors"
Pharmacological Review, Vol. 52, No. 1);
[0541] Glycine and NMDA Receptors (Danysz et al., "Glycine and
N-Methyl-D-Aspartate Receptors: Physiological Significance and
Possible Therapeutic Applications," Pharmacological Reviews, Vol.
50, No. 4);
[0542] Glutamate receptor ion channels (Dingledine et al., "The
Glutamate Receptor Ion Channels", Pharmacological Reviews, Vol. 51,
No. 1);
[0543] Purine and pyrimidine receptors including purinergic (e.g.,
P2) receptors (Ralevic et al., "Receptors for Purines and
Pyrimidines", Pharmacological Reviews, Vol. 50, No. 3); CNS
receptors and membrane transporters (E. Sylvester Vizi, "Role of
High-Affinity Receptors and Membrane Transporters in Nonsynaptic
Communication and Drug Action in the Central Nervous System,"
Pharmacological Reviews, Vol. 52, No. 1);
[0544] Opoid receptors, including but not limited to the -opioid
receptor (Quock et al., "The -Opioid Receptor: Molecular
Pharmacology, Signal Transduction and the Determination of Drug
Efficacy", Pharmacological Review, Col. 51, No. 3);
[0545] Angiotensin II receptors (Gasparo et al., "International
Union of Pharmacology. XXIII. The Angiotensin II Receptors"
Pharmacological Review, Vol. 52, No. 3);
[0546] Cholecystokinin receptors (Noble et al., "International
Union of Pharmacology. XXI. Structure, Distribution, and Functions
of Cholecystokinin Receptors", Pharmacological Reviews, Vol. 51,
No. 4)
[0547] Hormone receptors, including but not limited to, the
estrogen receptor; the glucocorticoid receptor; and the insulin
receptor;
[0548] Receptors found predominantly in the central nervous system,
including but not limited to, neuronal nicotinic acetylcholine
receptors; the dopamine D2/D3 receptor; GABA receptors; central
cannabinoid receptor CB1; opoid receptors, e.g., the kappa opioid
receptor, and the methadone-specific opioid receptor; nicotinic
acetylcholine receptors; serotonin receptors, e.g., the serotonin
5-HT3 receptor, the serotonin 5-HT4 receptor, and the serotonin-2
receptor; and dopamine receptors, e.g., the dopamine D2/D3
receptor; and the neurotensin receptor;
[0549] Receptors for growth factors, including but not limited to,
the erythropoietin receptor; the FGF receptor; the EGF receptor;
the VEGF receptor; VEGF receptor-2 protein; VEGF-receptor protein
(KDR); fibroblast growth factor receptor; the p75 nerve growth
factor receptor; epidermal growth factor receptor; IGF-1 receptor;
platelet factor-4 receptor; alpha platelet-derived growth factor
receptor; hepatocyte growth factor receptor; and human fibroblast
growth factor receptor;
[0550] Receptors for sphingolipids and lysophospholipids such as
the Edg family of GPCRs;
[0551] Receptors for interleukins, e.g., receptors for
interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,
IL-9, IL-10, IL-11, IL-12, IL-13, et seq.; and
[0552] Various receptors, including by way of non-limiting example,
receptors described in U.S. Pat. Nos. 6,210,967 (DNA encoding a
mammalian LPA receptor and uses thereof); 6,210,921 (CAR: a novel
coxsackievirus and adenovirus receptor; 6,211,343 (Lactoferrin
receptor protein; 6,218,509 (LH/CG receptor, DNA and use thereof;
6,214,972 (DNA encoding prostaglandin receptor DP); 6,221,613 (DNA
encoding a human melanin concentrating hormone receptor (MCH1) and
uses thereof); 6,221,660 (DNA encoding SNORF25 receptor); 6,225,080
(Mu-subtype opioid receptor); 6,222,015 (Estrogen receptor);
6,228,610 (Human metabotropic glutamate receptor subtypes (hmR4,
hmR6, hmR7) and related DNA compounds); 6,235,496 (Nucleic acid
encoding mammalian mu opioid receptor); 6,258,556 (cDNA and genomic
clones encoding human .mu. opiate receptor and the purified gene
product); 6,245,531 (Polynucleotide encoding insect ecdysone
receptor); 6,225,531 Glucan elicitor receptor, DNA molecule coding
therefor, fungus-resistant plants transformed with the DNA molecule
and method for creating the plants); 6,245,893 (Receptor that binds
anti-convulsant compounds); 6,248,712 (Urokinase-type plasminogen
activator receptor; 6,248,554 (DNA sequence coding for a BMP
receptor); 6,248,520 (Nucleic acid molecules encoding nuclear
hormone receptor coactivators and uses thereof); 6,242,251 (Rhesus
neuropeptide Y5 receptor); 6,252,056 (Human lysophosphatidic acid
receptor and use thereof); 6,255,472 (Isolated nucleic acid
molecule encoding a human skeletal muscle-specific receptor);
6,291,207 (Herpes virus entry receptor protein); 6,291,206 (BMP
receptor proteins); 6,291,195 (DNA encoding a human melanin
concentrating hormone receptor (MCH1) and uses thereof); 6,344,200
(Lactoferrin receptor protein); 6,335,180 (Nucleic acid sequences
encoding capsaicin receptor and uses thereof); 6,265,184
(Polynucleotides encoding chemokine receptor 88C); 6,207,799
(Neuropeptide Y receptor Y5 and nucleic acid sequences); 6,290,970
(Transferrin receptor protein of Moraxella); 6,326,350 (Transferrin
receptor subunit proteins of Neisseria meningitidis); 6,313,279
(Human glutamate receptor and related DNA compounds); 6,313,276
(Human endothelin receptor); 6,307,030 (Androgen receptor proteins,
recombinant DNA molecules coding for such, and use of such
compositions); 6,306,622 (cDNA encoding a BMP type II receptor);
6,300,087 (DNA encoding a human serotonin receptor (5-HT4B) and
uses thereof); 6,297,026 (Nucleic acids encoding the C140
receptor); 6,277,976 (Or-1, an orphan receptor belonging to the
nuclear receptor family); 6,274,708 (Mouse interleukin-11
receptor); 6,271,347 (Eosinophil eotaxin receptor); 6,262,016
(Transferrin receptor genes); 6,261,838 (Rat melanocortin receptor
MC3-R); 6,258,943 (Human neurokinin-3 receptor); 6,284,870 (Gamma
retinoic acid receptor); 6,258,944 (OB receptor isoforms and
nucleic acids encoding them); 6,261,801 (Nucleic acids encoding
tumor necrosis factor receptor 5); 6,261,800 (Luteinizing
hormone/choriogonadotropin (LH/CG) receptor); 6,265,563 (Opioid
receptor genes); 6,268,477 (Chemokine receptor 88-C); 6,316,611
(Human N-methyl-D-aspartate receptor subunits, nucleic acids
encoding same and uses therefor); 6,316,604 (Human C3b/C4b receptor
(CR1)); 6,287,855 (Nucleic acid encoding rat galanin receptor
(GALR2)); 6,268,221 (Melanocyte stimulating hormone receptor and
uses); and 6,268,214 (Vectors encoding a modified low affinity
nerve growth factor receptor).
[0553] X.A.3. Other Membrane Proteins
[0554] Other membrane proteins are within the scope of the
invention and include but are not limited to channels (e.g.,
potassium channels, sodium channels, calcium channels), pores
(e.g., nuclear pore proteins, water channels), ion and other pumps
(e.g., calcium pumps, proton pumps), exchangers (e.g.,
sodium/potassium exchangers, sodium/hydrogen exchangers,
potassium/hydrogen exchangers), electron transport proteins (e.g.,
cytochrome oxidase), enzymes and kinases (e.g., protein kinases,
ATPases, GTPases, phosphatases, proteases), structural/linker
proteins (e.g., Caveolins, clathrin), adapter proteins (e.g., TRAD,
TRAP, FAN),
[0555] X.A.3.a. Cellular Adhesion Molecules
[0556] Cellular adhesion molecules, including but not limited to
human rhinovirus receptor (ICAM-1), ICAM-2, ICAM-3, and PECAM-1,
and chemotactic/adhesion proteins (e.g., selectins, CD34, VCAM-1,
LFA-1, VLA-1) are within the scope of the invention. See also Alpin
et al., "Signal Transduction and Signal Modulation by Cell Adhesion
Receptors: The Role of Integrins, Cadherins, Immunoglobulin-Cell
Adhesion Molecules, and Selectins", Pharmacological Reviews, Vol.
50, No. 2.
[0557] X.A.3.b. Cytochrome P450 Enzymes
[0558] The family of enzymes known as "cytochrome P450" enzymes
(since they absorb light in the 450 nanometer range), or as
"cytochrome oxidase" enzymes (since they oxidize a wide range of
compounds that do not naturally occur in circulating blood), are
included within the scope of the invention. P450 enzymes
encompasses a variety of enzymes, many of which are involved in
xenobiotic metabolism, including by way of non-limiting example the
metabolism of drugs, prodrugs and toxins. Directories and databases
of P450s, and information regarding their substrates, are available
on-line (Fabian et al., The Directory of P450-containing Systems in
1996, Nucleic Acids Research 25:274-277, 1997). In humans, at least
about 200 different P450s are present (for a review, see Hasler et
al., Human cytochromes P450, Molecular Aspects of Medicine
20:1-137, 1999). There are multiple forms of these P450s and each
of the individual forms exhibit degrees of specificity towards
individual compounds or sets of compounds. In some cases, a
substrate, whether it is a drug or a carcinogen, is metabolized by
more than one cytochrome P450.
[0559] Members of the cytochrome P450 family are present in varying
levels and their expression and activities are controlled by
variables such as chemical environment, sex, developmental stage,
nutrition and age. The cytochrome P450s are found at high
concentrations in liver cells, and at lower concentrations in other
organs and tissues such as the lungs (e.g., Forme-Pfister et al.,
Xenobiotic and endobiotic inhibitors of cytochrome P-450 db1
function, the target of the debrisoquine/sparteine type
polymorphism, Biochem. Pharmacol. 37:3829-35, 1988). By oxidizing
lipophilic compounds, which makes them more water-soluble,
cytochrome oxidase enzymes help the body eliminate (via urine, or
in aerosols exhaled out of the lungs) compounds that might
otherwise act as toxins or accumulate to undesired levels.
[0560] In humans, several cytochrome P450s have been identified as
being involved in xenobiotic metabolism. These include CYP1A1,
CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6,
CYP2E1, CYP3A4, and CYP3A5 (Crespi et al., The use of
heterologously expressed drug metabolizing enzymes--state of the
art and prospects for the future, Pharm Ther 84:121-131, 1999).
[0561] X.A.3.c. Miscellaneous Membrane Proteins
[0562] In addition to the preceding non-limiting examples, the
invention can be applied to the membrane proteins described in U.S.
Pat. Nos. 6,335,018 (High molecular weight major outer membrane
protein of moraxella); 6,264,954 (Haemophilus outer membrane
protein); 6,197,543 (Human vesicle membrane protein-like proteins);
6,121,427 (Major outer membrane protein CD of branhamella);
6,083,743 and 6,013,514 (Haemophilus outer membrane protein);
6,004,562 (Outer membrane protein B1 of Moraxella catarrhalis);
5,863,764 (DNA encoding a human membrane protein); 5,861,283 (DNA
encoding a limbic system-associated membrane protein); 5,824,321
(Cloned leptospira outer membrane protein); 5,821,085 (Nucleotide
sequences of a T. pallidum rare outer membrane protein); 5,821,055
(Chlamydia major outer membrane protein); 5,808,024 (Nucleic acids
encoding high molecular weight major outer membrane protein of
moraxella); 5,770,714 (Chlamydia major outer membrane protein);
5,763,589 (Human membrane protein); 5,753,459 (Nucleotide sequences
of T. pallidum rare outer membrane protein); 5,607,920
(Concanavalin a binding proteins and a 76 kD chondrocyte membrane
protein (CMP) from chondrocytes and methods for obtaining same);
and 5,503,992 (DNA encoding the kD outer membrane protein of
Haemophilus influenzae).
[0563] X.B. Membrane Anchoring Domains
[0564] A membrane-anchoring domain can be incorporated into a
fusion protein of the invention. Non-limiting examples of membrane
anchoring domains include those derived from Prostaglandin H2
synthases (PGHS-1 and -2) (Nina et al., Anchoring of a monotopic
membrane protein: the binding of prostaglandin H2 synthase-1 to the
surface of a phospholipid bilayer, Eur. Biophys. J. 29:439-54,
2000; Otto and Smith, Photolabeling of prostaglandin endoperoxide H
synthase-1 with 3-trifluoro-3-(m-[125I]iodophenyl)diazirine as a
probe of membrane association and the cyclooxygenase active site, J
Biol Chem 271:9906-10, 1996; and Otto and Smith, The orientation of
prostaglandin endoperoxide synthases-1 and -2 in the endoplasmic
reticulum, J Biol Chem 269:19868-75, 1994; those derived from
carboxypeptidase E (EC 3.4.17.10) (Fricker et al., Identification
of the pH-dependent membrane anchor of carboxypeptidase E (EC
3.4.17.10), J. Biol. Chem., 265, 2476-2482, 1990); and peptide
convertase 3 (PC3) (Smeekens et al., Identification of a cDNA
encoding a second putative prohormone convertase related to PC2 in
AtT20 cells and islets of Langerhans, Proc Natl Acad Sci USA 88,
340-344, 1990).
[0565] X.C. Transmembrane Domains
[0566] A variety of types and examples of transmembrane domain are
known. Proteins with up to 12 transmembrane domains are known
(Fujiwara et al., Identification of thyroid hormone transporters in
humans: different molecules are involved in a tissue-specific
manner, Endocrinology 2001 142:2005-12; Sharina et al., Mutational
analysis of the functional role of conserved arginine and lysine
residues in transmembrane domains of the murine reduced folate
carrier, Mol Pharmacol 2001 59:1022-8). However, the invention is
not limited to any particular number of transmembrane domains.
[0567] Monotropic ("single pass") domains, which traverse a
membrane once, include by way of non-limiting example, those found
in receptors for epidermal growth factor (EGF), receptors for tumor
necrosis factor (TNF) and the like. Polytropic ("multipass")
proteins traverse a membrane two or more times. Non-limiting
examples of polytropic proteins are as follows.
[0568] Biotropic ("2 passes") membrane proteins include, but are
not limited to: EnvZ of E. coli; the peroxisomal membrane protein
Pex11-1p (Anton et al., ARF- and coatomer-mediated peroxisomal
vesiculation, Cell Biochem Biophys 2000; 32 Spring:27-36);
pleitropic drug ABC transporters of S. cervisiae (Rogers et al.,
The pleitropic drug ABC transporters from Saccharomyces cerevisiae,
J Mol Microbiol Biotechnol 2001 3:207-14); and human and rate urate
transporters hUAT and rUAT (Lipkowitz et al., Functional
reconstitution, membrane targeting, genomic structure, and
chromosomal localization of a human urate transporter, J Clin
Invest 2001 107:1103-15).
[0569] Tritropic ("3 pass") membrane proteins include, but are not
limited to: the ethylene receptor ETR1 of Arabidopsis; the
Cauliflower Card Expression protein CC1 (Palmer et al., A Brassica
oleracea Gene Expressed in a Variety-Specific Manner May Encode a
Novel Plant Transmembrane Receptor, Plant Cell Physiol 2001
42:404-413); and a splice variant of the mitochondrial membrane
protein hMRS3/4 (Li et al., Characterization of a novel human
putative mitochondrial transporter homologous to the yeast
mitochondrial RNA splicing proteins 3 and 4, FEBS Lett 2001
494:79-84).
[0570] Tetraspanins or tetraspans are non-limiting examples of
membrane proteins with four transmembrane domains. (Levy et al., J.
Biol. Chem., 226:14597-14602, 1991; Tomlinson et al., J. 1 mmol.
23:136-40, 1993; and Barclay et al., (In) The Leucocyte antigen
factbooks, Academic press, London, 1993). These proteins are
collectively known as the `transmembrane 4 superfamily` (TM4)
because they span the plasma membrane four times. The proteins
known to belong to this family include, but are not limited to:
mammalian antigen CD9 (MIC3), a protein involved in platelet
activation and aggregation; mammalian leukocyte antigen CD37,
expressed on B lymphocytes; mammalian leukocyte antigen CD53
(OX-44), which may be involved in growth regulation in
hematopoietic cells; mammalian lysosomal membrane protein CD63
(Melanoma-associated antigen ME491; antigen AD1); mammalian antigen
CD81 (cell surface protein TAPA-1), which may play an important
role in the regulation of lymphoma cell growth; mammalian antigen
CD82 (Protein R2; Antigen C33; Kangai 1 (KAI1)), which associates
with CD4 or CD8 and delivers costimulatory signals for the TCR/CD3
pathway; mammalian antigen CD151 (SFA-1); Platelet-endothelial
tetraspan antigen 3 (PETA-3); mammalian TM4SF2 (Cell surface
glycoprotein A15; TALLA-1; MXS1); mammalian TM4SF3
(Tumor-associated antigen CO-029); mammalian TM4SF6 (Tspan-6;
TM4-D); mammalian TM4SF7 (Novel antigen 2 (NAG-2); Tspan-4);
mammalian Tspan-2; Mammalian Tspan-3 (TM4-A); mammalian Tetraspan
NET-5; and Schistosoma mansoni and japonicum 23 Kd surface antigen
(SM23/SJ23).
[0571] Non-limiting examples of membrane proteins with six
transmembrane domains include the EBV integral membrane protein
LMP-1, and a splice variant of the mitochondrial protein hMRS3/4
(Li et al., Characterization of a novel human putative
mitochondrial transporter homologous to the yeast mitochondrial RNA
splicing proteins 3 and 4, FEBS Lett 2001 Apr. 6; 494(1-2):79-84).
Proteins with six transmembrane domains also include STEAP (six
transmembrane epithelial antigens of the prostate) proteins (Afar
et al., U.S. Pat. No. 6,329,503). The prototype member of the STEAP
family, STEAP-1, appears to be a type Ma membrane protein expressed
predominantly in prostate cells in normal human tissues.
Structurally, STEAP-1 is a 339 amino acid protein characterized by
a molecular topology of six transmembrane domains and intracellular
N- and C-termini, suggesting that it folds in a "serpentine" manner
into three extracellular and two intracellular loops.
[0572] Literally hundreds of 7-pass membrane proteins are known.
G-protein coupled receptors (GPCRs), including without limitation
beta-adreno receptors, adrenergic receptors, EDG receptors,
adenosine receptors, B receptors for kinins, angiotensin receptors,
and opiod receptors are of particular interest. GPCRs are described
in more detail elsewhere herein.
[0573] A non-limiting example of a protein with 9 transmembrane
domains is Lipocalin-1 interacting membrane receptor (Wojnar et
al., Molecular cloning of a novel Lipocalin-1 interacting human
cell membrane receptor (LIMR) using phage-display, J Biol Chem 2001
3; [epub ahead of print]).
[0574] Proteins with both transmembrane and anchoring domains are
known. For example, AMPA receptor subunits have transmembrane
domains and one membrane-anchoring domain.
[0575] A variety of databases that describe known, and software
programs that predict, membrane anchoring and transmembrane domains
are available to those skilled in the art. See, for example
Gcrdb.dba GCRDb [G Protein Coupled Receptor database], Tmbase.dba
Tmbase [database of transmembrane domains], Prodom.srv Propom
[Protein domains], Tmap.srv TMAP [Protein transmembrane segments
prediction], Tm7.srv TM7 [Retrieval of data on G protein-coupled
receptors], and Memsat.sof MEMSAT [transmembrane structure
prediction program].
[0576] Quentin and Fichant (J Mol Microbiol Biotechnol 2000
2:501-4, ABCdb: an ABC transporter database) have described a
database devoted to the ATP-binding cassette (ABC) protein domains
(ABCdb), the majority of which energize the transport of compounds
across membranes. In bacteria, ABC transporters are involved in the
uptake of a wide range of molecules and in mechanisms of virulence
and antibiotic resistance. In eukaryotes, most ABC transporters are
involved in drug resistance, and many are associated with diseases.
ABCdb can be accessed via the World Wide Web
(http://ir21cb.cnrs-mrs.fr/ABCdb/). See also Sanchez-Fernandez et
al., The Arabidopsis thaliana ABC protein superfamily: a complete
inventory, J Biol Chem 2001 May 9; [epub ahead of print], and
Rogers et al., The pleitropic drug ABC transporters from
Saccharomyces cerevisiae, J Mol Microbiol Biotechnol 2001 April;
3(2):207-14.
[0577] X.D. Functions and Activities of Membrane Proteins
[0578] Non-limiting examples of membrane proteins include
membrane-associated enzymes. Membrane-associated enzymes include
but not limited to certain enzymes of the electron transport chain
(ETC), antigenic proteins such as the major histocompatability
(MHC) antigens, transport proteins, channels, hormone receptors,
cytokine receptors, glucose permeases, gap junction proteins and
bacteriorhodopsins.
[0579] A "transport protein" or "transporter" is a type of membrane
protein that allows substances to cross plasma membranes at a rate
that is faster than what is found by diffusion alone. Some
transport proteins expend energy to move substances (active
transport). Many active transport proteins are ATPases (e.g., the
Na.sup.+--K.sup.+ ATPase), or at least bind ATP by virtue of
comprising an ATP-binding cassette (ABC) (see, e.g., Rogers et al.,
The pleitropic drug ABC transporters from Saccharomyces cerevisiae,
J Mol Microbiol Biotechnol 3:207-14, 2001). Nucleobase transporters
are reviewed by De Koning and Diallinas (Nucleobase Transporters,
Mol Membr Biol 17:75-94, 2000).
[0580] A "channel protein" is a protein that facilitates the
diffusion of molecules/ions across lipid membranes by forming a
hydrophilic pore or "channel" that provides molecules/ions access
through lipid membranes, which are generally hydrophobic. Channels
are often multimeric, with the pore being formed by subunit-subunit
interactions.
[0581] A "receptor" is a molecular entity, typically a protein,
that is displayed on the surface of a cell. A receptor is
characterized by high affinity, often a specific binding of a
specific substance, typically resulting in a specific biochemical
or physiological effect.
[0582] A "hormone" is a naturally occurring substance secreted by
specialized cells that affects the metabolism or behavior of other
cells having receptors for the hormone. Non-limiting examples of
hormones having receptors include but are not limited to insulin,
cytokines, steroid hormones, histamines, glucagon, angiotensin,
catecholamines, low density lipids (LDLs), tumor necrosis factor
alpha, tumor necrosis factor beta, estrogen, and testosterone.
[0583] X.E. G-Protein-Coupled Receptors
[0584] G protein-coupled receptors (GPCRs) constitute the most
prominent family of validated drug targets within biomedical
research and are thought to be involved in such diseases and
disorders as heart disease, hypertension, cancer, obesity, and
depression and other mental illnesses. Over half of approved drugs
elicit their therapeutic effects by selectively addressing members
of this target family and more than 1000 sequences of the human
genome encode for GPCRs containing the classical 7-pass membrane
structure characteristic of this family of proteins (Marinissen, M.
and J. S. Gutkind, G-protein-coupled receptors and signaling
networks: emerging paradigms (Review), Trends. Phamacol. Sci. 22:
368-376, 2001). Many pharmacological drug companies are interested
in the study of G-coupled proteins. It is possible to co-express a
G-coupled protein receptor and its associated G-protein to study
their pharmacological characteristics (Strosberg and Marullo,
Functional expression of receptors in microorganisms. TiPS, 1992.
13: 95-98).
[0585] G-protein-coupled receptors (GPCRs) are reviewed by
Marinissen, M. and J. S. Gutkind, G-protein-coupled receptors and
signaling networks: emerging paradigms. Trends. Phamacol. Sci. 22:
368-376, 2001; Sautel and Milligan, Molecular manipulation of
G-protein-coupled receptors: a new avenue into drug discovery, Curr
Med Chem 2000 889-96; Hibert et al., This is not a G
protein-coupled receptor, Trends Pharmacol Sci 1993, 14:7-12;
Wilson et al., Orphan G-protein-coupled receptors: the next
generation of drug targets?, Br J Pharmacol 1998, 125:1387-92; Roth
et al., G protein-coupled receptor (GPCR) trafficking in the
central nervous system: relevance for drugs of abuse, Drug Alcohol
Depend 1998, 51:73-85; Ferguson and Caron, G protein-coupled
receptor adaptation mechanisms, Semin Cell Dev Biol 1998, 9:119-27;
Wank, G protein-coupled receptors in gastrointestinal physiology.
I. CCK receptors: an exemplary family, Am J Physiol 1998,
274:G607-13; Rohrer and Kobilka, G protein-coupled receptors:
functional and mechanistic insights through altered gene
expression. (Review), Physiol Rev 1998, 78:35-52; and Larhammar et
al., The receptor revolution--multiplicity of G-protein-coupled
receptors. (Review), Drug Des Discov 1993, 9:179-88.
[0586] GPCR localization and regulation has been studied using
GFP-comprising fusion proteins (Kallal and Benovic, Using green
fluorescent proteins to study G-protein-coupled receptor
localization and trafficking. (Review), Trends Pharmacol Sci 2000
21:175-80; and Ferguson, Using green fluorescent protein to
understand the mechanisms of G-protein-coupled receptor regulation.
(Review), Braz J Med Biol Res 1998, 31:1471-7); and by using
chimeric GPCRs (Milligan and Rees, Chimaeric G alpha proteins:
their potential use in drug discovery. (Review), Erratum in: Trends
Pharmacol Sci 1999 June; 20(6):252.
[0587] GPCRs belong to a superfamily of at least 6 families of
receptors, the most important of which is the main family, A.
Members of the membrane protein gene superfamily of GPCRs have been
characterized as having seven putative transmembrane domains. The
transmembrane domains are believed to represent transmembrane
alpha-helices connected by extracellular or cytoplasmic loops. A
functional G-protein is a trimer which consists of a variable alpha
subunit coupled to much more tightly-associated and constant beta
and gamma subunits, although G-protein independent actions have
been postulated (Marinissen, M. and J. S. Gutkind,
G-protein-coupled receptors and signaling networks: emerging
paradigms. Trends. Phamacol. Sci. 22: 368-376, 2001 Review). A
variety of ligands have been identified which function through
GPCRs. In general, binding of an appropriate ligand (e.g.,
bioactive lipids, ions, bioactive amines, photons, odorants,
hormones, neurotransmitters, peptides, nucleosides, etc.) to a GPCR
leads to the activation of the receptor. G-protein coupled
receptors include a wide range of biologically active receptors,
such as hormone, viral, growth factor and neuroreceptors.
Typically, activation of a GPCR initiates the regulatory cycle of a
corresponding G-protein. This cycle consists of GTP exchange for
GDP, dissociation of the alpha and beta/gamma subunits, activation
of the second messenger pathway by a complex of GTP and the alpha
subunit of the G-protein, and return to the resting state by GTP
hydrolysis via the innate GTPase activity of the G-protein alpha
subunit A.
[0588] GPCRs include, without limitation, dopamine receptors which
bind to neuroleptic drugs used for treating psychotic and
neurological disorders. Other examples of members of this family
include calcitonin, adrenergic, endothelin, cAMP, adenosine,
muscarinic, acetylcholine, serotonin, histamine, thrombin, kinin,
follicle stimulating hormone, opsins and rhodopsins, odorant,
cytomegalovirus receptors, and the like.
[0589] Most GPCRs have single conserved cysteine residues in each
of the first two extracellular loops which form disulfide bonds
that are believed to stabilize functional protein structure. The
seven transmembrane regions, each comprising conserved hydrophobic
stretches of about 20 to 30 amino acids, are designated as TM1,
TM2, TM3, TM4, TMS, TM6, and TM7. TM3 is also implicated in signal
transduction.
[0590] Although not wishing to be bound by any particular theory,
it is believed that GPCRs participate in cell signaling through
their interactions with heterotrimetric G-proteins composed of
alpha, beta and gamma subunits (Marinissen, M. and J. S. Gutkind,
G-protein-coupled receptors and signaling networks: emerging
paradigms. Trends. Phamacol. Sci. 22:368-376, 2001). In some
aspects of the invention, GPCRs and homologs are displayed on the
surfaces of minicells.
[0591] X.F. EDG Receptors and Other Sphingolipid-Binding
Receptors
[0592] The Endothelial Differentiation Gene (EDG) receptor family
includes but is not limited to eight presently known GPCRs that
have a high affinity to lipid ligands (Lynch et al., Life on the
edg. Trends Pharmacol. Sci., 1999. 20: 273-5). These transmembrane
receptors are found in several different tissues in different
species. EDG receptors have been shown to be involved in calcium
mobilization, activation of mitogen-activated protein kinase,
inhibition of adenylate cyclase activation, and alterations of the
cytoskelaton. The EDG family is divided into two different groups
based on homology and ligand specificity. The EDG 2, 4, and 7
receptors are specific for the ligand lysophosphatidic acid (LPA)
(An et al., Signaling Mechanism and molecular characteristics of G
protein-coupled receptors for lysophosphatidic acid and sphingosine
1-phosphate. J. Cell Biochem, 30/31:147-157, 1998; Goetzl et al.,
Distinctive expression and functions of the type 4 endothelial
differentiation gene-encoded G protein-coupled receptor for
lysophosphatidic acid in ovarian cancer. Cancer Res., 59:5370-5,
1999). In contrast, EDG 1, 3, and 5 bind sphingosine-1-phosphate
(S1P) (Zhang et al., Comparative analysis of three murine G-protein
coupled receptors activated by sphingosine-1-phosphate. Gene,
227:89-99, 1999). EDG-6 is believed to interact with S1P (Yamazaki
et al., Edg-6 as a putative sphingosine 1-phosphate receptor
coupling to Ca2.sup.++ signaling pathway. Biochem Phys Res Com,
268:583-589, 2000).
[0593] Receptors that bind S1P and other sphingolipids are used in
one aspect of the invention (for a review of some S1P-binding
receptors, see Spiegel et al., Biochim. Biophys. Acta 1484:107-116,
2000). Such receptors include but are not limited to members of the
EDG family of receptors (a.k.a. 1pA receptors, Chun, Crit. Rev.
Neuro. 13:151-168, 1999), and isoforms and homologs thereof such as
NRG1 and AGR16.
[0594] EDG-1 was the first identified member of a class of G
protein-coupled endothelial-derived receptors (EDG). Non-limiting
examples of other EDG family members that also bind S1P include
EDG-3 (a.k.a. ARG16; the rat homolog of EDG-3 is designated H218),
EDG-5, EDG-6 and EDG-8. For reviews, see Goetzl et al., Adv. Exp.
Med. Biol. 469:259-264, 1999; and Chun et al., Cell. Biochem.
Biophys. 30:213-242, 1999).
[0595] EDG-1 is described by Lee et al., (Ann. NY Acad. Sci.
845:19-31, 1998). Liu and Hla, The mouse gene for the inducible
G-Protein-coupled receptor edg-1. Genomics, 1997, 43: p. 15-24.
Human EDG-1c genes and proteins are described in published PCT
application WO 99/46277 to Bergsma et al.
[0596] EDG-3 is described by Okamoto et al. (Biochem. Biophys. Res.
Commun. 260:203-208, 1999) and An et al. (FEBS Letts. 417:279-282,
1997). See also An et al., J. Biol. Chem. 275:288-296, 2000.
[0597] EDG-5 human and mammalian genes are described in U.S. Pat.
No. 6,057,126 to Munroe et al. and published PCT application WO
99/33972 to Munroe et al. The rat homolog, H218, is described in
U.S. Pat. No. 5,585,476 to MacLennan et al. Van Brocklyn et al., J.
Biol. Chem. 274:4626-4632, 1999; and Gonda et al., Biochem. J.
337:67-75, 1999. See also An et al., J. Biol. Chem. 275:288-296,
2000.
[0598] EDG-6 is described by Graler et al. (Genomics 53:164-169,
1998), Yamazaki et al. (Biochem. Biophys. Res. Commun. 268:583-589,
2000), and Van Brocklyn et al. (Sphingosine-1-phosphate is a ligand
for the G protein-coupled receptor EDG-6, Blood 95:2624-9,
2000).
[0599] EDG-8 from rat brain is described by Im et al., (J. Biol.
Chem. 275:14281-14286, 2000). Homologs of EDG-8 from other species,
including humans, may also be used in the present invention.
[0600] The Mil receptor (Mil is an abbreviation for "miles apart")
binds SIT and regulates cell migration during vertebrate heart
development. The Mil receptor of Zebrafish is described by Mohler
et al. (J. Immunol. 151:1548-1561, 1993). Another S1P receptor is
NRG1 (nerve growth factor regulated gene-1), the rat version of
which has been identified (Glickman et al., Mol. Cel. Neurosci.
14:141-152, 1999).
[0601] Receptors that bind sphingosylphosphoryl choline (SPC) are
also used in this aspect of the invention. Such receptors include
but are not limited to members of the SCaMPER family of receptors
(Mao et al., Proc. Natl. Acad. Sci. U.S.A. 93:1993-1996, 1996;
Betto et al., Biochem. J. 322:327-333, 1997). Some evidence
suggests that EDG-3 may bind SPC in addition to S1P (Okamoto et
al., Biochem. Biophys. Res. Commun. 260:203-208, 1999). Derivatives
of EDG-3 that bind both SIT and SPC are used in one aspect of the
invention.
[0602] Receptors that bind lysophophatidic acid may be used in the
present invention. These include EDG-2 (LPA1), EDG-4 (LPA2), EDG-7
(LPA3). See Moller et al., Expression and function of
lysophosphatidic acid receptors in cultured rodent microglial
cells, J Biol Chem 2001 May 4 [epub ahead of print]; Fukushima and
Chun, The LPA receptors, Prostaglandins 64(1-4):21-32, 2001; Contos
and Chun, The mouse 1p(A3)/Edg7 lysophosphatidic acid receptor
gene: genomic structure, chromosomal localization, and expression
pattern, Gene 267:243-53, 2001; Schulte et al., Lysophosphatidic
acid, a novel lipid growth factor for human thyroid cells:
over-expression of the high-affinity receptor edg4 in
differentiated thyroid cancer, Int J Cancer 92249-56, 2001; Kimura
et al., Two novel Xenopus homologs of mammalian LP(A1)/EDG-2
function as lysophosphatidic acid receptors in Xenopus oocytes and
mammalian cells, J Biol Chem 276:15208-15, 2001; and Swarthout and
Walling, Lysophosphatidic acid: receptors, signaling and survival
(Review), Cell Mol Life Sci 57:1978-85, 2000.
[0603] Examples of lysophospholipid receptors including, but not
limited to EDG proteins, are disclosed in Fukushima et al.
(Lysophospholipid receptors. Annu. Rev. Pharmacol. Toxicol.
41:507-534, 2001) Malek and Lee (Nrg-1 Belongs to the Endothelial
Differentiation Gene Family of G Protein-coupled
Sphingosine-1-phosphate Receptors, J. Biol. Chem. 276:5692-5699,
2001), Hla et al. (Sphingosine-1-phosphate signaling via the EDG-1
family of G-protein-coupled receptors (Review), Ann N Y Acad Sci
905:16-24, 2000; Chun, Lysophospholipid receptors: implications for
neural signaling (Review), Crit. Rev Neurobiol 13:151-68, 1999);
and Chun et al. (A growing family of receptor genes for
lysophosphatidic acid (LPA) and other lysophospholipids (LPs)
(Review), Cell Biochem Biophys 30:213-42, 1999).
[0604] XI. Recombinant DNA Expression
[0605] In order to achieve recombinant expression of a fusion
protein, an expression cassette or construct capable of expressing
a chimeric reading frame is introduced into an appropriate host
cell to generate an expression system. The expression cassettes and
constructs of the invention may be introduced into a recipient
eubacterial or eukaryotic cell either as a nonreplicating DNA or
RNA molecule, which may be a linear molecule or, more preferably, a
closed covalent circular molecule. Since such molecules are
incapable of autonomous replication, the expression of the gene may
occur through the transient expression of the introduced sequence.
Alternatively, permanent expression may occur through the
integration of the introduced DNA sequence into the host
chromosome.
[0606] XI.A. Recombinant DNA Expression Systems
[0607] A variety of eubacterial recombinant DNA expression systems
may be used to produce the fusion proteins of the invention. Host
cells that may be used in the expression systems of the present
invention are not strictly limited, provided that they are suitable
for use in the expression of the fusion protein of interest and can
produce minicells. Non-limiting examples of recognized eubacterial
hosts that may be used in the present invention include bacteria
such as E. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella,
Serratia, and the like.
[0608] Eubacterial expression systems utilize plasmid and viral
(bacteriophage) expression vectors that contain replication sites
and control sequences derived from a species compatible with the
host may be used. Suitable phage or bacteriophage vectors include
gt10, gt11 and the like. Suitable virus vectors may include
pMAM-neo, pKRC and the like. Appropriate eubacterial plasmid
vectors include those capable of replication in E. coli (such as,
by way of non-limiting example, pBR322, pUC118, pUC119, ColE1,
pSC101, pACYC 184, OVX. See "Molecular Cloning: A Laboratory
Manual" 1989). Bacillus plasmids include pC194, pC221, pT127, and
the like (Gryczan, In: The Molecular Biology of the Bacilli,
Academic Press, NY, pp. 307-329, 1982). Suitable Streptomyces
plasmids include p1J101 (Kendall et al., J. Bacteriol.
169:4177-4183, 1987), and Streptomyces bacteriophages such as C31
(Chapter et al., In: Sixth International Symposium on
Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary, pp.
45-54, 1986). Pseudomonas plasmids are reviewed by John et al.
(Rev. Infect. Dis. 8:693-704, 1986), and Izaki (Jpn. J. Bacteriol.
33:729-742, 1978). See also Brent et al., "Vectors Derived From
Plasmids," Section II, and Lech et al. "Vectors derived from Lambda
and Related Bacteriophages" Section III, in Chapter 1 of Short
Protocols in Molecular Biology, 2nd Ed., Ausubel et al., eds., John
Wiley and Sons, New York, 1992, pages 1-13 to 1-27; Lech et al.
"Vectors derived from Lambda and Related Bacteriophages" Section
III and Id. pages 1-28 to page 1-52.
[0609] To express a protein, including but not limited to a fusion
protein, in a eubacterial cell, it is necessary to operably link
the ORF encoding the protein to a functional eubacterial or viral
promoter. Such promoters may be either constitutive or, more
preferably, regulatable (i.e., inducible or derepressible).
Examples of constitutive promoters include the int promoter of
bacteriophage lambda, the bla promoter of the beta-lactamase gene
sequence of pBR322, and the cat promoter of the chloramphenicol
acetyl transferase gene sequence of pPR325, and the like. Examples
of inducible eubacterial promoters include the major right and left
promoters of bacteriophage lambda (P.sub.L and P.sub.R), the trp,
recA, lacZ, lacI, and gal promoters of E. coli, the alpha-amylase
(Ulmanen et al., J. Bacteriol. 162:176-182, 1985) and the
sigma-28-specific promoters of B. subtilis (Gilman et al., Gene
Sequence 32:11-20, 1984), the promoters of the bacteriophages of
Bacillus (Gryczan, in: The Molecular Biology of the Bacilli,
Academic Press, Inc., NY, 1982), and Streptomyces promoters (Ward
et al., Mol. Gen. Genet. 203:468-478, 1986). Eubacterial promoters
are reviewed by Glick (Ind. Microbiot. 1:277-282, 1987), Cenatiempo
(Biochimie 68:505-516, 1986), and Gottesman (Ann. Rev. Genet.
18:415-442, 1984).
[0610] Proper expression also requires the presence of a
ribosome-binding site upstream of the gene sequence-encoding
sequence. Such ribosome-binding sites are disclosed, for example,
by Gold et al. (Ann. Rev. Microbiol. 35:365-404, 1981). The
selection of control sequences, expression vectors, transformation
methods, and the like, are dependent on the type of host cell used
to express the gene. As used herein, "cell", "cell line", and "cell
culture" may be used interchangeably and all such designations
include progeny. Thus, the words "transformants" or "transformed
cells" include the primary subject cell and cultures derived
therefrom, without regard to the number of transfers. It is also
understood that all progeny may not be precisely identical in DNA
content, due to deliberate or inadvertent mutations. However, as
defined, mutant progeny have the same functionality as that of the
originally transformed cell.
[0611] Mammalian expression systems utilize host cells such as HeLa
cells, cells of fibroblast origin such as VERO or CHO-K1, or cells
of lymphoid origin and their derivatives. Preferred mammalian host
cells include SP2/0 and J558L, as well as neuroblastoma cell lines
such as IMR 332, which may provide better capacities for correct
post-translational processing. Non-limiting examples of mammalian
extrachromosomal expression vectors include pCR3.1 and pcDNA3.1,
and derivatives thereof including but not limited to those that are
described by and are commercially available from Invitrogen
(Carlsbad, Calif.).
[0612] Several expression vectors are available for the expression
of polypeptides in mammalian host cells. A wide variety of
transcriptional and translational regulatory sequences may be
employed, depending upon the nature of the host. The
transcriptional and translational regulatory signals may be derived
from viral sources, such as adenovirus, bovine papilloma virus,
cytomegalovirus (CMV), simian virus, or the like, where the
regulatory signals are associated with a particular gene sequence
which has a high level of expression. Alternatively, promoters from
mammalian expression products, such as actin, collagen, myosin, and
the like, may be employed. Transcriptional initiation regulatory
signals may be selected which allow for repression or activation,
so that expression of the gene sequences can be modulated. Of
interest are regulatory signals that are temperature-sensitive
since, by varying the temperature, expression can be repressed or
initiated, or are subject to chemical (such as metabolite)
regulation.
[0613] Preferred eukaryotic plasmids include, for example, BPV,
vaccinia, SV40, 2-micron circle, and the like, or their
derivatives. Such plasmids are well known in the art (Botstein et
al., Miami Wntr. Symp. 19:265-274, 1982; Broach, in: The Molecular
Biology of the Yeast Saccharomyces: Life Cycle and Inheritance,
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., p.
445-470, 1981; Broach, Cell 28:203-204, 1982; Bollon et al., J.
Clin. Hematol. Oncol. 10:39-48, 1980; Maniatis, In: Cell Biology: A
Comprehensive Treatise, Vol. 3, Gene Sequence Expression, Academic
Press, NY, pp. 563-608, 1980).
[0614] Expression of polypeptides in eukaryotic hosts generally
involves the use of eukaryotic regulatory regions. Such regions
will, in general, include a promoter region sufficient to direct
the initiation of RNA synthesis. Preferred eukaryotic promoters
include, for example, the promoter of the mouse metallothionein I
gene sequence (Hamer et al., J. Mol. Appl. Gen. 1:273-288, 1982);
the TK promoter of Herpes virus (McKnight, Cell 31:355-365, 1982);
the SV40 early promoter (Benoist et al., Nature (London)
290:304-31, 1981); and the yeast gal4 gene sequence promoter
(Johnston et al., Proc. Natl. Acad. Sci. (USA) 79:6971-6975, 1982;
Silver et al., Proc. Natl. Acad. Sci. (USA) 81:5951-5955,
1984).
[0615] Expression sequences and elements are also required for
efficient expression. Non-limiting examples include Kozak and IRES
elements in eukaryotes, and Shine-Delgarno sequences in
prokaryotes, which direct the initiation of translation (Kozak,
Initiation of translation in prokaryotes and eukaryotes. Gene,
1999. 234: 187-208; Martinez-Salas et al., Functional interactions
in internal translation initiation directed by viral and cellular
IRES elements, Jour. of Gen. Virol. 82:973-984, 2001); enhancer
sequences; optional sites for repressor and inducers to bind; and
recognition sites for enzymes that cleave DNA or RNA in a
site-specific manner. Translation of mRNA is generally initiated at
the codon which encodes the first methionine; if so, it is
preferable to ensure that the linkage between a eukaryotic promoter
and a preselected ORF does not contain any intervening codons that
encode a methionine (i.e., AUG). The presence of such codons
results either in the formation of a fusion protein with an
uncharacterized N-terminal extension (if the AUG codon is in the
same reading frame as the ORF) or a frame-shift mutation (if the
AUG codon is not in the same reading frame as the ORF).
[0616] XI.B. Expression of Membrane Proteins Presently, the most
commonly used expression systems for the expression of integral
membrane proteins are eukaryotic and eubacterial whole cell
expression systems. Although minicells have been used to express
several eubacterial membrane proteins, the production of
non-eubacterial membrane proteins has not been reported. One aspect
of the invention is the discovery that the minicell expression
system can be made to express and preferably display integral
membrane proteins from non-eubacterial organisms.
[0617] Some commonly used expression systems include in vitro
systems, such as the Rabbit Reticulocyte Lysate System and E. coli
S30 Extract System (both available from Promega) (Zubay, Methods
Enz. 65:856, 1980) and in vivo systems, such as eukaryotic cell
culture expression, and bacterial expression systems. Although this
is not an exhaustive list, these systems are representative.
[0618] The Rabbit Reticulocyte Lysate system utilizes a cell lysate
that contains all the enzymes required for transcription and
translation to drive protein expression, and is a good in vitro
system for producing small amounts of labeled and unlabeled
protein. However, this system is not well-suited for the production
of large quantities of proteins and is limited to soluble proteins
as there are no membranes in which to incorporate membrane
proteins.
[0619] In eukaryotic cell culture systems, expression vectors
suited for expression in host eukaryotic cells are transfected into
cultured cells and protein is translated from mRNA produced from
the vector DNA template Kaufman, Overview of vector design for
mammalian gene expression. Mol Biotechnol, 2001. 16: 151-160; Lee,
et al., Heterologous gene expression in avian cells: Potential as a
producer of recombinant proteins. J Biomed Sci, 1999. 6: 8-17;
Voorma et al., Initiation of protein synthesis in eukaryotes. Mol
Biol Rep, 1994. 19: 139-45). Cells can then either be harvested to
prepare at least partially purified proteins or proteins produced
from the expression element can be studied in the host cell
environment.
[0620] Regarding membrane proteins, such systems have limitations.
Primary cell lines are difficult to maintain and are short lived.
Immortalized cell lines divide indefinitely, but have been altered
in many ways and can be unpredictable. The transfection efficiency
is very low in most eukaryotic cells and some cell types are
refractory to transformation. Moreover, other proteins are
expressed in these cells along with the protein of interest. This
can cause difficulties when performing certain experiments and when
attempting to immunoprecipitate the protein. Good experimental data
are difficult to obtain from studies such as binding assays
(because of high background due to endogenous proteins), and
crystal determination of protein structure (because it is difficult
to obtain enough purified protein to efficiently form
crystals).
[0621] Bacterial expression systems are generally similar to that
of the eukaryotic expression systems in that they both use the host
cell enzymes to drive protein expression from recombinant
expression vectors (Cornelis, P., Expressing genes in different
Escherichia coli compartments. Curr Opin Biotechnol, 2000. 11: p.
450-454; Laage and Langosch, Strategies for prokaryotic expression
of eukaryotic membrane proteins. Traffic, 2001. 2: 99-104; Pines,
O. and M. Inouye, Expression and secretion in E. coli. Mol
Biotechnol, 1999. 12: 25-34).
[0622] In bacterial expression systems, bacterial cells are
transformed with expression elements, and transcription and
translation is driven from a bacterial promoter. Bacteria divide
very rapidly and are easy to culture; it is relatively easy to
produce a large number of bacteria in a short time. Moreover,
incorporation of expression elements vector into bacterial cells is
efficient. Transformed cells can be isolated that arise from a
single bacterium. Cultures of transformed cells are thus
genetically identical and all cells in the culture will contain the
expression element. However, there are proteins that are not
suitable for expression in bacteria because of differences between
eukaryotic cells and bacterial cells in transcription, translation,
and post-translational modification.
[0623] The E. coli whole cell expression system has been used to
express functional integral membrane proteins. For a review, see
Strosberg, Functional expression of receptors in microorganisms.
TiPS, 1992. 13: 95-98. Examples of mammalian integral membrane
proteins that have been expressed in Escherichia coli include rat
alpha-2B-adrenoceptors (Xia et al., Functional expression of rat
.beta.2B-adrenoceptor in E. coli. Euro J. Pharma, 1993. 246:
129-133) and the human beta2-adrenergic receptor (Marullo et al.,
Human 2-adrenergic receptors expressed in Escherichia coli
membranes retain their pharmacological properties. Proc. Natl.
Acad. Sci. USA, 1988. 85: 7551-7555). In some of these studies, the
integral membrane proteins were not only expressed in E. coli
expression systems, but also retained their pharmacological
properties. This allows for binding studies to be performed with
minimal background signal ("noise") from host cell proteins. It has
also been shown that signal sequences (the short hydrophobic amino
acid sequence at the N-terminus of integral membrane proteins that
signals the transport of the protein to the membrane) from
mammalian cells may be functional in the E. coli system.
[0624] As is discussed herein, the expression of membrane proteins
such as GPCRs, ion channels, and immuno-receptors in minicells, and
their incorporation into the membranes thereof, allows for the
study and use of such non-eubacterial membrane proteins. The
minicell system of the invention is particularly well-suited for
the study and expression of EDG proteins because of the lipid
nature of the ligands for these receptors. The identification of
ligand binding kinetics and biochemistry of these receptors because
of the physiochemical properties of the lipid ligands (LPA and
S1P), which results in high non-specific binding (Lee et al.,
Sphingosine-1-phosphate is a ligand for the G protein-coupled
receptor EDG-1. Science, 1998. 279: 1552-1555; Van Brocklyn et al.,
Sphingosine-1-phosphate is a ligand for the G protein-coupled
receptor EDG-6. Blood, 2000. 95: 2624-2629; Liu et al., Edg-1, the
G protein-coupled receptor for sphingosine-1-phosphate, is
essential for vascular maturation. J. Clin. Investigation, 2000.
106: 951-961).
[0625] It is believed, for example, that in the case of the ion
channels, the minicell expression system is less cumbersome then
procedures that are presently used to study properties of ion
channels, such as, e.g., reconstitution studies (Montal, Molecular
anatomy and molecular design of channel proteins. FASEB J., 1990.
4: p. 2623-2635). Ionic conditions both inside and outside of
minicells can be manipulated in various ways, and the properties of
an ion channel that is expressed in a minicell, and factors that
activate or modulate the activities of the channel, can be studied.
Binding and kinetic studies are performed on ligand mediated ion
channels. This type of study is enhanced when the ion channel is
able to interact specifically with its ligand and has a low
background of non-specific binding from the endogenous proteins.
This can be accomplished by making the minicells into protoplasts
or poroplasts in which the ligand-activated ion channels in the
inner membrane are exposed to the external environment and have
better access to their specific ligand.
[0626] A "recombinant expression system" (or simply "expression
system") is one that directs the production of exogenous gene
products in a host cell or minicell of choice. By "expressed" it is
meant that a gene product of interest (which can be a protein or
nucleic acid) is produced in the expression system of choice.
[0627] Host cells (and/or minicells) harboring an expression
construct are components of expression systems. An "expression
vector" is an artificial nucleic acid molecule into which an
exogenous ORF encoding a protein, or a template of a bioactive
nucleic acid can be inserted in such a manner so as to be operably
linked to appropriate expression sequences that direct the
expression of the exogenous gene. By the term "operably linked" it
is meant that the part of a gene that is transcribed is correctly
aligned and positioned with respect to expression sequences that
promote, are needed for and/or regulate this transcription. The
term "gene product" refers to either a nucleic acid (the product of
transcription, reverse transcription, or replication) or a
polypeptide (the product of translation) that is produced using the
non-vector nucleic acid sequences as a template.
[0628] In some applications, it is preferable to use an expression
construct that is an episomal element. If the episomal expression
construct expresses (or, preferably in some applications,
over-expresses) a an ORF that has been incorporated into the
episomal expression construct, the minicells will direct the
production of the polypeptide encoded by the ORF. At the same time,
any mRNA molecules transcribed from a chromosomal gene prior to
minicell formation that have been transferred to the minicell are
degraded by endogenous RNases without being replaced by new
transcription from the (absent) bacterial chromosome.
[0629] Chromosomally-encoded mRNAs will not be produced in
minicells and will be "diluted" as increasing amounts of mRNAs
transcribed from the episomal element are generated. A similar
dilution effect is expected to increase the relative amount of
episomally-generated proteins relative to any chromosomally-encoded
proteins present in the minicells. It is thus possible to generate
minicells that are enriched for proteins encoded by and expressed
from episomal expression constructs.
[0630] Although by no means exhaustive, a list of episomal
expression vectors that have been expressed in eubacterial
minicells is presented in Table 4.
[0631] It is also possible to transform minicells with exogenous
DNA after they have been prepared or separated from their parent
cells. For example, phage RNA is produced in minicells after
infection by lambda phage (Witkiewicz and Taylor, Ribonucleic acid
synthesis after adsorption of the bacteriophage lambda on
Escherichia coli minicells, Acta Microbiol Pol A 7:21-4, 1975),
even though replication of lambda phage may not occur in minicells
(Witkiewicz and Taylor, The fate of phage lambda DNA in
lambda-infected minicells, Biochim Biophys Acta 564:31-6,
1979).
[0632] Because it is the most characterized minicell-producing
species, many of these episomal elements have been examined in
minicells derived from E. coli. It is understood by practitioners
of the art, however, that many episomal elements that are expressed
in E. coli also function in other eubacterial species, and that
episomal expression elements for minicell systems in other species
are available for use in the invention disclosed herein.
[0633] In one aspect of the invention, eukaryotic and
archeabacterial minicells are used for expression of membrane
proteins, particularly in instances where such desirable proteins
have enhanced or altered activity after they undergo
post-translational modification processes such as phosphorlyation,
proteolysis, mystrilation, GPI anchoring and glycosylation.
Expression elements comprising expression sequence operably linked
to ORFs encoding the membrane proteins of interest are transformed
into eukaryotic cells according to methods and using expression
vectors known in the art. By way of non-limiting example, primary
cultures of rat cardiomyocytes have been used to produce exogenous
proteins after transfection of expression elements therefor by
electroporation (Nakajima et al., Expression and characterization
of Edg-1 receptors in rat cardiomyocytes: Calcium deregulation in
response to sphingosine-1-phosphate, Eur. J. Biochem. 267:
5679-5686, 2000).
[0634] Yeast cells that produce minicells are transformed with
expression elements comprising an ORF encoding a membrane protein
operably linked to yeast expression sequences. Cells that harbor a
transferred expression element may be selected using a gene that is
part of the expression element that confers resistant to an
antibiotic, e.g., neomycin.
[0635] Alternatively, in one aspect of the invention, bacterial
minicells are prepared that contain expression elements that are
prepared from shuttle vectors. A "shuttle vector" has sequences
required for its replication and maintenance in cells from two
different species of organisms, as well as expression elements, at
least one of which is functional in bacterial cells, and at least
one of which is functional in yeast cells. For example, E.
coli-yeast shuttle vectors are known in the art and include, by way
of non-limiting example, those derived from Yip, Yrp, Ycp and Yep.
Preferred E. coli-yeast shuttle vectors are episomal elements that
can segregate into yeast minicells (i.e., Yrp, Ycp and Yep.
Particularly preferred are expression vectors of the Yep (yeast
episomal plasmid) class, and other derivatives of the naturally
occurring yeast plasmid known as the 2 .mu.m circle. The latter
vectors have relatively high transformation frequencies and are
stably maintained through mitosis and meiosis in high copy
number.
TABLE-US-00005 TABLE 4 Episomal Elements That Segregate Into
Escherichia coli Minicells EPISOMAL ELEMENT REFERENCES Plasmids
R6K, Nesvera et al., Folia Microbiol. (Praha) 23: 278-285 R1DRD19
(1978) PSC101 Fox et al., Blood 69: 1394-1400 (1987) PBR322 Fox et
al., Blood 69: 1394-1400 (1987) F element Cohen et al., Proc. Natl.
Acad. Sci. 61: 61-68 (1968); Khachatourians G.G., Biochim. Biophys.
Acta. 561: 294-300 (1979) NR1 Hochmannova et al., Folia Microbiol.
(Praha) 26: 270-276 R6.delta.1 Hochmannova et al., Folia Microbiol.
(Praha) 26: 270-276 PTTQ18 Rigg et al., Arch. Oral. Biol. 45: 41-52
(2000) PGPR2.1 Rigg et al., Arch. Oral. Biol. 45: 41-52 (2000);
expresses cell surface antigen of P. gingivalis "mini-plasmid"
Firshein et al., J. Bacteriol. 150: 1234-1243 (1982) derivative of
RK2 ColE1 Rashtchian et al., J. Bacteriol. 165: 82-87 (1986);
Witkiewicz et al., Acta. Microbiol. Pol. A 7: 21-24 (1975) PSC101
Rashtchian et al., J. Bacteriol. 165: 82-87 (1986); Curtiss, Roy,
III, U.S. Pat. No. 4,190,495; Issued Feb. 26, 1980 pACYC184 Chang
et al., J. Bacteriol. 134: 1141-1156 (1978); Rose, Nucleic Acids
Res 16: 355 (1988) Co1Ib, Co1Ib7 Skorupska et al., Acta. Microbiol.
Pol. A 8: 17-26 (1976) DRD& pUC19 Heighway et al., Nucleic
Acids Res. 17: 6893-6901 (1989) R-plasmid Hochmannova et al., Folia
Microbiol. (Praha) 25: 11-15 (1980) PCR1 Hollenberg et al., Gene 1:
33-47 (1976); yeast shuttle vector Bacteriophage Lambda Witkiewicz
et al., Acta. Microbiol. Pol. A 7: 21-24 (1975) M13 Staudenbauer et
al., Mol. Gen. Genet. 138: 203-212 (1975) T7 Libby, Mech Ageing
Dev. 27: 197-206 (1984) P1 Curtiss, Roy, III, U.S. Pat. No.
4,190,495; Issued Feb. 26, 1980; J Bacteriol 1995; 177: 2381-6,
Partition of P1 plasmids in Escherichia coli mukB chromosomal
partition mutants, Funnell and Gagnier.
[0636] For expression of membrane proteins, and/or other proteins
of interest in the recipient cell, ORFs encoding such proteins are
operably linked to eukaryotic expression sequences that are
appropriate for the recipient cell. For example, in the case of E.
coli-yeast shuttle vectors, the ORFs are operably linked to
expression sequences that function in yeast cells and/or minicells.
In order to assess the effectiveness of a gene delivery vehicle, or
a gene therapy expression element, an ORF encoding a detectable
polypeptide (e.g., GFP, beta-galactosidase) is used. Because the
detectable polypeptide is operably linked to eukaryotic expression
elements, it is not expressed unless it has been transferred to its
recipient (eukaryotic) cell. The signal from the detectable
polypeptide thus correlates with the efficiency of gene transfer by
a gene delivery agent, or the degree of expression of a eukaryotic
expression element.
[0637] Gyuris and Duda (High-efficiency transformation of
Saccharomyces cells by bacterial minicell protoplast fusion, Mol
Cel Biol 6:329507, 1986) allegedly demonstrated the transfer of
plasmid molecular by fusing minicell protoplasts with yeast
protoplasts. Gyuris and Duda state that 10% of Saccharomyces
cerevisiae cells were found to contain transforming DNA sequences.
However, the plasmids did not contain eukaryotic expression
elements, were not shuttle vectors, and genetic expression of the
plasmids in yeast cells was not examined.
[0638] XII. Uses of Minicells in Research
[0639] XII.A. In General
[0640] The minicells of the invention can be used in research
applications such as, by way of non-limiting example, proteomics,
physiology, chemistry, molecular biology, physics, genetics,
immunology, microbiology, proteomics, virology, pathology, botany,
and neurobiology. Research applications include but are not limited
to protein-ligand binding studies, competitive inhibition studies,
structural studies, protein interaction studies, transfection,
signaling studies, viral interaction studies, ELISA, antibody
studies, gel electrophoresis, nucleotide acid) applications,
peptide production, cell culture applications, cell transport
studies, isolation and separation studies, chromatography, labeling
studies, synthesis of chemicals, chemical cross linking, flow
cytometry, nanotechnology, micro switches, micro-machines,
agricultural studies, cell death studies, cell-cell interactions,
proliferation studies, and protein-drug interactions. Minicells are
applicable to research applications involving, by way of
non-limiting example, the elucidation, manipulation, production,
replication, structure, modeling, observations, and
characterization of proteins.
[0641] The types of proteins that can be involved in research
applications of minicells can be either soluble proteins or
membrane bound proteins, and include but are not limited to
receptors (e.g., GPCRs, sphingolipid receptors, neurotransmitter
receptors, sensory receptors, growth factor receptors, hormone
receptors, chemokine receptors, cytokine receptors, immunological
receptors, and compliment receptors, FC receptors), channels (e.g.,
potassium channels, sodium channels, calcium channels), pores
(e.g., nuclear pore proteins, water channels), ion and other pumps
(e.g., calcium pumps, proton pumps), exchangers (e.g.,
sodium/potassium exchangers, sodium/hydrogen exchangers,
potassium/hydrogen exchangers), electron transport proteins (e.g.,
cytochrome oxidase), enzymes and kinases (e.g., protein kinases,
ATPases, GTPases, phosphatases, proteases), structural/linker
proteins (e.g., Caveolins, clathrin), adapter proteins (e.g., TRAD,
TRAP, FAN), chemotactic/adhesion proteins (e.g., ICAM11, selectins,
CD34, VCAM-1, LFA-1, VLA-1), and chimeric/fusion proteins (e.g.,
proteins in which a normally soluble protein is attached to a
transmembrane region of another protein).
[0642] Research products are designed for any specific type of
application. These products may be packaged and distributed as, by
way of non-limiting example, kits, chemicals, solutions, buffers,
powders, solids, filters, columns, gels, matrixes, emulsions,
pellets, capsules, and aerosols. Kits and reagents for certain
research applications may be required by regulatory agency to be
labeled "research use only" in order to indicate that the reagents
are not intended for use in humans.
[0643] XII.B. Transfection
[0644] Transfection is the process of introducing genetic material
into eukaryotic and archaebacterial cells using biological,
biochemical or physical methods. This process allows researchers to
express and study target proteins in cultured cells (research use)
as well as to deliver genetic material to cells in vivo or ex vivo
systems (gene therapy). There are a variety of techniques which
allow for the introduction and expression of proteins into target
cells. These include mechanical transfection (Biolistic particles
and Electroporation), calcium phosphate, DEAE-dextran/polybrene,
viral based techniques and lipid based techniques.
[0645] The genetic material and/or nucleic acid to be delivered can
be, by way of non-limiting example, nucleic acids that repair
damaged or missing genes, nucleic acids for research applications,
nucleic acids that kill a dysfunctional cell such as a cancer cell,
antisense oligonucleotides to reduce or inhibit expression of a
gene product, genetic material that increases expression of another
gene, nucleotides and nucleotide analogs, peptide nucleic acids
(PNAs), tRNAs, rRNAs, catalytic RNAs, RNA:DNA hybrid molecules, and
combinations thereof.
[0646] The genetic material may comprise a gene expressing a
protein. exemplary proteins include, but are not limited to,
receptors (e.g., GPCRs, sphingolipid receptors, neurotransmitter
receptors, sensory receptors, growth factor receptors, hormone
receptors, chemokine receptors, cytokine receptors, immunological
receptors, and compliment receptors, FC receptors), channels (e.g.,
potassium channels, sodium channels, calcium channels), pores
(e.g., nuclear pore proteins, water channels), ion and other pumps
(e.g., calcium pumps, proton pumps), exchangers (e.g.,
sodium/potassium exchangers, sodium/hydrogen exchangers,
potassium/hydrogen exchangers), electron transport proteins (e.g.,
cytochrome oxidase), enzymes and kinases (e.g., protein kinases,
ATPases, GTPases, phosphatases, proteases), structural/linker
proteins (e.g., Caveolins, clathrin), adapter proteins (e.g., TRAD,
TRAP, FAN), chemotactic/adhesion proteins (e.g., ICAM11, selectins,
CD34, VCAM-1, LFA-1, VLA-1), and chimeric/fusion proteins (e.g.,
proteins in which a normally soluble protein is attached to a
transmembrane region of another protein).
[0647] A minicell that is used to deliver therapeutic agents may
comprise and display a binding moiety. By way of non-limiting
example, binding moieties used for particular purposes may be a
binding moiety directed to a compound or moiety displayed by a
specific cell type or cells found predominantly in one type of
tissue, which may be used, among other things, to target minicells
and their contents to specific cell types or tissues. A preferred
binding moiety is an antibody or antibody derivative. Other binding
moieties include, but are not limited to, receptors, enzymes,
ligands, binding peptides, fusion proteins, small molecules
conjugated to transmembrane proteins, ligands conjugated to
transmembrane proteins, viral fusion proteins, and fusion/chimeric
proteins.
[0648] A minicell containing genetic material may be to a target
cell by methods including, but not limited to, receptor mediated
endocytosis, cell fusion, or phagocytosis (Aderem et al., Mechanism
of Phagocytosis in Macrophages, Annu. Rev. Immunol. 17:593-623,
1999). The minicell gene delivery system is used to deliver genetic
material in culture for research applications as well as to cells
in vivo as part of gene therapy or other therapeutic
applications.
[0649] By way of non-limiting example, a minicell may express a
protein such as invasin to induce receptor mediated endocytosis
(Pepe et al., "Yersinia enterocolitica invasin: A primary role in
the initiation of infection," Proc. Natl. Acad. Sci. U.S.A.
90:6473-6477, 1993; Alrutz et al., "Involvement of focal adhesion
kinase in invasin-mediated uptake," Proc. Natl. Acad. Sci. U.S.A.
95:13658-13663, 1998). Invasin interacts with the Beta2 Integrin
protein and causes it to dimerize. Upon dimerization the Beta2
Integrin signals for an endocytotic event. Thus a minicell
expressing the invasin protein will be taken up by cells expressing
Beta2 Integrin via endocytosis.
[0650] Another non-limiting example of the minicell gene delivery
and transfection system using invasin involves the expression of
invasin following a targeting event. In this example, a minicell
expresses a targeting protein that is capable of bringing the
minicell in contact with a specific target cell. Upon contact with
the target cell, the minicell will be induced to transcribe and
translate invasin. The induction is accomplished via signaling
events or with a transcription factor dimerization event. The
minicells can be engineered to contain targeting proteins that
induce protein expression only upon contact with a specific target
cell. By way of non-limiting example, the invasin is expressed only
at the target cell where it induces endocytosis, thus preventing
the minicell from entering any cell but the target cell.
[0651] Proteins can be induced and expressed post contact with
target cells include but are not limited to antibodies and antibody
derivatives, receptors, enzymes, ligands, binding peptides, fusion
proteins, small molecules conjugated to transmembrane proteins,
ligands conjugated to transmembrane proteins, viral fusion
proteins, antibiotics, apoptotic proteins, hormones, toxins,
poisons, and fusion/chimeric proteins.
[0652] Another non-limiting example of gene delivery or
transfection using the minicell involves the use of the type III
secretion apparatus of bacteria. The type III secretion apparatus
is expressed in the minicell and used to transfer genetic material
to a target cell.
[0653] Another non-limiting example of gene delivery and
transfection using minicells involves minicells that have been
engineered to contain anionic lipids or cationic lipids (Axel et
al., "Toxicity, Uptake Kinetics and Efficacy of New Transfection
Reagents: Increase of Oligonucleotide Uptake," Jour. of Vasc. Res.
040:1-14, 2000). Many types of lipids have been shown to induce or
enhance transfection and gene delivery in a variety of cell types.
Minicells containing such lipids could be used to transfer genetic
material to specific cell types. Minicells can also be engineered
to express targeting proteins that would allow the minicell to
associate tightly with a target cell, which will facilitate the
lipid interactions and gene transfer.
[0654] Another non-limiting example of gene delivery or
transfection using minicells involves the use of ligands to induce
receptor mediated endocytosis. By way of non-limiting example, the
ligand is expressed on the surface of the minicell, or is attached
to the surface of the minicell. A minicell containing genetic
material is then able to associate with a target cell expressing
the target receptor for the ligand. The receptor/ligand interaction
will result in the endocytosis of the minicell into the target cell
where the minicell would release and deliver the genetic
material.
[0655] Another non-limiting example of gene delivery or
transfection using minicells involves the use of fusion proteins,
such as but not limited to viral capsid proteins. In this example
the fusion protein would be expressed or attached to the outside of
the minicell. The fusion protein would then induce fusion of a
target cell with the minicell upon contact. The contact could be
initiated via random non-targeting events or via the use of
specific targeting proteins. In both cases the end result would be
the fusion of the minicell with a target cell and the delivery of
the genetic material.
[0656] XII.C. Non-Limiting Examples of Research Applications of
Minicells
[0657] XII.C.1. Phage Interactions With Bacterial Membranes
[0658] One non-limiting example of a research application for
minicells would be the study of phage interactions with a bacterial
membrane. The minicells could be used to study how phage associate
and enter into a host bacterium. Another non-limiting example is
the research application of minicells is to study isolated cell
signaling pathways. The proteins of a signaling pathway could be
expressed in the minicell and the signal cascade could be
monitored. Another non-limiting example of research applications is
the use of minicells to determine how recombination events occur.
In this example the minicell is used to provide an environment to
study the recombination event between two episomal plasmid DNA
units.
[0659] XII.C.2. Matrices
[0660] Another non-limiting example of a research application of
minicells is to form chromatography matrices for
immunoprecipitation, isolation and separation techniques. The
minicell can express and display target proteins with binding
activity, including but not limited to antibodies and antibody
derivatives. The minicell is then used to generate a matrix and
loaded in a column or tube. The solution to be separated is mixed
or passed through the column allowing the minicell to bind its
target. The minicells are then separated away with the attached
substance.
[0661] XII.C.3. Mutagenesis
[0662] Another non-limiting example of a research application for
minicells involves site directed mutagenesis studies of target
proteins. In this application minicells are generated to express
target proteins with various mutations and deletions to study if
function is compromised, enhanced or has an altered specificity for
ligand binding.
[0663] XII.C.4. Metabolic Pathways
[0664] Another non-limiting example of research applications for
minicells involves the study of metabolic rates of proteins and
metabolites. The minicell can be generated to express metabolic
pathways and the kinetics and function of that pathway can be
studied.
[0665] XII.C.5. Cell Free Production of Proteins
[0666] Another non-limiting example of a research application for
minicells involves uses in cell free production of functional
proteins (Jermutus et al., Recent advances in producing and
selecting functional proteins by using cell-free translation,
Current Opinion in Biotechnology 9:534-548, 1999). Minicells can be
prepared as a reagent used to prepare compositions for in vitro
translation. As is described in detail elsewhere herein, the
composition of minicells can be manipulated so as to be enriched
for particular proteins or nucleic acids, including those involved
in protein translation and folding and/or modification of the
proteins so produced into functional forms, i.e., forms having the
activity of the corresponding protein as it is isolated from
natural sources. Non-limiting examples of such proteins and nucleic
acids are ribosomal RNAs, ribosomal proteins, tRNAs, and the
like.
[0667] XII.C.6. Assays
[0668] Minicells could also be used in manual, semi-automated,
automated and/or robotic assays for the in vitro determinations of
the compounds of interest including, by way of non-limiting
example, ligands, proteins, small molecules, bioactive lipids,
drugs, heavy metals, and the like in environmental samples (e.g.,
air, water, soil), blood, urine or tissue of humans or samples from
non-human organisms (e.g., plants, animals, protists) for the
purpose of quantifying one or more compounds in a sample. A
non-limiting example of this type of research applications is the
expression on the surfaces of the minicells of a receptor such as
the receptor that binds a toxin produced by Bacillus anthracis. The
protein, protective antigen (PA), is a 82.7 kDa protein that binds
one of the secreted anthrax toxins, lethal factor (LF) (see Price,
B. et al., Infection and Immunity 69: 4509-4515. 2001). Minicells
expressing the PA protein could be used to detect LF in an
environmental sample or in human blood, urine or tissue for the
purposes of determining the presence of anthax. As a non-limiting
example, a competitive binding assay or an antibody-based assay
could be used to indicate binding of LF in the environmental or
tissue sample. Another non-limiting example is the use of
PA-expressing minicells in a lateral flow diagnostic where
interaction between the minicells and the LF-containing sample is
indicated by the presence of a colored reaction product on a test
strip.
[0669] XIII. Minicell-Based Delivery of Biologically Active
Agents
[0670] XIII.A. General Considerations
[0671] The minicells of the invention are capable of encapsulating
and/or loading into a membrane a variety of substances, including
but not limited to biologically active agents, including but not
limited to diagnostic and therapeutic agents. Biologically active
agents include, but are not limited to, nucleic acids, e.g., DNA,
RNA, gene therapy constructs, ribozymes, antisense and other
synthetic oligonucleotides including those with chemical
modifications; peptide nucleic acids (PNAs); proteins; synthetic
oligopeptides; peptomimetics; small molecules; radioisotopes;
antibiotics; antibodies and antibody derivatives; and combinations
and/or prodrugs of any of the preceding.
[0672] The surface of a minicell may be chemically altered in order
to have certain properties that are desirable for their use as drug
delivery agents. By way of non-limiting example, minicells may be
chemically conjugated to polyethylene glycol (PEG), which provides
for "stealth" minicells that are not taken as well and/or as
quickly by the reticuloendothelial system (RES). Other compounds
that may be attached to minicells include without limitation
polysaccharides, polynucleotides, lipopolysaccharides,
lipoproteins, glycosylated proteins, synthetic chemical compounds,
and/or combinations of any of the preceding.
[0673] A minicell that is used to deliver therapeutic agents may
comprise and display a binding moiety. By way of non-limiting
example, binding moieties used for particular purposes may be a
binding moiety directed to a compound or moiety displayed by a
specific cell type or cells found predominantly in one type of
tissue, which may be used, among other things, to target minicells
and their contents to specific cell types or tissues. A preferred
binding moiety is an antibody or antibody derivative, which are
described in detail elsewhere herein. Other binding moieties
include, but are not limited to, receptors, enzymes, ligands,
binding peptides, fusion proteins, small molecules conjugated to
transmembrane proteins, ligands conjugated to transmembrane
proteins, viral fusion proteins, and fusion/chimeric proteins.
[0674] XIII.B. Cellular Uptake
[0675] In addition to binding moieties, proteins and other
compounds that induce or enhance the uptake or fusion of the
minicell with the target gene can be displayed on the surface of a
minicell for applications involving the delivery of therapeutic
agents, gene therapy, and/or transfection or other research
applications. See, generally, Adhesion Protein Protocols, Vol. 96,
Dejana, E. and Corada, M., eds., Humana Press, 1999.
[0676] XIII.B.1. Cellular Uptake Sequences from Eukaryotic
Cells
[0677] Eukaryotic adhesion receptors, which mediate intercellular
adhesion, can be used as agents or targets for cellular uptake.
There are at least three distinct classes of adhesive molecules
that leukocytes employ during their adhesive interactions (a)
integrins, including but not limited to LEC-CAMS/Selectins (ELAM-1,
LAM-1/Leu8/TQ1, and GMP140/PADGEM); (b) those belonging to the
immunoglobulin superfamily including but not limited to CD2(LFA-2),
CD3/TCR, CD4, CD8, CD28, CD44, CD54 (ICAM-1), ICAM-2, CD58 (LFA-3),
VCAM-1,B7; and (c) Class I and II Major Histocompatability Antigens
(MHC).
[0678] The adhesion receptors that belong to the integrin family
and control intercellular interactions are of particular interest.
At least ten different structurally related cell surface
heterodimeric (alpha and beta complexes) molecules have been
defined as integrins and further classified into subfamilies
(Springer T. A., 1990, Nature 346:425-434; Hynes, R. O., 1987, Cell
48:549-554; Moller, G. Editor, 1990, Immunol. Rev. 114.:1-217).
Each subfamily has a unique beta subunit, designated integrin beta1
(CD29), integrin beta2 (CD18), and integrin beta3 (CD61), each of
which can associate with multiple alpha subunits, each with at
least one di-valent cation binding site. The integrin family
includes receptors for extracellular matrix components such as
fibronectin, laminin, vitronectin, and collagen which recognize
Arg-Gly-Asp in their ligands and utilize the beta1 or beta3
subunits (Springer T. A., 1990, Nature 346:425-434; Hynes, R. 0.,
1987, Cell 48:549-554; Hemler, M. E., 1988, Immunol. Today
9:109-113; Patarroyo, M., and Makgoba, M. W., 1989, Scand. J.
Immunol. 30:129-164; Moller, G. Editor, 1990, Immunol. Rev.
11.4:1-217). There are at least six distinct alpha subunits alpha1
(CD49a), alpha2 (CD49b), alpha3 (CD49c), alpha4 (CD49d), alpha5
(CD49e), and alpha6 (CD49f) capable of associating with beta1
(CD29). The beta1 integrins are expressed on many nonhematopoietic
and leukocyte cell types and are thought to play an active role in
tissue organization by binding to extracellular matrix components
found in many tissues and in the basement membranes underlying
muscles, nervous system, epithelium and endothelium. While the
expression of many beta1 integrins on leukocytes requires
consistent activation, their expression on nonhematopoietic cells
does not (Hemler, M. E., 1988, Immunol. Today 9:109-113; Patarroyo,
M., and Makgoba, M. W., 1989, Scand. J. Immunol. 30:129-164). The
complexity of the integrin family has been increased by the
discovery of novel beta subunits beta3 (CD61), beta4 and beta5 that
can associate with alpha 4, alpha 6, and alpha V subunits (Springer
T. A., 1990, Nature 346:425-434; Hemler, M. E., 1988, Immunol.
Today 9:109-113). This combinatorial use of alpha and beta subunits
confers considerable diversity in ligand recognition and also helps
regulate communications between the inside and outside of the
cell.
[0679] By way of non-limiting example, a minicell display an
adhesion receptor, or a fusion protein that has a transmembrane
domain linked to a functional portion of an adhesion receptor. Such
minicells will bind to cells displaying the ligand for the adhesion
receptor.
[0680] XIII.B.2. Cellular Uptake Sequences from Prokaryotes
[0681] Bacterial adhesion proteins are another source of
polypeptides that are used to stimulate uptake of minicells. See,
generally, Handbook of Bacterial Adhesion: Priniciples, Methods,
and Applications, Yuehuei H. An; Richard J. Friedman, eds., Humana
Press, 2000; and Hultgren et al., "Bacterial Adhesions and Their
Assembly," Chapter 150 in: Escherichia coli and Salmonella
typhimurium: Cellular and Molecular Biology, 2.sup.nd Ed.,
Neidhardt, Frederick C., Editor in Chief, American Society for
Microbiology, Washington, D.C., 1996, Volume 2, pages 1903-1999,
and references cited therein.
[0682] By way of non-limiting example, a minicell may express a
protein such as invasin to induce receptor mediated endocytosis
(Pepe et al., Yersinia enterocolitica invasin: A primary role in
the initiation of infection, Proc. Natl. Acad. Sci. U.S.A.
90:6473-6477, 1993; Alrutz et al., Involvement of focal adhesion
kinase in invasin-mediated uptake, Proc. Natl. Acad. Sci. U.S.A.
95:13658-13663, 1998). Invasin interacts with the Beta2 Integrin
protein and causes it to dimerize. Upon dimerization the Beta2
Integrin signals for an endocytotic event. Thus a minicell
expressing the invasin protein will be taken up by cells expressing
Beta2 Integrin via endocytosis.
[0683] As another non-limiting example, the pneumococcal adhesin
protein CpbA interacts with the human polyimmunoglobulin receptor
(hpIgR) as either a part of the outer surface of a bacterial cell
or as a free molecule Zhang et al. (Cell 102:827-837, 2000). The
regions of CpbA:hpIgR interaction were mapped using a series of
large peptide fragments derived from CpbA. CpbA (Swiss-Prot
Accession No. O30874) contains a choline binding domain containing
residues 454-663 and two N-terminal repetitive regions called R1
and R2 that are contained in residues 97-203 and 259-365,
respectively. Polypeptides containing R1 and R2 interact with
hpIgR, whereas polypeptides containing other sequences from CpbA do
not bind to hpIgR. The R1 and/or R2 sequences of the CpbA
polypeptide, and/or essentially identical, substantially identical,
or homologous amino acid sequences, are used to facilitate the
uptake of minicells by cells.
[0684] Another non-limiting example of gene delivery or
transfection using the minicell involves the use of the type III
secretion apparatus of bacteria. The type III secretion apparatus
is expressed in the minicell and used to transfer genetic material
to a target cell.
[0685] Other non-limiting examples of a minicell gene delivery and
transfection targeting moiety are ETA (detoxified exotoxin a)
protein delivery element described in U.S. Pat. No. 6,086,900 to
Draper; Interalin and related proteins from Listeria species
(Galan, Alternative Strategies for Becoming an Insider: Lessons
from the Bacterial World, Cell 103:363-366, 2000); Intimin from
pathogenic E. coli strains (Frankel et al., Intimin and the host
cell--is it bound to end in Tir(s)? Trends in Microbiology
9:214-218); and SpeB, streptococcal pyrogenic exotoxin B
(Stockbauer et al., A natural variant of the cysteine protease
virulence factor of group A Streptococcus with an
arginine-glycine-aspartic acid (RGD) motif preferentially binds
human integrins .alpha..sub.v.beta..sub.3 and
.alpha..sub.IIb.beta..sub.3 Proc. Natl. Acad. Sci. U.S.A.
96:242-247, 1999).
[0686] XIII.B.3. Cellular Uptake Sequences from Viruses
[0687] Cellular uptake sequences derived from viruses include, but
are not limited to, the VP22 protein delivery element derived from
herpes simplex virus-1 and vectors containing sequences encoding
the VP22 protein delivery element are commercially available from
Invitrogen (Carlsbad, Calif.; see also U.S. Pat. No. 6,017,735 to
Ohare et al.); and the Tat protein delivery element derived from
the amino acid sequence of the Tat protein of human
immunodeficiency virus (HIV). See U.S. Pat. Nos. 5,804,604;
5,747,641; and 5,674,980.
[0688] XIII.B.4. Lipids
[0689] Another non-limiting example of gene delivery and
transfection using minicells involves minicells that have been
engineered to contain anionic lipids or cationic lipids (Axel et
al., Toxicity, Uptake Kinetics and Efficacy of New Transfection
Reagents: Increase of Oligonucleotide Uptake, Jour. of Vasc. Res.
040:1-14, 2000). Many types of lipids have been shown to induce or
enhance transfection and gene delivery in a variety of cell types.
Minicells containing such lipids could be used to transfer genetic
material to specific cell types. Minicells can also be engineered
to express targeting proteins that would allow the minicell to
associate tightly with a target cell, which will facilitate the
lipid interactions and gene transfer.
[0690] Another non-limiting example of gene delivery or
transfection using minicells involves the use of ligands to induce
receptor mediated endocytosis. By way of non-limiting example, the
ligand is expressed on the surface of the minicell, or is attached
to the surface of the minicell. A minicell containing genetic
material is then able to associate with a target cell expressing
the target receptor for the ligand. The receptor/ligand interaction
will result in the endocytosis of the minicell into the target cell
where the minicell would release and deliver the genetic
material.
[0691] Another non-limiting example of gene delivery or
transfection using minicells involves the use of fusion proteins,
such as but not limited to viral capsid proteins. In this example
the fusion protein would be expressed or attached to the outside of
the minicell. The fusion protein would then induce fusion of a
target cell with the minicell upon contact. The contact could be
initiated via random non-targeting events or via the use of
specific targeting proteins. In both cases the end result would be
the fusion of the minicell with a target cell and the delivery of
the genetic material.
[0692] XIII.C. Post-Targeting Expression of Cellular Uptake
Sequences
[0693] Another non-limiting example of the minicell gene delivery
and transfection system using invasin involves the expression of
invasin following a targeting event. In this example, a minicell
expresses a targeting protein that is capable of bringing the
minicell in contact with a specific target cell. Upon contact with
the target cell, the minicell will be induced to transcribe and
translate invasin. The induction is accomplished via signaling
events or with a transcription factor dimerization event. The
minicells can be engineered to contain targeting proteins that
induce protein expression only upon contact with a specific target
cell. By way of non-limiting example, the invasin is expressed only
at the target cell where it induces endocytosis, thus preventing
the minicell from entering any cell but the target cell.
[0694] Proteins can be induced and expressed post contact with
target cells include but are not limited to antibodies and antibody
derivatives, receptors, enzymes, ligands, binding peptides, fusion
proteins, small molecules conjugated to transmembrane proteins,
ligands conjugated to transmembrane proteins, viral fusion
proteins, antibiotics, apoptotic proteins, hormones, toxins,
poisons, and fusion/chimeric proteins.
[0695] XIII.D. Intracellular Targeting and Organellar Delivery
[0696] After delivery to and entry into a targeted cell, a minicell
may be designed so as to be degraded, thereby releasing the
therapeutic agent it encapsulates into the cytoplasm of the cell.
The minicell and/or therapeutic agent may include one or more
organellar delivery elements, which targets a protein into or out
of a specific organelle or organelles. For example, the ricin A
chain can be included in a fusion protein to mediate its delivery
from the endosome into the cytosol. Additionally or alternatively,
delivery elements for other organelles or subcellular spaces such
as the nucleus, nucleolus, mitochondria, the Golgi apparatus, the
endoplasmic reticulum (ER), the cytoplasm, etc. are included
Mammalian expression constructs that incorporate organellar
delivery elements are commercially available from Invitrogen
(Carlsbad, Calif.: pShooter.TM. vectors). An H/KDEL (i.e.,
His/Lys-Asp-Glu-Leu sequence) may be incorporated into a fusion
protein of the invention, preferably at the carboxy-terminus, in
order to direct a fusion protein to the ER (see Andres et al., J.
Biol. Chem. 266:14277-142782, 1991; and Pelham, Trends Bio. Sci.
15:483-486, 1990).
[0697] Another type of organellar delivery element is one which
directs the fusion protein to the cell membrane and which may
include a membrane-anchoring element. Depending on the nature of
the anchoring element, it can be cleaved on the internal or
external leaflet of the membrane, thereby delivering the fusion
protein to the intracellular or extracellular compartment,
respectively. For example, it has been demonstrated that mammalian
proteins can be linked to i) myristic acid by an amide-linkage to
an N-terminal glycine residue, to ii) a fatty acid or
diacylglycerol through an amide- or thioether-linkage of an
N-terminal cysteine, respectively, or covalently to iii) a
phophotidylinositol (PI) molecule through a C-terminal amino acid
of a protein (for review, see Low, Biochem. J. 244:1-13, 1987). In
the latter case, the PI molecule is linked to the C-terminus of the
protein through an intervening glycan structure, and the PI then
embeds itself into the phopholipid bilayer; hence the term "GPI"
anchor. Specific examples of proteins know to have GPI anchors and
their C-terminal amino acid sequences have been reported (see Table
1 and FIG. 4 in Low, Biochemica et Biophysica Acta, 988:427-454,
1989; and Table 3 in Ferguson, Ann. Rev. Biochem., 57:285-320,
1988). Incorporation of GPI anchors and other membrane-targeting
elements into the amino- or carboxy-terminus of a fusion protein
can direct the chimeric molecule to the cell surface.
[0698] XIII.E. Minicell-Based Gene Therapy
[0699] The delivery of nucleic acids to treat diseases or disorders
is known as gene therapy (Kay et al., Gene Therapy, Proc. Natl.
Acad. Sci. USA 94:12744-12746, 1997). It has been proposed to use
gene therapy to treat genetic disorders as well as pathogenic
diseases. For reviews, see Desnick et al., Gene Therapy for Genetic
Diseases, Acta Paediatr. Jpn. 40:191-203, 1998; and Bunnell et al.,
Gene Therapy for Infectious Diseases, Clinical Microbiology Reviews
11:42-56, 1998).
[0700] Gene delivery systems use vectors that contain or are
attached to therapeutic nucleic acids. These vectors facilitate the
uptake of the nucleic acid into the cell and may additionally help
direct the nucleic acid to a preferred site of action, e.g., the
nucleus or cytoplasm (Wu et al., "Delivery Systems for Gene
Therapy," Biotherapy 3:87-95, 1991). Different gene delivery
vectors vary with regards to various properties, and different
properties are desirable depending on the intended use of such
vectors. However, certain properties (for example, safety, ease of
preparation, etc.) are generally desirable in most
circumstances.
[0701] The minicells of the invention may be used as delivery
agents for any therapeutic or diagnostic agent, including without
limitation gene therapy constructs. Minicells that are used as
delivery agents for gene therapy constructs may, but need not be,
targeted to specific cells, tissues, organs or systems of an
organism, of a pathogen thereof, using binding moieties as
described in detail elsewhere herein.
[0702] In order to enhance the effectiveness of gene delivery
vectors in, by way of non-limiting example, gene therapy and
transfection, it is desirable in some applications of the invention
to target specific cells or tissues of interest (targeted cells or
tissues, respectively). This increases the effective dose (the
amount of therapeutic nucleic acid present in the targeted cells or
tissues) and minimizes side effects due to distribution of the
therapeutic nucleic acid to other cells. For reviews, see Peng et
al., "Viral Vector Targeting," Curr. Opin. Biotechnol. 10:454-457,
1999; Gunzburg et al., "Retroviral Vector Targeting for Gene
Therapy," Cytokines Mol. Ther. 2:177-184, 1996.; Wickham,
"Targeting Adenovirus," Gene Ther. 7:110-114, 2000; Dachs et al.,
"Targeting Gene Therapy to Cancer: A Review," Oncol. Res.
9:313-325, 1997; Curiel, "Strategies to Adapt Adenoviral Vectors
for Targeted Delivery," Ann NY Acad. Sci. 886:158-171, 1999;
Findeis et al., "Targeted Delivery of DNA for Gene Therapy via
Receptors," Trends Biotechnol. 11:202-205, 1993.
[0703] Some targeting strategies make use of cellular receptors and
their natural ligands in whole or in part. See, for example,
Cristiano et al., "Strategies to Accomplish Gene Delivery Via the
Receptor-Mediated Endocytosis Pathway," Cancer Gene Ther., Vol. 3,
No. 1, pp. 49-57, January-February 1996.; S.C. Philips,
"Receptor-Mediated DNA Delivery Approaches to Human Gene Therapy,"
Biologicals, Vol. 23, No. 1, pp. 13-6, March 1995; Michael et al.,
"Strategies to Achieve Targeted Gene Delivery Via the
Receptor-Mediated Endocytosis Pathway," Gene Ther., Vol. 1, No. 4,
pp. 223-32, July 1994; Lin et al., "Antiangiogenic Gene Therapy
Targeting The Endothelium-Specific Receptor Tyrosine Kinase Tie2,"
Proc. Natl. Acad. Sci., USA, Vol. 95, pp. 8829-8834, 1998. Sudimack
et al., "Targeted Drug Delivery Via the Folate Receptor," Adv. Drug
Deliv., pp. 147-62, March 2000; Fan et al., "Therapeutic
Application of Anti-Growth Factor Receptor Antibodies," Curr. Opin.
Oncol., Vol. 10, No. 1, pp. 67-73, January 1998; Wadhwa et al.,
"Receptor Mediated Glycotargeting," J. Drug Target, Vol. 3, No. 2,
pp. 111-27, 1995; Perales et al., "An Evaluation of
Receptor-Mediated Gene Transfer Using Synthetic DNA-Ligand
Complexes," Eur. J. Biochem, Vol. 1, No 2, pp. 226, 255-66,
December 1994; Smith et al., "Hepatocyte-Directed Gene Delivery by
Receptor-Mediated Endocytosis," Semin Liver Dis., Vol. 19, No. 1,
pp. 83-92, 1999.
[0704] Antibodies, particularly single-chain antibodies, to surface
antigens specific for a particular cell type may also be used as
targeting elements. See, for example, Kuroki et al., "Specific
Targeting Strategies of Cancer Gene Therapy Using a Single-Chain
Variable Fragment (scFv) with a High Affinity for CEA," Anticancer
Res., pp. 4067-71, 2000; U.S. Pat. No. 6,146,885, to Dornburg,
entitled "Cell-Type Specific Gene Transfer Using Retroviral Vectors
Containing Antibody-Envelope Fusion Proteins"; Jiang et al., "In
Vivo Cell Type-Specific Gene Delivery With Retroviral Vectors That
Display Single Chain Antibodies," Gene Ther. 1999, 6:1982-7;
Engelstadter et al., "Targeting Human T Cells By Retroviral Vectors
Displaying Antibody Domains Selected From A Phage Display Library,"
Hum. Gene Ther. 2000, 11:293-303; Jiang et al., "Cell-Type-Specific
Gene Transfer Into Human Cells With Retroviral Vectors That Display
Single-Chain Antibodies," J. Virol 1998,72:10148-56; Chu et al.,
"Toward Highly Efficient Cell-Type-Specific Gene Transfer With
Retroviral Vectors Displaying Single-Chain Antibodies," J. Virol
1997, 71:720-5; Chu et al., "Retroviral Vector Particles Displaying
The Antigen-Binding Site Of An Antibody Enable Cell-Type-Specific
Gene Transfer," J. Virol 1995, 69:2659-63; and Chu et al., "Cell
Targeting With Retroviral Vector Particles Containing
Antibody-Envelope Fusion Proteins," Gene Ther. 1994, 1:292-9.
[0705] Minicells are used to deliver DNA-based gene therapy to
targeted cells and tissues. Double minicell transformants are used
not only to target a particular cell/tissue type (e.g. HIV-infected
T-cells) but are also engineered to fuse with and enter targeted
cells and deliver a protein-based toxin (e.g., antibiotic, or
pro-apoptotic gene like Bax), an antisense expression construct
(e.g., antisense to a transcription factor), or antisense
oligonucleotides (e.g., antisense to an anti-apoptotic gene such as
Bcl-2. The doubly-transformed minicells express not only these cell
death promoters, but also only target particular cells/tissues,
thus minimizing toxicity and lack of specificity of gene therapy
vectors. By "doubly-transformed" it is meant that the minicell
comprises 2 expression elements, one eubacterial, the other
eukaryotic. Alternatively, shuttle vectors, which comprise
eubacterial and eukaryotic expression elements in one vector, may
be used.
[0706] Minicell-based gene therapy is used to deliver expression
plasmids that could correct protein expression deficiencies or
abnormalities. As a non-limiting example, minicell inhalants are
targeted to pulmonary alveolar cells and are used to deliver
chloride transporters that are deficient or otherwise material in
cystic fibrosis. Protein hormone deficiencies (e.g., dwarfism) are
corrected by minicell expression systems (e.g., growth hormone
expression in pituitary cells). Duchene's muscular dystrophy is
characterized by a mutation in the dystrophin gene; this condition
is corrected by minicell-based gene therapy. Angiogenesis treatment
for heart patients is made effective by FGF or VGEF-producing
minicells targeted to the heart. In this case, plasmid-driven
over-expression of these grown factors is preferred.
[0707] XIV. Therapeutic Uses of Minicells
[0708] In addition to minicell-based gene therapy, minicells can be
used in a variety of therapeutic modalities. Non-limiting examples
of these modalities include the following applications.
[0709] XIV.A. Diseases and Disorders
[0710] Diseases and disorders to which the invention can be applied
include, by way of non-limiting example, the following.
[0711] Diseases and disorders that involve the respiratory system,
such as cystic fibrosis, lung cancer and tumors, asthma, pathogenic
infections, allergy-related diseases and disorders, such as asthma;
allergic bronchopulmonary aspergillosis; hypersensitivity
pneumonia, eosinophilic pneumonia; emphysema; bronchitis; allergic
bronchitis bronchiectasis; cystic fibrosis; hypersensitivity
pneumotitis; occupational asthma; sarcoid, reactive airway disease
syndrome, interstitial lung disease, hyper-eosinophilic syndrome,
parasitic lung disease and lung cancer, asthma, adult respiratory
distress syndrome, and the like;
[0712] Diseases and disorders of the digestive system, such as
those of the gastrointestinal tract, including cancers, tumors,
pathogenic infections, colitis; ulcerative colitis, diverticulitis,
Crohn's disease, gastroenteritis, inflammatory bowel disease, bowel
surgery ulceration of the duodenum, a mucosal villous disease
including but not limited to coeliac disease, past infective
villous atrophy and short gut syndromes, pancreatitis, disorders
relating to gastroinstestinal hormones, Crohn's disease, and the
like;
[0713] Diseases and disorders of the skeletal system, such as
spinal muscular atrophy, rheumatoid arthritis, osteoarthritis,
osteoporosis, multiple myeloma-related bone disorder,
cortical-striatal-spinal degeneration, and the like;
[0714] Autoimmune diseases, such as Rheumatoid arthritis (R A),
multiple sclerosis (MS), Sjogren's syndrome, sarcoidosis, insulin
dependent diabetes mellitus (IDDM), autoimmune thyroiditis,
reactive arthritis, ankylosing spondylitis, scleroderma,
polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener's
granulomatosis, Crohn's disease and ulcerative colitis amyotrophic
lateral sclerosis, multiple sclerosis, autoimmune gastritis,
systemic lupus erythematosus, autoimmune hemolytic anemia,
autoimmune neutropenia, systemic lupus erythematosus, graft vs.
host disease, bone marrow engraftment, some cases of Type I
diabetes, and the like;
[0715] Neurological diseases and disorders, such as depression,
bipolar disorder, schizophrenia, Alzheimer's disease, Parkinson's
disease, familial tremors, Gilles de la Tourette syndrome, eating
disorders, Lewy-body dementia, chronic pain and the like;
[0716] Pathological diseases and resultant disorders such as
bacterial infections such as infection by Escherichia, Shigella,
Salmonella; sepsis, septic shock, and bacteremia; infections by a
virus such as HIV, adenovirus, smallpox virus, hepatovirus, and the
like; and AIDS-related encephalitis, HIV-related encephalitis,
chronic active hepatitis, and the like;
[0717] Proliferative disease and disorders, such as acute
lymphoblastic leukemia, acute myelogenous leukemia, chronic
myelogenous leukemia, metastatic melanoma, Kaposi's sarcoma,
multiple myeloma, breast cancer, anal cancer, vulvar cancer, and
the like; and
[0718] Various diseases, disorders and traumas including, but not
limited to, apoptosis mediated diseases, inflammation, cerebral
ischemia, myocardial ischemia, aging, sarcoidosis, granulomatous
colitis, scleroderma, degenerative diseases, necrotic diseases,
alopecia, neurological damage due to stroke, diffuse cerebral
cortical atrophy, Pick disease, mesolimbocortical dementia,
thalamic degeneration, Huntington chorea, cortical-basal ganglionic
degeneration, cerebrocerebellar degeneration, familial dementia
with spastic paraparesis, polyglucosan body disease, Shy-Drager
syndrome, olivopontocerebellar atrophy, progressive supranuclear
palsy, dystonia musculorum deformans, Hallervorden-Spatz disease,
Meige syndrome, acanthocytic chorea, Friedreich ataxia, Holmes
familial cortical cerebellar atrophy,
Gerstmann-Straussler-Scheinker disease, progressive spinal muscular
atrophy, progressive balbar palsy, primary lateral sclerosis,
hereditary muscular atrophy, spastic paraplegia,
glomeralonephritis, chronic thyroiditis, Grave's disease,
thrombocytopenia, myasthenia gravis, psoriasis, peroneal muscular
atrophy, hypertrophic interstitial polyneuropathy, heredopathia
atactica polyneuritiformis, optic neuropathy, and
ophthalmoplegia.
[0719] A variety of diseases and disorders caused or exacerbated by
pathogens may be treated using the invention. For a comprehensive
description of pathogens and associated diseases and disorders, see
Zinsser Microbiology, 20th Ed., Joklik, ed.,
Appelton-Century-Crofts, Norwalk, Conn., 1992, and references cited
therein.
[0720] Minicells could also be used for replacement therapy (via
gene therapy) in a variety of conditions known to be caused by
protein or proteins that are either absent (e.g. Duchene's Muscular
Dystrophy), reduced in level (Dwarfism) or abberant (Sickle-cell
anemia).
[0721] For a comprehensive description of diseases and disorders
that may be treated using the invention, see The Merck Manual of
Diagnosis and Therapy, 17th Ed., Beers et al., eds.; published
edition, Merck and Co., Rahway, N.J., 1999; on-line edition,
Medical Services, Usmedsa, USHH,
http://www.merck.com/pubs/mmanual/, and references cited
therein.
[0722] XIV.B. Removal of Toxins and Pathogens by Selective
Absorption
[0723] When introduced into the bloodstream of an animal,
receptor-displaying minicells bind and absorb toxic compounds,
thereby removing such compounds from the general circulation. A
therapeutic benefit ensues as the bound toxic compound cannot
access the cells upon which it would otherwise exert its toxic
effect.
[0724] Minicells expressing receptors for toxic substances are
introduced IV in order to remove those toxins from the blood. One
non-limiting example is in the treatment of sepsis. In one
embodiment, a fusion protein is formed from the transmembrane
domain of the EGF receptor or toxR and a known soluble receptor for
LPS (lipopolysaccharide), such as the LBP (lipopolysaccharide
binding protein) or the extracellular domain of CD14 receptor
protein, both of which bind the LPS bacterial endotoxin. These
minicells inactivate LPS by initially binding to it and preventing
LPS binding to naturally occurring CD14 receptors on heart cells
and other cells involved in the endotoxic shock response.
Eventually, the minicell-LPS complex is cleared from the blood by
macrophages and other components of the immune system.
[0725] In another embodiment, minicells expressing receptors for
toxic drugs (e.g., morphine) are used to treat drug overdoses. In
other embodiments, minicells of the invention are used to express
receptors to venoms (e.g., snake venom) or poisons (e.g.,
muscarinic receptor expression for the treatment of muscarine
poisoning). In other embodiments, minicells of the invention
expressing EDGRs are used to clear the blood of toxins and other
undesirable compounds.
[0726] As another non-limiting example, minicells that bind
pathogens are used to treat disease. Minicells, and pathogens bound
thereto, may be ingested by human neutrophils and thus serve as an
adjuvant to therapeutic processes mediated by neutrophils (Fox et
al., Fate of the DNA in plasmid-containing Escherichia coli
minicells ingested by human neutrophils, Blood 69:1394-400, 1987).
In a related modality, minicells are used to bind compounds
required for the growth of a pathogen.
[0727] XIV.C. Antiviral Therapy
[0728] In one modality, minicells of the invention are used as
"sponges" for the selective absorption of any viral particle in the
body. Without being limited to the following examples, minicells
expression receptors or antibodies selectively directed against
viruses such as HIV, Hepatitis B and smallpox are used.
[0729] For the treatment of viremia, viruses are cleared from the
blood by absorption during dialysis or by IV injection of minicells
expressing targets for viral receptors. As the minicells interact
with blood-borne virus particles, there is an initial reduction of
host cell infection by virtue of the minicell-viral complexes that
are formed. Since viral particles attach to and/or enter the
minicell, they are not active because of the lack of machinery
needed for viral replication in the minicells. The virus infected
minicells are then cleared from the system by macrophages and
processed by the immune system.
[0730] Certain retroviruses that infect particular host cells
express viral proteins on the surfaces of the infected cells. HIV
infection of T-cells is one non-limiting example of this. The viral
protein, GP120, is expressed on the surfaces of infected T-cells
(Turner et al., Structural Biology of HIV, J. Mol. Biol. 285:1-32,
1999). Minicells expressing CD4 act as anti-GP120 minicells not
only to target virus particles in an infected patient, but also to
identify infected T-cells. It may be desirable to also express
co-receptors such as CCR5, CXR4 or ARD (Dragic, An overview of the
determinants of CCR5 and CXCR4 co-receptor function, J. Gen. Virol.
82:1807-1814, 2001). The minicells are then used to kill the
infected T-cells, or to inhibit viral replication and/or virion
assembly.
[0731] In another non-limiting example of anti-pathogen therapy,
minicells can by used to express bacterial surface antigens on
their surfaces that facilitate cellular uptake of the minicell by
intracellular pathogens such as Mycobacterium tuberculosis (the
causative agent of tuberculosis), Rickettsiae, or viruses. In this
"smart sponge" approach, selective absorption is accompanied by
internalization of the pathogen by minicells. Destruction of the
pathogen follows as a result of a combination of intraminicell
digestion of pathogens and/or by the eventual processing of the
virus-containing minicell by the cellular immune system of the
patient.
[0732] XIV.D. Antibacterial and Antiparasitic Applications
[0733] Minicells may be used to kill pathogenic bacteria,
protozoans, yeast and other fungi, parasitic worms, viruses and
other pathogens by mechanisms that either do or do not rely on
selective absorption. Antibiotics can be delivered to pathogenic
organisms after first being targeted by the proteins or small
molecules on the surfaces of the minicells that promote binding of
the minicells to the surfaces of the pathogen. Fusion or injection
of minicell contents into the pathogenic cell can result in the
death or disablement of the pathogen and thus lower the effective
dose of an antibiotic or gene therapeutic agent. Delivery of
antibiotics tethered to or encapsulated by the minicells will
reduce the effective dose of an antibiotic and will reduce its
elimination by the renal system. In the case of delivering
encapsulated molecules (e.g., antibiotics), purified/isolated
minicells expressing membrane-bound proteins for targeting can be
incubated with the molecules in vitro prior to administration. This
would be particularly applicable to the use of protoplast minicells
or poroplast minicells that have their outer membrane and cell wall
or outer membrane only removed, respectively, thus facilitating the
diffusion of the small molecule into the intact minicell.
[0734] Without being limited by the following example, minicells
can be use as antibacterial agents by expressing on the surfaces of
the minicells antigens, receptors, antibodies, or other targeting
elements that will target the minicell to the pathogenic organism
and facilitate the entry of plasmids, proteins, small molecules in
order to gain access to or entry into the organism. Antibiotics may
be encapsulated by minicells post isolation from the parent strain
so that the antibiotic will not be effective against the
minicell-producing bacteria itself. Since minicells are not able to
reproduce, the antibiotic will not be lethal to the minicell
delivery vehicle, but only to the targeted pathogen. In another
non-limiting example, lyosgenic factors e.g., complement may be
expressed on the surfaces of the minicells or encapsulated by same
as to promote lysis of the pathogen.
[0735] Minicells can also be engineered to express toxic proteins
or other elements upon binding to the pathogen. Induction of
minicell protein expression can be an event that is coincident with
targeting or triggered by minicell binding to the target pathogen,
thus making minicells toxic only when contact is made with the
pathogenic organism. Minicells can be engineered to express
fusion/chimeric proteins that are tethered to the membrane by
transmembrane domains that have signaling moieties on the
cytoplasmic surfaces of the minicells, such as kinases or
transcription factors. In one non-limiting example, a minicell
fusion membrane-bound protein could be expressed containing an
extracellular domain with a receptor, scFv, or other targeting
protein that binds to the pathogen. The second segment of the
chimera could be a transmembrane domain of a protein such as the
EGF receptor or ToxR (that would tether the fusion protein to the
membrane). Importantly, the cytoplasmic domain of the fusion
protein could be a kinase that phosphorylates a bacterial
transcription factor present in the minicell or could be fused to a
transcription factor that would be expressed on the cytoplasmic
surface of the minicell. The expression plasmid that was previously
introduced into the minicells would then be activated by promoters
utilizing the activated bacterial transcription factor pre-existing
in the minicells or that which may be introduced by the minicell.
Without being limited to the following example, the binding event
could be signaled by a fusion protein containing elements of a
receptor (e.g., EGF) or by an adhesion protein (e.g., an integrin)
that require oligomerization. In the example of the use of
integrins, bacterial or other transcription factors that also
require dimerization could be cloned as fusion proteins such that
the binding event would be signaled by a dimerization of two or
more identical recombinant chimeric proteins that have
association-dependent transcription factors tagged to the
C-terminus of the fusion protein. The minicells would only be toxic
when contact is made with the pathogen.
[0736] The proposed mechanism of induction coincident with
targeting is not limited to the antiparasitic uses of minicells but
can be used in other therapeutic situations where minicells are
used to express proteins of therapeutic benefit when directed
against eucaryotic cells of the organism (e.g., kill cancer cells
with protein toxins expressed only after binding of the minicell to
the cancer cell).
[0737] Transfer of DNA-containing plasmids or other expression
element, antisense DNA, etc. may be used to express toxic proteins
in the target cells or otherwise inhibit transcription and/or
translation in the pathogenic organism or would otherwise be toxic
to the cell. Without being limited by the following example,
minicells can be used to transfer plasmids expressing growth
repressors, DNAses, or other proteins or peptides (e.g.,
pro-apoptotic) that would be toxic to the pathogen.
[0738] XIV.E. Cancer Therapy
[0739] Fusion proteins expressed in minicells are used for cancer
therapy. In a non-limiting example, phage display antibody
libraries are used to clone single chain antibodies against
tumor-associated (tumor-specific) antigens, such as MUCH-1 or
EGFvIII. Fusion proteins expressing these antibodies, and further
comprising a single-pass transmembrane domain of an integral
membrane protein, are used to "present" the antibody to the surface
of the minicells. Injected minicells coated with anti-tumor
antibodies target the tumor and deliver pro-apoptotic genes or
other toxic substances to the tumor. The minicells are engulfed by
the tumor cells by processes such receptor-mediated endocytosis
(by, e.g., macrophages). By way of non-limiting example,
toxR-invasin could be expressed on the surfaces of the minicells to
promote endocytosis through the interaction between invasin and
beta2-integrins on the surfaces of the target cells.
[0740] Fusion proteins possessing viral fusion-promoting proteins
facilitate entry of the minicell to the tumor cell for gene therapy
or for delivery of chemotherapy bioactive proteins and nucleic
acids. In these and similar applications, the minicell may contain
separate eukaryotic and eubacterial expression elements, or the
expression elements may be combined into a single "shuttle
vector."
[0741] XV. Diagnostic Uses of Minicells
[0742] Minicells are transformed with plasmids expressing
membrane-bound proteins, such as receptors, that bind to specific
molecules in a particular biological sample such as blood, urine,
feces, sweat, saliva or a tissue such as liver or heart. Minicells
can also be used for delivery of therapeutic agents across the
blood-brain barrier to the brain. This modality is used, by way of
non-limiting example, for imaging purposes, and for the delivery of
therapeutic agents, e.g., anti-depressants, and agents for the
treatment of cancer, obesity, insomnia, schizophrenia, compulsive
disorders and the like. Recombinant expression systems are
incorporated into minicells where the plasmid-driven protein
expression construct could be the produce a single gene product or
a fusion protein, such as a soluble protein for the particular
ligand fused with a transmembrane domain of a different gene. The
fusion protein then acts as a membrane bound receptor for a
particular ligand or molecule in the sample. Conventional cloning
techniques (e.g., PCR) are used to identify genes for binding
proteins, or phage display is used to identify a gene for a
single-stranded variable antibody gene expressing binding protein
for a particular ligand. The protein product is preferably a
soluble protein. By constructing a plasmid containing this gene
plus the transmembrane domain of a known single-pass membrane
protein such as that of the EGF receptor, a fusion protein may be
expressed on the surfaces of the minicells as an integral membrane
protein with an extracellular domain that is preferably capable of
binding ligand.
[0743] In another type of fusion protein, the transmembrane domain
of the EGF receptor is fused to a known soluble receptor for a
particular ligand, such as the LBP (lipopolysaccharide binding
protein) or the extracellular domain of CD14 receptor protein, both
of which bind the bacterial endotoxin, LPS (lipopolysaccharide).
The LBP/EGF or CD14/EGF fusion protein is used to measure LPS in
the serum of patients suspected of sepsis.
[0744] The minicell system is used to express receptors such as
those of the EDG (endothelial cell differentiation gene) family
(e.g., EDG 1-9) that recognize sphingolipids such as
sphingosine-1-phosphate (S1P), sphingosylphosphoryl choline (SPC)
and the lysophospholipid, lysophosphatidic acid (LPA). Since these
proteins are 7-pass integral membrane proteins, no additional
transmembrane domains of another protein are needed, and the
receptor protein is thus not a fusion protein.
[0745] Truncated or mutant forms of a protein of interest are
useful in a diagnostic assay. For example, a protein that is an
ligand-binding enzyme can be altered so as to bind its substrate of
interest but can no longer convert substrate into product. One
example of this application of minicell technology is the
expression of a truncated or mutant lactic dehydrogenase which is
able to bind lactic acid, but is not able to convert lactic acid to
pyruvate. Similarly, hexokinase deriviatives are used in minicells
for glucose monitoring.
[0746] Minicells as diagnostic tools can be used either in vitro or
in vivo. In the in vitro context, the minicells are used in an
ELISA format or in a lateral flow diagnostic platform to detect the
presence and level of a desired analyte. A sample (tissue, cell or
body fluid sample) is taken and then tested in vitro. One advantage
of the minicell system in detecting substances in tissue, cells or
in body fluids is that the minicells can be used in vitro assays
where the minicell is labeled with either a radioactive or
fluorescent compound to aid in its detection in a an ELISA format
or lateral flow platform. Thus, the use of secondary antibody
detection systems is obviated.
[0747] As an in vivo diagnostic, minicells can be radiolabeled. One
method of labeling is to incubate minicells for a short time (about
8 hr) with a T.sub.1/2 tracer (e.g., Tn99M) that is useful for
detecting tumor metastases. The Tn99M accumulates in cells and
loads into minicells after isolation or into the parent bacteria
during growth phase. As Tn99M is oxidized by either the parent E.
coli strain or by the minicells after isolation, the Tn99M is
retained by the cell. Iodine-labeled proteins may also be used
(Krown et al., TNF-alpha receptor expression in rat cardiac
myocytes: TNF-alpha inhibition of L-type Ca2+ transiets, FEBS
Letters 376:24-30, 1995).
[0748] One non-limiting example of in vivo detection of cancer
making use of radiolabeled minicells is the use of the minicells to
express chimeric membrane-bound single-chain antibodies against
tumor-specific antigens (TSA) expressed on malignant melanoma or
other transformed cells. Such TSAs include, but are not limited to,
the breast cancer associated MUC1 antigen and variant forms of the
EGFR (EGFvIII). By way of non-limiting example, minicells
expressing antibodies to melanoma cells can be injected (IV) into a
patient and then subjected to CAT scan of the lymphatic drainage in
order to determine if a metastasis has occurred. This diagnostic
technique obviates the need for lymph node dissection.
[0749] Another example of an in vivo diagnostic is to use the
minicell system to express antibodies against oxidized low-density
lipoproteins (LDL). Oxidized LDLs are associated with atherogenic
plaques. Radiolabeled minicells (prepared as above) are injected IV
into a person prior to nuclear imaging for image enhancement. MRI
image contrast enhancement is performed by preparing minicells
complexed (loaded) with contrast enhancers such as paramagnetic
relaxivity agents and magnetic susceptibility agents.
[0750] In diagnostic as well as other applications, minicells
preferentially detect a diagnostic marker, i.e., a marker
associated with a disease or disorder. A diagnostic marker is
statistically more like to occur in individuals suffering from a
disease than in those who are not diseased. Preferably, a
diagnostic marker directly causes or is produced during a disease;
however, the association may be no more than a correlation.
[0751] XVI. Drug Discovery (Screening) with Minicells
[0752] XVI.A. Assays
[0753] Minicells can be used in assays for screening
pharmacological agents. By way of non-limiting example, the
minicell system provides an environment for the expression of GPCRs
and studies of their ligand binding kinetics. Such GPCR's include
any member the Endothelial Differentiation Gene (EDG) receptor
family. GPCRs may participate in neoplastic cell proliferation,
angiogenesis and cell death. Small molecules that either activate
or inhibit the action of these GPCRs can be used in therapeutic
interaction.
[0754] Assays are performed to determine protein expression and
protein function. For example, the production of the protein can be
followed using protein .sup.35S-Met labeling. This is performed by
providing the cell only methionine that is labeled with .sup.35S.
The cells are treated with IPTG to induce protein expression, and
the .sup.35S-Met is incorporated into the protein. The cells are
then lysed, and the resulting lysates were electrophoresed on an
SDS gel and exposed to autoradiography film.
[0755] Another technique for assessing protein expression involves
the use of western blots. Antibodies directed to various expressed
proteins of interest have been generated and many are commercially
available. Techniques for generating antibodies to proteins or
polypeptides derived therefrom are known in the art (see, e.g.,
Cooper et al., Section III of Chapter 11 in: Short Protocols in
Molecular Biology, 2nd Ed., Ausubel et al., eds., John Wiley and
Sons, New York, 1992, pages 11-22 to 11-46). Standard western blot
protocols, which may be used to show protein expression from the
expression vectors in minicells and other expression systems, are
known in the art. (see, e.g., Winston et al., Unit 10.7 of Chapter
10 in: Short Protocols in Molecular Biology, 2nd Ed., Ausubel et
al., eds., John Wiley and Sons, New York, 1992, pages 10-32 to
10-35).
[0756] The amount of functional protein produced from a minicell
expression system is determined through the use of binding studies.
Ligands for the proteins of interest are used to show specific
binding in the minicell system. Radiolabeled ligand is incubated
with cells expressing the protein, in this case, a receptor for
TNF-alpha. The cells are then centrifuged and the radioactivity
counted in a scintillation counter. The amount of ligand that is
bound reflects the amount of functional protein that is present in
the sample.
[0757] By way of non-limiting example, the minicell system can be
made to express EDGRs for the purpose of screening combinatorial
chemistry libraries for molecules that enhance EDG activity. EDG
activity is assayed in the minicell environment in several ways.
One way is to crystallize minicells expressing an EDG protein (or
any membrane-bound protein of choice) and then measure changes in
the crystal structure to detect novel ligands. Circular dichroism
(CD), x-ray diffraction, electron spin resonance (EPR) or other
biophysical approaches are used to probe the structure of proteins
in the minicell context. Additionally or alternately, minicells are
produced that express not only the EDGR, but also express
G-proteins (i.e., double transformants). An assay system involving
GTP binding and hydrolysis is used to identify and assess which
small molecules in the combinatorial chemistry library serve as
activating ligands for EDG. The minicell expression system is used
in in vitro binding assays and in high throughput drug screenings.
The expression of mutant or truncated isoforms of proteins are used
for functional analyses in order to discover inactive or overactive
proteins for potential use in diagnostics or therapeutics.
[0758] XVI.B. High-Throughput Screening (HTS)
[0759] HTS typically uses automated assays to search through large
numbers of compounds for a desired activity. Typically HTS assays
are used to find new drugs by screening for chemicals that act on a
particular enzyme or molecule. For example, if a chemical
inactivates an enzyme it might prove to be effective in preventing
a process in a cell that causes a disease. High throughput methods
enable researchers to try out thousands of different chemicals
against each target very quickly using robotic handling systems and
automated analysis of results.
[0760] As used herein, "high throughput screening" or "HTS" refers
to the rapid in vitro screening of large numbers of compounds
(libraries); generally tens to hundreds of thousands of compounds,
using robotic screening assays. Ultra high-throughput Screening
(uHTS) generally refers to the high-throughput screening
accelerated to greater than 100,000 tests per day.
[0761] To achieve high-throughput screening, it is best to house
samples on a multicontainer carrier or platform. A multicontainer
carrier facilitates measuring reactions of a plurality of candidate
compounds simultaneously. Multi-well microplates may be used as the
carrier. Such multi-well microplates, and methods for their use in
numerous assays, are both known in the art and commercially
available.
[0762] Screening assays may include controls for purposes of
calibration and confirmation of proper manipulation of the
components of the assay. Blank wells that contain all of the
reactants but no member of the chemical library are usually
included. As another example, a known inhibitor (or activator) of
an enzyme for which modulators are sought, can be incubated with
one sample of the assay, and the resulting decrease (or increase)
in the enzyme activity determined according to the methods herein.
It will be appreciated that modulators can also be combined with
the enzyme activators or inhibitors to find modulators which
inhibit the enzyme activation or repression that is otherwise
caused by the presence of the known the enzyme modulator.
Similarly, when ligands to a sphingolipid target are sought, known
ligands of the target can be present in control/calibration assay
wells.
[0763] The minicells of the invention are readily adaptable for use
in high-throughput screening assays for screening candidate
compounds to identify those which have a desired activity, e.g.,
inhibiting an enzyme that catalyzes a reaction that produces an
undesirable compound, inhibiting function of a receptor independent
of ligand interference, or blocking the binding of a ligand to a
receptor therefor. The compounds thus identified can serve as
conventional "lead compounds" or can themselves be used as
therapeutic agents.
[0764] The methods of screening of the invention comprise using
screening assays to identify, from a library of diverse molecules,
one or more compounds having a desired activity. A "screening
assay" is a selective assay designed to identify, isolate, and/or
determine the structure of, compounds within a collection that have
a preselected activity. By "identifying" it is meant that a
compound having a desirable activity is isolated, its chemical
structure is determined (including without limitation determining
the nucleotide and amino acid sequences of nucleic acids and
polypeptides, respectively) the structure of and, additionally or
alternatively, purifying compounds having the screened activity).
Biochemical and biological assays are designed to test for activity
in a broad range of systems ranging from protein-protein
interactions, enzyme catalysis, small molecule-protein binding,
agonists and antagonists, to cellular functions. Such assays
include automated, semi-automated assays and HTS (high throughput
screening) assays.
[0765] In HTS methods, many discrete compounds are preferably
tested in parallel by robotic, automatic or semi-automatic methods
so that large numbers of test compounds are screened for a desired
activity simultaneously or nearly simultaneously. It is possible to
assay and screen up to about 6,000 to 20,000, and even up to about
100,000 to 1,000,000 different compounds a day using the integrated
systems of the invention.
[0766] Typically in HTS, target molecules are contained in each
well of a multi-well microplate; in the case of enzymes, reactants
are also present in the wells. Currently, the most widely
established techniques utilize 96-well microtiter plates. In this
format, 96 independent tests are performed simultaneously on a
single 8 cm.times.12 cm plastic plate that contains 96 reaction
wells. One or more blank wells contains all of the reactants except
the candidate compound. Each of the non-standard wells contain at
least one candidate compound.
[0767] These wells typically require assay volumes that range from
50 to 500 ul. In addition to the plates, many instruments,
materials, pipettors, robotics, plate washers and plate readers are
commercially available to fit the 96-well format to a wide range of
homogeneous and heterogeneous assays. Microtiter plates with more
wells, such as 384-well microtiter plates, are also used, as are
emerging methods such as the nanowell method described by Schullek
et al. (Anal Biochem., 30 246, 20-29, 1997).
[0768] In one modality, screening comprises contacting a
sphingolipid target with a diverse library of member compounds,
some of which are ligands of the target, under conditions where
complexes between the target and ligands can form, and identifying
which members of the libraries are present in such complexes. In
another non limiting modality, screening comprises contacting a
target enzyme with a diverse library of member compounds, some of
which are inhibitors (or activators) of the target, under
conditions where a product or a reactant of the reaction catalyzed
by the enzyme produce a detectable signal. In the latter modality,
inhibitors of target enzyme decrease the signal from a detectable
product or increase a signal from a detectable reactant (or
vice-versa for activators).
[0769] Minicells of the invention expressing and/or displaying a
protein are used for screening assays designed to identify agents
that modulate the activity of the target protein. Such assays
include competitive inhibition binding assays for high throughput
assays. Competitive inhibition assays include but are not limited
to assays that screen agents against a specific target protein to
identify agents that inhibit, interfere, block, or compete with
protein-ligand interactions, protein-protein interactions,
enzymatic activity, or function of a specific protein. Examples of
competitive inhibition include but are not limited to the
development of neutral inhibitors of the serine protease factor Xa
that were discovered using a high throughput screening assay using
a compound library (Carr et al, Neutral inhibitors of the serine
protease factor Xa, Bioorg Med Chem Lett 11, 2001), the design and
characterization of potent inhibitors for the human oxytocin
receptor (Seyer et al, Design, synthesis and pharmacological
characterization of a potent radio iodinated and photoactivatable
peptidic oxytocin antagonist, J Med. Chem. 44:3022-30, 2001), and
the identification of non-peptide somatostatin antagonists of the
sst(3) protein (Thurieau et al, Identification of potent
non-peptide somatostatin antagonists with sst(3) selectivity, J
Med. Chem. 44:2990-3000, 2001).
[0770] High throughput competitive inhibition assays are designed
to identify agents that inhibit a specific target protein. Such
assays include but are not limited to ones that measure enzymatic
activity, protein-ligand interactions, protein-protein interactions
and other functions of proteins. Minicells that express and/or
display a specific protein could be used in all types of
competitive inhibition assays.
[0771] One non-limiting example of high throughput competitive
inhibition screening using minicells for the purpose of this patent
involves the competitive inhibition of known ligands. The ligand is
attached to but not limited to a fluorophore, fluorescent protein,
tags such as 6.times.His tag or FLAG tag, chromophores,
radiolabeled proteins and molecules, binding moieties such as
avidin and strepavidin, voltage sensitive dies and proteins,
bioluminescent proteins and molecules, or fluorescent peptides. The
target protein, which binds the tagged ligand, is expressed and
stably displayed by the minicell. When the ligand is added to the
minicell solution the ligand binds to the target protein. Following
a wash the interaction is detected via the fluorophore, fluorescent
protein, tag, or fluorescent peptide. The ligand-bound minicells
could either be centrifuged (taking advantage of the sedimentation
properties of the minicell particle) or immunoprecipitated with an
antibody against an antigen expressed on the minicell membrane or
the minicells can be adsorbed/fixed to a substrate such as a
standard 96 well plate. The competitive inhibition assay is carried
out by adding agents to the minicell mix either before, together or
after the ligand is added. Thus if the agent is a competitive
inhibitor of the ligand to the target protein the ligand will be
washed away from the minicell because it is not associated with the
target protein. The agent prevents binding and thus eliminated the
detection signal from the minicell.
[0772] Minicells of this invention are used in "functional
screening HTS assays". Functional screening assays are defined as
assays that provide information about the function of a specific
target protein. Functional assays screen agents against specific
target proteins to identify agents that either act as antagonist or
as an agonist against the protein. Functional assays require that
the target protein be in an environment that allows it to carry out
its natural function. Such functions include but are not limited to
G-proteins coupling with a GPCR, enzymatic activity such as
phosphorlyation or proteolysis, protein-protein interaction, and
transport of molecules and ions.
[0773] Functional assays screen agents against proteins which are
capable of natural function. Target proteins used in functional
studies must carry out a function that is measurable. Examples of
protein functions that are measurable include but are not limited
to the use of Fluorescent Resonance Energy Transfer (FRET) to
measure the G-protein coupling to a GPCR (Ruiz-Velasco et al.,
Functional expression and FRET analysis of green fluorescent
proteins fused to G-protein subunits in rat sympathetic neurons, J.
Physiol. 537:679-692, 2001; Janetopoulos et al., Receptor-mediated
activation of heterotrimeric G-proteins in living cells, Science
291:2408-2411, 2001); Bioluminescence Resonance Energy Transfer
(BRET) to assay for functional ligand induced G-protein coupling to
a target GPCR (Menard, L. Bioluminescence Resonance Energy Transfer
(BRET): A powerful platform to study G-protein coupled receptors
(GPCR) activity in intact cells, Assay Development, Nov. 28-30,
2001), the use of florescent substrates to measure the enzymatic
activity of proteases (Grant, Designing biochemical assays for
proteases using fluorogenic substrates, Assay Development, Nov.
28-30, 2001); and the determination of ion channel function via the
use of voltage sensitive dies (Andrews et al, Correlated
measurements of free and total intracellular calcium concentration
in central nervous system neurons, Microsc Res Tech. 46:370-379,
1999).
[0774] One non-limiting example of high throughput functional
screening assay using minicells for the purpose of this patent
involves the functional coupling of GPCRs to their respective
G-protein. Upon ligand binding, voltage polarization, ion binding,
light interaction and other stimulatory events activate GPCRs and
cause them to couple to their respective G-protein. In a minicell,
both the GPCR and its respective G-proteins can be simultaneously
expressed. Upon activation of the GPCR the coupling event will
occur in the minicell. Thus by detecting this coupling in the
minicell, one could screen for agents that bind GPCRs to identify
antagonists and agonists. The antagonists are identified using
inhibition assays that detect the inhibition of function of the
GPCR. Thus the agent interacts with the GPCR in a way that it
inhibits the GPCR from being activated. The agonists are identified
by screening for agents that activate the GPCR in the absence of
the natural activator.
[0775] The detection of GPCR activation and coupling in a minicell
is accomplished by using systems that generate a signal upon
coupling. One non-limiting example involves the use of BRET or
FRET. These systems require that two fluorescent or bioluminescent
molecules or proteins be brought into close contact. Thus by
attaching one of these molecules or proteins to the GPCR and one to
the G-protein, they will be brought together upon coupling and a
signal will be generated. This signal can be detected using
specific detection equipment and the coupling event can be
monitored. Thus the function of the GPCR can be assayed and used in
functional assays in minicells.
[0776] Another non-limiting functional assay for GPCRs and other
proteins in minicells involves the use of transcription factors.
Many bacterial transcription factors and eukaryotic transcription
factors require dimerization for activation. By attaching one
subunit of a transcription factor to a GPCR and the other subunit
to a G-protein, the subunits will dimerize upon coupling of the
GPCR to the G-protein because they will be brought into close
contact. The dimerized transcription factor will then be activated
and will act on its target episomal DNA. In the minicell system the
episomal DNA target will be a plasmid that encodes for proteins
that provide a signal for detection. Such proteins include but are
not limited to luciferase; green fluorescent protein (GFP), and
derivatives thereof such as YFP, BFP, etc.; alcohol dehydrogenase,
and other proteins that can be assayed for expression. The
activation of the GPCR will result in coupling and activation of
the transcription factor. The transcription factor will then induce
transcription and translation of specific detector proteins. Thus
the activation of the GPCR will be monitored via the expression of
the detector protein.
[0777] In another modality, the transcription factor can inhibit
expression in the minicell system and thus allowing for the
screening of constitutively active GPCRs and proteins. For example
if the GPCR were constitutively active then the transcription
factor to use would be one that inhibits transcription and
translation. Thus agents could be screened against the
constitutively active GPCR to identify agents that caused the
constitutively active GPCR to uncouple. The uncoupling will result
in the inactivation of the transcription factor. The inhibition
caused by the transcription factor will be removed and
transcription and translation will occur. Thus a detectable protein
will be made and a signal will be received.
[0778] The transcription dimerization assay can be used for any
protein function that involves a protein-protein interaction,
protein-ligand interaction and protein-drug interaction. Thus any
assay involving such interactions can be carried out in the
minicell.
[0779] Another non-limiting functional screening assay involves the
use of enzymatic function to screen for functionality. In this
modality the receptor or other protein performs a specific
enzymatic function. This function is then carried out in the
minicell and monitored using biochemical and other techniques. For
example if the target protein was a protease then fluorescent
peptides with the cleavage site of the protease could be used to
monitor the activity of the protease. If the protease was
functioning then the peptide would be cleaved and the fluorescents
would change. Thus agents can be screened against the protease in
the minicell system and the fluorescents can be monitored using
specific detection systems. In another non-limiting example, a
membrane-bound enzyme such as sphingomyelinase could be expressed
in minicells and the minicell particles adsorbed to a standard
substrate such as a 96 well plate. The enzymatic activity could be
assessed by a standard in vitro assay involving the release of
product (phosphocholine) (e.g., Amplex.TM. kit A-12220 sold by
Molecular Probes). Sphingomyelinase inhibitors could be screened by
measuring the reduction of phosphocholine production in the well
when presented with substrate (sphingomyelin) in a coupled
fluorescence assay.
[0780] Another non-limiting example of minicells used for
functional assays involves the screening of agonists/antagonists
for ion channels. In this example the calcium channel, SCaMPER, is
encoded on a polycistronic episomal plasmid, which also encodes for
a luminescent soluble protein, aequorin. In this assay, the
minicell will contain aequorin proteins in its cytoplasm and
SCaMPER proteins expressed on the minicell membrane. Thus upon
activation of SCaMPER by its ligand, SPC, or by an analog thereof,
calcium will flow into the minicell and will be bound by the
aequorin which will luminescence. Thus a detection signal for the
functional activation of the calcium channel is obtained.
[0781] Minicell can also be employed for expression of target
proteins and the preparation of membrane preparations for use in
screening assays. Such proteins include but are not limited to
receptors (e.g., GPCRs, sphingolipid receptors, neurotransmitter
receptors, sensory receptors, growth factor receptors, hormone
receptors, chemokine receptors, cytokine receptors, immunological
receptors, and compliment receptors, FC receptors), channels (e.g.,
potassium channels, sodium channels, calcium channels), pores
(e.g., nuclear pore proteins, water channels), ion and other pumps
(e.g., calcium pumps, proton pumps), exchangers (e.g.,
sodium/potassium exchangers, sodium/hydrogen exchangers,
potassium/hydrogen exchangers), electron transport proteins (e.g.,
cytochrome oxidase), enzymes and kinases (e.g., protein kinases,
ATPases, GTPases, phosphatases, proteases), structural/linker
proteins (e.g., Caveolins, clathrin), adapter proteins (e.g., TRAD,
TRAP, FAN), chemotactic/adhesion proteins (e.g., ICAM11, selectins,
CD34, VCAM-1, LFA-1, VLA-1), and chimeric/fusion proteins (e.g.,
proteins in which a normally soluble protein is attached to a
transmembrane region of another protein). In such assays the
membrane preparations are used to screen for agents that are either
antagonists or agonists. These assays use various formats including
but not limited to competitive inhibition.
[0782] The format for the screening of minicells includes but is
not limited to the use of test tubes, 6 well plates, 12 well
plates, 24 well plates, 96 well plates, 384 well plates, 1536 well
plates, and other microtiter well plates. In these systems the
minicells can be immobilized, attached, bound, or fused with the
above test tubes or plates. The minicells can also be free in
solution for use in tubes and plates. The detection systems for the
minicell assay include but are not limited to fluorescent plate
readers, scintillation counters, spectrophotometers, Viewlux CCD
Imager, Luminex, ALPHAQuest, BIAcore, FLIPR and F-MAT. Minicell
assays can be carried out with but not limited to techniques such
as manual handling, liquid handlers, robotic automated systems and
other formats.
[0783] XVI.C. Chemical Libraries
[0784] Developments in combinatorial chemistry allow the rapid and
economical synthesis of hundreds to thousands of discrete
compounds. These compounds are typically arrayed in moderate-sized
libraries of small organic molecules designed for efficient
screening. Combinatorial methods, can be used to generate unbiased
libraries suitable for the identification of novel inhibitors. In
addition, smaller, less diverse libraries can be generated that are
descended from a single parent compound with a previously
determined biological activity. In either case, the lack of
efficient screening systems to specifically target therapeutically
relevant biological molecules produced by combinational chemistry
such as inhibitors of important enzymes hampers the optimal use of
these resources.
[0785] A combinatorial chemical library is a collection of diverse
chemical compounds generated by either chemical synthesis or
biological synthesis, by combining a number of chemical "building
blocks," such as reagents. For example, a linear combinatorial
chemical library, such as a polypeptide library, is formed by
combining a set of chemical building blocks (amino acids) in a
large number of combinations, and potentially in every possible
way, for a given compound length (i.e., the number of amino acids
in a polypeptide compound). Millions of chemical compounds can be
synthesized through such combinatorial mixing of chemical building
blocks.
[0786] A "library" may comprise from 2 to 50,000,000 diverse member
compounds. Preferably, a library comprises at least 48 diverse
compounds, preferably 96 or more diverse compounds, more preferably
384 or more diverse compounds, more preferably, 10,000 or more
diverse compounds, preferably more than 100,000 diverse members and
most preferably more than 1,000,000 diverse member compounds. By
"diverse" it is meant that greater than 50% of the compounds in a
library have chemical structures that are not identical to any
other member of the library. Preferably, greater than 75% of the
compounds in a library have chemical structures that are not
identical to any other member of the collection, more preferably
greater than 90% and most preferably greater than about 99%.
[0787] The preparation of combinatorial chemical libraries is well
known to those of skill in the art. For reviews, see Thompson et
al., Synthesis and application of small molecule libraries, Chem
Rev 96:555-600, 1996; Kenan et al., Exploring molecular diversity
with combinatorial shape libraries, Trends Biochem Sci 19:57-64,
1994; Janda, Tagged versus untagged libraries: methods for the
generation and screening of combinatorial chemical libraries, Proc
Natl Acad Sci USA. 91:10779-85, 1994; Lebl et al.,
One-bead-one-structure combinatorial libraries, Biopolymers
37:177-98, 1995; Eichler et al., Peptide, peptidomimetic, and
organic synthetic combinatorial libraries, Med Res Rev. 15:481-96,
1995; Chabala, Solid-phase combinatorial chemistry and novel
tagging methods for identifying leads, Curr Opin Biotechnol.
6:632-9, 1995; Dolle, Discovery of enzyme inhibitors through
combinatorial chemistry, Mol. Divers. 2:223-36, 1997; Fauchere et
al., Peptide and nonpeptide lead discovery using robotically
synthesized soluble libraries, Can J Physiol Pharmacol. 75:683-9,
1997; Eichler et al., Generation and utilization of synthetic
combinatorial libraries, Mol Med Today 1:174-80, 1995; and Kay et
al., Identification of enzyme inhibitors from phage-displayed
combinatorial peptide libraries, Comb Chem High Throughput Screen
4:535-43, 2001.
[0788] Such combinatorial chemical libraries include, but are not
limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175,
Furka, Int. J. Pept. Prot. Res., 37:487-493 (1991) and Houghton, et
al., Nature, 354:84-88 1991). Other chemistries for generating
chemical diversity libraries can also be used. Such chemistries
include, but are not limited to, peptoids (PCT Publication No. WO
91/19735); encoded peptides (PCT Publication WO 93/20242); random
bio-oligomers (PCT Publication No. WO 92/00091); benzodiazepines
(U.S. Pat. No. 5,288,514); diversomers, such as hydantoins,
benzodiazepines and dipeptides (Hobbs, et al., Proc. Nat. Acad.
Sci. USA, 90:6909-6913 1993); vinylogous polypeptides (Hagihara, et
al., J. Amer. Chem. Soc. 114:6568 1992); nonpeptidal
peptidomimetics with .beta.-D-glucose scaffolding (Hirschmann, et
al., J. Amer. Chem. Soc., 114:9217-9218 1992); analogous organic
syntheses of small compound libraries (Chen, et al., J. Amer. Chem.
Soc., 116:2661 1994); oligocarbamates (Cho, et al., Science,
261:1303 1993); and/or peptidyl phosphonates (Campbell, et al., J.
Org. Chem. 59:658 1994); nucleic acid libraries (see, Ausubel,
Berger and Sambrook, all supra); peptide nucleic acid libraries
(see, e.g., U.S. Pat. No. 5,539,083); antibody libraries (see,
e.g., Vaughn, et al., Nature Biotechnology, 14(3):309-314 (1996)
and PCT/US96/10287); carbohydrate libraries (see, e.g., Liang, et
al., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853);
small organic molecule libraries (see, e.g., benzodiazepines, Baum
C&E News, Jan. 18, page 33 (1993); isoprenoids (U.S. Pat. No.
5,569,588); thiazolidinones and metathiazanones (U.S. Pat. No.
5,549,974); pyrrolidines (U.S. Pat. Nos. 5,525,735 and 5,519,134);
morpholino compounds (U.S. Pat. No. 5,506,337); benzodiazepines
(U.S. Pat. No. 5,288,514); and the like.
[0789] Devices for the preparation of combinatorial libraries are
commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem.
Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied
Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford,
Mass.). In addition, numerous combinatorial libraries are
themselves commercially available (see, e.g., ComGenex, Princeton,
N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar,
Ltd., Moscow, RU, 3D Pharmaceuticals, Exton, Pa., Martek Bio
sciences, Columbia, Md., etc.).
[0790] XVI.D. Measuring Enzymatic and Binding Reactions During
Screening Assays
[0791] Techniques for measuring the progression of enzymatic and
binding reactions in multicontainer carriers are known in the art
and include, but are not limited to, the following.
[0792] Spectrophotometric and spectrofluorometric assays are well
known in the art. Examples of such assays include the use of
colorimetric assays for the detection of peroxides, as disclosed in
Example 1(b) and Gordon, A. J. and Ford, R. A., The Chemist's
Companion: A Handbook Of Practical Data, Techniques, And
References, John Wiley and Sons, N.Y., 1972, Page 437.
[0793] Fluorescence spectrometry may be used to monitor the
generation of reaction products. Fluorescence methodology is
generally more sensitive than the absorption methodology. The use
of fluorescent probes is well known to those skilled in the art.
For reviews, see Bashford et al., Spectrophotometry and
Spectrofluorometry: A Practical Approach, pp. 91-114, IRL Press
Ltd. (1987); and Bell, Spectroscopy In Biochemistry, Vol. I, pp.
155-194, CRC Press (1981).
[0794] In spectrofluorometric methods, enzymes are exposed to
substrates that change their intrinsic fluorescence when processed
by the target enzyme. Typically, the substrate is nonfluorescent
and converted to a fluorophore through one or more reactions. As a
non-limiting example, SMase activity can be detected using the
Amplex.RTM. Red reagent (Molecular Probes, Eugene, Oreg.). In order
to measure sphingomyelinase activity using Amplex Red, the
following reactions occur. First, SMase hydrolyzes sphingomyelin to
yield ceramide and phosphorylcholine. Second, alkaline phosphatase
hydrolyzes phosphorylcholine to yield choline. Third, choline is
oxidized by choline oxidase to betaine. Finally, H.sub.2O.sub.2, in
the presence of horseradish peroxidase, reacts with Amplex Red to
produce the fluorescent product, Resorufin, and the signal
therefrom is detected using spectrofluorometry.
[0795] Fluorescence polarization (FP) is based on a decrease in the
speed of molecular rotation of a fluorophore that occurs upon
binding to a larger molecule, such as a receptor protein, allowing
for polarized fluorescent emission by the bound ligand. FP is
empirically determined by measuring the vertical and horizontal
components of fluorophore emission following excitation with plane
polarized light. Polarized emission is increased when the molecular
rotation of a fluorophore is reduced. A fluorophore produces a
larger polarized signal when it is bound to a larger molecule (i.e.
a receptor), slowing molecular rotation of the fluorophore. The
magnitude of the polarized signal relates quantitatively to the
extent of fluorescent ligand binding. Accordingly, polarization of
the "bound" signal depends on maintenance of high affinity
binding.
[0796] FP is a homogeneous technology and reactions are very rapid,
taking seconds to minutes to reach equilibrium. The reagents are
stable, and large batches may be prepared, resulting in high
reproducibility. Because of these properties, FP has proven to be
highly automatable, often performed with a single incubation with a
single, premixed, tracer-receptor reagent. For a eview, see Owicki
et al., Application of Fluorescence Polarization Assays in
High-Throughput Screening, Genetic Engineering News, 17:27,
1997.
[0797] FP is particularly desirable since its readout is
independent of the emission intensity (Checovich, W. J., et al.,
Nature 375:254-256, 1995; Dandliker, W. B., et al., Methods in
Enzymology 74:3-28, 1981) and is thus insensitive to the presence
of colored compounds that quench fluorescence emission. FP and FRET
(see below) are well-suited for identifying compounds that block
interactions between receptors and their ligands. See, for example,
Parker et al., Development of high throughput screening assays
using fluorescence polarization: nuclear receptor-ligand-binding
and kinase/phosphatase assays, J Biomol Screen 5:77-88, 2000.
[0798] Exemplary normal-and-polarized fluorescence readers include
the POLARION fluorescence polarization system (Tecan A G,
Hombrechtikon, Switzerland). General multiwell plate readers for
other assays are available, such as the VERSAMAX reader and the
SPECTRAMAX multiwell plate spectrophotometer (both from Molecular
Devices).
[0799] Fluorescence resonance energy transfer (FRET) is another
useful assay for detecting interaction and has been described
previously. See, e.g., Heim et al., Curr. Biol. 6:178-182, 1996;
Mitra et al., Gene 173:13-17 1996; and Selvin et al., Meth.
Enzymol. 246:300-345, 1995. FRET detects the transfer of energy
between two fluorescent substances in close proximity, having known
excitation and emission wavelengths. As an example, a protein can
be expressed as a fusion protein with green fluorescent protein
(GFP). When two fluorescent proteins are in proximity, such as when
a protein specifically interacts with a target molecule, the
resonance energy can be transferred from one excited molecule to
the other. As a result, the emission spectrum of the sample shifts,
which can be measured by a fluorometer, such as a fMAX multiwell
fluorometer (Molecular Devices, Sunnyvale Calif.).
[0800] Scintillation proximity assay (SPA) is a particularly useful
assay for detecting an interaction with the target molecule. SPA is
widely used in the pharmaceutical industry and has been described
(Hanselman et al., J. Lipid Res. 38:2365-2373 (1997); Kahl et al.,
Anal. Biochem. 243:282-283 (1996); Undenfriend et al., Anal.
Biochem. 161:494-500 (1987)). See also U.S. Pat. Nos. 4,626,513 and
4,568,649, and European Patent No. 0,154,734. An exemplary
commercially available system uses FLASHPLATE scintillant-coated
plates (NEN Life Science Products, Boston, Mass.).
[0801] The target molecule can be bound to the scintillator plates
by a variety of well known means. Scintillant plates are available
that are derivatized to bind to fusion proteins such as GST, His6
or Flag fusion proteins. Where the target molecule is a protein
complex or a multimer, one protein or subunit can be attached to
the plate first, then the other components of the complex added
later under binding conditions, resulting in a bound complex.
[0802] In a typical SPA assay, the gene products in the expression
pool will have been radiolabeled and added to the wells, and
allowed to interact with the solid phase, which is the immobilized
target molecule and scintillant coating in the wells. The assay can
be measured immediately or allowed to reach equilibrium. Either
way, when a radiolabel becomes sufficiently close to the
scintillant coating, it produces a signal detectable by a device
such as a TOPCOUNT NXT microplate scintillation counter (Packard
BioScience Co., Meriden Conn.). If a radiolabeled expression
product binds to the target molecule, the radiolabel remains in
proximity to the scintillant long enough to produce a detectable
signal.
[0803] In contrast, the labeled proteins that do not bind to the
target molecule, or bind only briefly, will not remain near the
scintillant long enough to produce a signal above background. Any
time spent near the scintillant caused by random Brownian motion
will also not result in a significant amount of signal. Likewise,
residual unincorporated radiolabel used during the expression step
may be present, but will not generate significant signal because it
will be in solution rather than interacting with the target
molecule. These non-binding interactions will therefore cause a
certain level of background signal that can be mathematically
removed. If too many signals are obtained, salt or other modifiers
can be added directly to the assay plates until the desired
specificity is obtained (Nichols et al., Anal. Biochem.
257:112-119, 1998).
[0804] XVI.E. Screening for Novel Antibiotics
[0805] As bacteria and other pathogens acquire resistance to known
antibiotics, there is an ongoing interest in identifying novel
antibiotics. See, e.g., Powell W A, Catranis C M, Maynard Calif.
Synthetic antimicrobial peptide design. Mol Plant Microbe Interact
1995 September-October; 8(5):792-4. Minicells can be used to assay,
identify and purify novel antibiotics to eubacteria. By way of
non-limiting example, a minicell that comprises a detectable
compound can be contacted with a candidate antibiotic to see if the
minicell is lysed by a candidate compound, which would release the
detectable compound from the interior of the minicell into
solution, this producing a signal that indicates that the candidate
antibiotic is effective at lysing bacteria. In such assays, the
detectable compound is such that it produces less or more of the
same signal, or a different signal, inside the minicell as compared
to in solution post-lysis. By way of non-limiting example, the
minicell could comprise a fluorescent compounds that, when
contacted with a second fluorescent compound in solution, produces
FRET.
[0806] XVI. F. Reverse Screening
[0807] In one version of minicell display, the invention provides
methods for screening libraries of minicells in which each minicell
comprises an expression element that encodes a few, preferably one,
membrane proteins in order to identify a membrane protein that
interacts with a preselected compound. By way of non-limiting
example, sequences encoding membrane proteins, fusion proteins, or
cytoplasmic proteins are cloned into an expression vector, either
by "shotgun" cloning or by directed cloning, e.g., by screening or
selecting for cDNA clones, or by PCR amplification of DNA
fragments, that encode a protein using one or more oligonucleotides
encoding a highly conserved region of a protein family. For a
non-limiting example of such techniques, see Krautwurst, D., et al.
1998. Identification of ligands for olfactory receptors by
functional expression of a receptor library. Cell 95:917-926. By
way of non-limiting example, a minicell expressing a receptor binds
a preselected ligand, which may be a drug. Various assays for
receptor binding, enzymatic activity, and channeling events are
known in the art and may include detectable compounds; in the case
of binding assays, competition assays may also be used
(Masimirembwa, C. M., et al. 2001. In vitro high throughput
screening of compounds for favorable metabolic properties in drug
discovery. Comb. Chem. High Throughput Screen. 4:245-263;
Mattheakis, L. C., and A. Saychenko. 2001. Assay technologies for
screening ion channel targets. Curr. Opin. Drug Discov. Devel.
4:124-134; Numann, R., and P. A. Negulescu. 2001. High-throughput
screening strategies for cardiac ion channels. Trends Cardiovasc.
Med. 11:54-59; Le Poul, E., et al. 2002. Adaptation of aequorin
functional assay to high throughput screening. J. Biomol. Screen.
7:57-65; and Graham, D. L., et al. 2001. Application of
beta-galactosidase enzyme complementation technology as a high
throughput screening format for antagonists of the epidermal growth
factor receptor. J. Biomol. Screen. 6:401-411).
[0808] Once a minicell has been identified by an assay and
isolated, DNA is prepared from the minicell. The cloned DNA present
in the minicell encodes the receptor displayed by the minicell.
Having been cloned, the receptor is used as a therapeutic target.
For example, the receptor is produced via recombinant DNA
expression and used in minicell-based or other assays to identify
and characterize known and novel compounds that are ligands,
antagonists and/or agonists of the cloned receptor. The ligands,
antagonists and agonists may be used as lead compounds and/or drugs
to treat diseases in which the receptor plays a role. In
particular, when the preselected ligand is a drug, diseases for
which that drug is therapeutic are expected to be treated using the
novel ligands, antagonists and agonists, or drugs and prodrugs
developed therefrom.
[0809] Preparations of minicells that express and secrete secretes
a soluble protein can be prepared in order to identify ligands,
including but not limited to small molecules, that interact with
the soluble protein. Soluble proteins include, but are not limited
to, known secreted or proteolytically cleaved proteins and
peptides, hormones and cytokines. In this format, minicells are
placed in, or adhered to, the wells of a microtiter multiwell
plate. A different compound or group of compounds is placed in each
well, along with any reagents necessary to generate or squelch a
signal corresponding to a change in the soluble protein produced by
the minicell. Such changes include, by way of non-limiting example,
conformational changes in the protein that may occur as a result of
binding of a ligand or otherwise. A well that generates the
appropriate signal contains a compound that causes a change in the
soluble protein.
[0810] It is also possible to carry out procedures such as the one
described in the immediately preceding paragraph "in reverse." In
this format, a known ligand, which may be a drug, is used to
identify soluble proteins that bind to the ligand/drug. Libraries
of minicells wherein each minicell secretes a different soluble
protein are prepared, and each type of minicell is placed into, or
adhered to the wall of, a well of a microtiter plate, along with
reagents for generating a signal when the ligand/drug binds to a
soluble protein. Minicells that generate the appropriate signal
comprise a cloned DNA that encodes a soluble protein that interacts
with the known ligand/drug. Once cloned, the soluble protein is
prepared and used as a therapeutic target in order to identify
known or novel compounds that bind thereto. When the preselected
ligand is a drug, diseases for which that drug is therapeutic are
expected to be treated using the known and novel compounds so
identified, or using drugs and prodrugs developed from such
compounds.
[0811] Minicells expressing known membrane and soluble proteins can
also be used to help characterize lead compounds and accelerate the
generation of drugs therefrom. In particular, such studies may be
used identify potentially detrimental interactions that might occur
upon in vivo administration, e.g., ADME/Tox screening (Ekins, S.,
et al. 2002. In silico ADME/Tox: the state of the art. J. Mol.
Graph. Model. 20:305-309; and Li, A., et al. 2002. Early ADME/Tox
studies and in silico screening. Drug Discov. Today 7:25-27).
[0812] By way of non-limiting example, a human receptor that is
known to be important for the normal functioning of a cell may be
expressed in minicells, and various chemical derivatives of a lead
compound can be tested to ensure that they do not bind to the
receptor, as such binding would be expected to have adverse effects
in vivo. As another example, an enzyme that degrades a drug, such
as a cytochrome P450, is expressed in minicells and used to examine
the susceptibility of a candidate drug to such degradation. The
cytochrome P450 family of enzymes is primarily responsible for the
metabolism of xenobiotics such as drugs, carcinogens and
environmental chemicals, as well as several classes of endobiotics
such as steroids and prostaglandins. Exemplary P450 cytochromes
involved in drug degradation include, but are not limited to,
CYP2D6 (cytochrome P4502D6, also known as debrisoquine
hydroxylase), CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C18,
CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5.
[0813] XVI.G. Molecular Variants
[0814] In one aspect of the invention, minicells are used in
methods of screening to identify agents that improve, enhance, or
decrease the interaction of a protein with another compound. These
methods include, by way of non-limiting example, modification of
protein targets through directed or random mutagenic approaches to
identify critical interactions between a wild-type protein target
and a specific drug molecule. Information obtained from studies of
mutant proteins is used to specifically produce or modify a
therapeutic agent to interact more specifically and/or effectively
with the wild-type protein target, thus increasing the therapeutic
efficacy of the parental drug and/or decreasing non-specific,
potentially deleterious interactions. See, for example, Lietha, D.,
et al. 2001. Crystal structures of NK1-heparin complexes reveal the
basis for NK1 activity and enable engineering of potent agonists of
the MET receptor. EMBO J. 20:5543-5555; and Chen, Y. Z., et al. Can
an optimization/scoring procedure in ligand-protein docking be
employed to probe drug-resistant mutations in proteins? J. Mol.
Graph. Model. 19:560-570; Zhao, H. and F. H. Arnold. Combinatorial
protein design: Strategies for screening protein libraries. Current
Opinion in Structural Biology 7:480-485 (1997); and Carrupt P A, el
Tayar N, Karlen A, Testa B. Molecular electrostatic potentials for
characterizing drug-biosystem interactions. Methods Enzymol. 1991;
203:638-77. Martin Y C. Computer-assisted rational drug design.
Methods Enzymol. 1991; 203:587-613.
[0815] By way of non-limiting example, information obtained using
the methods of the invention may be in conjunction with x-ray
crystallographic structural determinations to characterize
receptor:ligand interactions (Muller, G. 2000. Towards 3D
structures of G protein-coupled receptors: a multidisciplinary
approach. Curr. Med. Chem. 7:861-888). By way of non-limiting
example, minicells may be used to display the family of molecular
variants to characterize the specific mutagenic changes on the
functional properties of the protein.
[0816] Studies of variant proteins can also be used to modify drugs
to fit natural variants of proteins, especially those associated
with pathogens. Pathogens such as viruses, including retroviruses
such as HIV, may acquire mutations that change a site where a drug
acts, thereby rendering the pathogen immune to the drug. Studies of
variant proteins can be used to quickly produce derivatives of a
drug that are active against a variant protein. See, for example,
Varghese J N, Smith P W, Sollis S L, Blick T J, Sahasrabudhe A,
McKimm-Breschkin J L, Colman P M. Drug design against a shifting
target: a structural basis for resistance to inhibitors in a
variant of influenza virus neuraminidase. Structure 1998 Jun. 15;
6(6):735-46; and Baldwin E T, Bhat T N, Liu B, Pattabiraman N,
Erickson J W. Structural basis of drug resistance for the V82A
mutant of HIV-1 proteinase. 78: Nat Struct Biol 1995 March;
2(3):244-9.
[0817] XVI.H. Directed Evolution
[0818] The minicells and methods described herein can be used in
directed evolution. Unlike natural variation, directed evolution
generates new protein variants in vitro (see, e.g., Arnold, F. H.
and A. A. Violkov. Directed Evolution of Biocatalysts. Curr Op Chem
Biol 1999. 3:54-59). Amino acid substitutions can be introduced
into a protein of interest by mutating the gene encoding the
protein. Mutations are introduced by, e.g., replicating DNA in
mutator strains, by chemical mutagenesis or radiation-induced
mutagenesis (Drake, J. W., The Molecular Basis of Mutation,
Holden-Day, San Francisco, 1970). Other methods include error-prone
PCR and "domain shuffling" (Moore, G. L. and C.D. Maranas. Modeling
DNA Mutation and Recombination for Directed Evolution Experiments.
J. Ther. Biol. 2000. 205:483-503). In the latter method, different
regions of members of the same gene family are recombined so that
the inherent variability of members of the family is used to
produce novel "isoforms" of genes.
[0819] A group of variants is screened to select for those variants
which have the desired activity. The activity of the initial
variants that are so isolated may be inadequate for a given
application, but the process can be repeated using these initial
members to generate a second group of variants, or reiterated as
many times as is necessary to produce one or more variants having
the desired activity or characteristics.
[0820] XVI.I. Isolation and Characterization of Components of
Signal Transduction Pathways
[0821] In one version of minicell display, the invention provides
methods for screening libraries of minicells, in which each
minicell comprises a preselected component of a signal transduction
pathway, in order to identify soluble and membrane proteins that
interact with the preselected component. By way of non-limiting
example, a plurality of minicells, each of which displays the same
G-protein-coupled receptor (GPCR), is used to prepare a minicell
library in which a different G-protein encoding sequence is present
and expressed in each minicell. Minicells comprising a G-protein
that interacts with the GPCR are identified, e.g., via
transactivation assays described in Example 18. Once a minicell has
been identified by an assay and isolated, DNA is prepared from the
minicell. The cloned DNA present in the minicell encodes a
G-protein that interacts with the GPCR of the displayed by the
minicells of the library. Having been cloned, the G-protein is used
as a therapeutic target that can be used in screening assays to
identify novel lead compounds and drugs that interfere or alter the
activity of the GPCR. In particular, when the GPCR of the minicell
library is known to be a therapeutic target for a specific disease,
it is expected that compounds that interfere or alter the activity
of a G-protein that interacts with the GPCR will be or lead to
therapeutics for that specific disease.
[0822] In addition to G-protein signal transduction pathways, other
non-limiting examples of signal transduction pathways include the
MAPK pathway, the SAPK pathway, the p38 pathway and/or the
ceramide-mediated stress response pathway. See Zhang, W., and L. E.
Samelson. 2000. The role of membrane-associated adaptors in T cell
receptor signalling. Semin. Immunol. 12:35-41; Liebmann, C. 2001.
Regulation of MAP kinase activity by peptide receptor signalling
pathway: paradigms of multiplicity. Cell Signal. 13:777-785; Lee,
Jr., J. T., and J. A. McCubrey. 2002. The Raf/MEK/ERK signal
transduction cascade as a target for chemotherapeutic intervention
in leukemia. Leukemia. 16:486-507; Tibbles, L. A., and J. R.
Woodgett. 1999. The stress-activated protein kinase pathways. Cell
Mol. Life. Sci. 55:1230-1254; Rao, K. M. 2001. MAP kinase
activation of macrophages. J. Leukoc. Biol. 69:3-10; Pelech, S. L.,
and D. L. Charest. 1995. MAP kinase-dependent pathways in cell
cycle control. Prog. Cell Cycle Res. 1:33-52; Lee, S. H., et al.
2001. BetaPix-enhanced p38 activation by
Cdc42/Rac/PAK/MKK3/6-mediated pathway. Implication in the
regulation of membrane ruffling. J. Biol. Chem. 276:25066-25072;
Ono, K., et al. 2000. The p38 signal transduction pathway
Activation and function. Cellular Signalling 12:1-13; You, A. 2001.
Differentiation, apoptosis, and function of human immature and
mature myeloid cells: intracellular signaling mechanism. Int. J.
Hematol. 73:438-452; Johnson, D. I. 1999. Cdc42: An essential
Rho-type GTPase controlling eukaryotic cell polarity. Microbiol.
Mol. Biol. Rev. 63:54-105; Williams, J. A. 2001. Intracellular
signaling mechanisms activated by cholecystokinin-regulating
synthesis and secretion of digestive enzymes in pancreatic acinar
cells. Annu. Rev. Physiol. 63:77-97; Mathias, S., et al. 1998.
Signal trasduction of stress via ceramide. Biochem J. 335:465-480;
and Hannun, Y. A., et al. 2000. Ceramide in the eukaryotic stress
response. Trends Cell Biol. 10:73-80.
[0823] XVII. Determining the Structures of Membrane Proteins
[0824] Three-dimensional (3D) structures of proteins may be used
for drug discovery. However, GPCRs and other membrane proteins
present challenging problems for 3D structure determination.
Muller, Towards 3D structures of G protein-coupled receptors: a
multidisciplinary approach. (Review), Curr Med Chem 2000 pp.
861-88; Levy et al., Two-dimensional crystallization on lipid
layer: A successful approach for membrane proteins, J Struct Biol
1999 127, 44-52. Although the three-dimensional structures of
hundreds of different folds of globular proteins have been
determined, fewer than 20 different integral membrane protein
structures have been determined. There are many reasons for this.
Extracting membrane proteins from the membrane can easily disrupt
their native structure, and membrane proteins are notoriously
difficult to crystallize.
[0825] Some membrane proteins readily form two-dimensional crystals
in membranes and can be used for structure determination using
electron diffraction spectroscopy (ED) instead of x-ray
crystallography. This is the technique that was used to determine
the structure of bacteriorhodopsin (see below).
[0826] Nuclear magnetic resonance (NMR) is an alternative method
for determining membrane protein structure, but most membrane
proteins are too large for high-resolution NMR at the present state
of the art. Furthermore, membrane proteins require special
conditions for NMR, e.g. deuterated lipids must be used to avoid
confusing the signal of the protein protons with the noise of
membrane lipid protons.
[0827] Membrane protein for which structures have been determined
include photosynthetic reaction center, porin, porin OmpF, plant
light-harvesting complex (chlorophyll a-b binding protein),
bacterial light-harvesting complex, cytochrome c oxidase,
glycophorin A, the Sec A translocation ATPase of Bacillus subtilis,
and a bacterial potassium channel. For details, see: Weinkauf et
al., (2001): Conformational stabilization and crystallization of
the Sec A translocation ATPase from Bacillus subtilis. Acta
Crystallogr D Biol Crystallogr 57:559-565; Cowan et al., (1992):
Crystal structures explain functional properties of two E. coli
porins. Nature 358:727-33; Deisenhofer et al., (1984): X-ray
structure analysis of a membrane protein complex. Electron density
map at 3 A resolution and a model of the chromophores of the
photosynthetic reaction center from Rhodopseudomonas viridis. J Mol
Biol 180:385-98; Deisenhofer et al., (1985): Structure of the
protein subunits in the photosynthetic reaction centre of
Rhodopseudomonas viridis at 3 Angstroms resolution. Nature 318:618;
Doyle et al., (1998): The structure of the potassium channel:
molecular basis of K.sup.+ conduction and selectivity. Science
280:69-77; Henderson et al., (1990): Model for the structure of
bacteriorhodopsin based on high-resolution electron
cryo-microscopy. J Mol Biol 213:899-929; Iwata et al., (1998):
Complete structure of the 11-subunit bovine mitochondrial
cytochrome bc1 complex. Science 281:64-71; Koepke et al., (1996):
The crystal structure of the light-harvesting complex II (B800-850)
from Rhodospirillum molischianum. Structure 4:581-97; Kuhlbrandt et
al., (1994): Atomic model of plant light-harvesting complex by
electron crystallography. Nature 367:614-21; Lemmon et al., (1992):
Sequence specificity in the dimerization of transmembrane
alpha-helices. Biochemistry 31:12719-25; MacKenzie et al., (1997):
A transmembrane helix dimer: structure and implications. Science
276:131-3; McDermott et al., (1995): Crystal structure of an
integral membrane light-harvesting complex from photosynthetic
bacteria, Nature 374:517-21; Michel (1982): Three-dimensional
crystals of a membrane protein complex. The photosynthetic reaction
centre from Rhodopseudomonas viridis. J Mol Biol 158:567-72;
Tsukihara et al., (1996): The whole structure of the .beta.-subunit
oxidized cytochrome c oxidase at 2.8 A. Science 272:1136-44; and
Weiss et al., (1991): The structure of porin from Rhodobacter
capsulatus at 1.8 A resolution. FEBS Lett 280:379-82. Table 5,
which is based upon Preusch et al. (1998) as revised by White &
Wimley (1999), lists membrane proteins whose crystallographic
structures have been determined.
TABLE-US-00006 TABLE 5 Structural Data Regarding Membrane Proteins
PROTEIN REFERENCES MONOTOPIC MEMBRANE PROTEINS Portaglandin H2
synthase-1. Sheep. 3.5 .ANG. Picot et al. (1994) Cyclooxygenase-2.
Mus Musculus. 3.0 .ANG. Kurumbail et al. (1996) Squalene-hopene
cyclase. Alicyclobacillus Wendt et al. (1999) acidocaldarius. 2.0
.ANG. TRANSMEMBRANE PROTEINS Bacterial Rhodopsins (Halobacterium
salinarium) Bacteriorhodopsin (BR) 2D xtals. EM. 3.5 .ANG.
Grigrorieff et al. (1996) 2D xtals. EM. 3.0 .ANG. Kimura et al.
(1997) 3D xtals. X-ray. 2.5 .ANG. Pebay-Peyroula et al. (1997) 3D
xtals. X-ray. 1.9 .ANG. Belhrhali et al. (1999) 3D xtals. X-ray 2.1
.ANG. K intermediate Edman et al. (1999) 3D xtals. X-ray. 2.3 .ANG.
Luecke et al. (1998) 3D xtals. X-ray. 1.55 .ANG. Luecke et al.
(1999) 3D xtals. X-ray. D96N mutant (BR) 1.80 .ANG. Luecke et al.
(1999) 3D xtals. X-ray. D96N mutant (M) 2.00 .ANG. 3D xtals. X-ray.
2.9 .ANG. Essen et al. (1998) Halorhodopsin (HR) 3D xtals. Xray.
1.8 .ANG. Kolbe et al. (2000) G PROTEIN-COUPLED RECEPTORS
Rhodopsin. Bovine Rod Outer Segment. 2.8 .ANG. Palczewski et al.
(2000) Photosynthetic Reaction Centers Rhodopseudomonas virdis. 2.3
.ANG. Deisenhofer et al. (1985) Rhodobacter sphaeroides. 3.0 .ANG.
Yeates et al. (1987) Rhodobacter sphaeroides. 3.1 .ANG. Chang et
al. (1991) Light Harvesting Complexes Rhodopseudomonas acidophila.
2.5 .ANG. McDermott et al. (1995) Rhodospirillum molischianum. 2.4
.ANG. Koepke et al. (1996) Photosystems Photosystem I.
Synechococcus elongates 4.0 .ANG. Schubert et al. (1997)
Photosystem II. Synechocoocus elongates 3.8 .ANG. Zouni et al.
(2001) Beta-Barrel Membrane Proteins-Multimeric (Porins and
Relatives) Porin. Rhodobacter capsulatus. 1.8 .ANG. Weiss &
Schulz (1992) Porin. Rhodopeudomonas blastica 1.96 .ANG. Kreutsch
et al. (1994) OmpF. E. coli. 2.4 .ANG. Cowan et al. (1992) PhoE. E.
coli. 3.0 .ANG. Cowan et al. (1992) Maltoporin. Salmonella
typhimurium. 2.4 .ANG. Meyer et al. (1997) Maltoporin. E. coli 3.1
.ANG. Schirmer et al. (1995) Beta-Barrel Membrane
Proteins-Monomeric/Dimeric TolC outer membrane protein. E. coli 2.1
.ANG. Protein is a Koronakis et al. (2000) trimer, each
contributing 4 strands to a single barrel. OmpA. E. coli. 2.5 .ANG.
Pautsch & Schulz (1998) OmpA E. coli. By NMR, in DPC micelles
Arora et al. (2001) OmpX. E. coli. 1.9 .ANG. Vogt & Schulz
(1990) OMPLA (outer membrane phospholipase A) E. coli. 2.1 .ANG..
Snijder et al. (1999) monomer (1QD5) and dimer (1QD6) FhuA. E.
coli. 2.5 .ANG. Ferguson et al. (1998); Lambert et al., 1999 FhuA +
ferrichrome-iron. E. coli. 2.7 .ANG. Buchanan et al. (1999) FepA.
E. coli. 2.4 .ANG. Ferguson et al. (1999) Glycophorin A. humanm.
MacKenzie et al. (1997) Non-constitutive Toxins, etc.
Alpha-hemolysin. Staphylococcus aureus. 1.9 .ANG. Song et al.
(1996) LukF. Staphylococcus aureus. 1.9 .ANG. Olson et al. (1999)
Ion Channels KcsA Potassium, H.sup.+ gated. Streptomyces lividans.
3.2 .ANG. Doyle et al. (1998) MscL Mechanosensitive. Mycobacterium
tuberculosis. Chang et al. (1998) 3.5 .ANG. Other Channels AQP1 -
aquaporin water channel. Red blood cell. Electron Murata et al.
(2000) crystallography in membrane plane. 3.8 .ANG. AQP1 - In
vitreous ice by electron microscopy. 3.7 .ANG. Ren et al. (2000)
GipF - glycerol facilitator channel. E. coli. 2.2 .ANG. Fu et al.
(2000) P-type ATPase Calcium ATPase. Sarcoplasmic reticulum.
Rabbit. 2.6 .ANG. Toyoshima et al. (2000) Respiratory Proteins
Fumerate Reductase Complex. Escherichia coli. 3.3 .ANG. Iverson et
al. (1999) Fumerate Reductase Complex. Wolinella succinogenes 2.2
.ANG. Lancaster et al. (1999) ATP synthase (F.sub.1c.sub.10). S.
cerevisiae. 3.9 .ANG.. X-ray structure Stock et al. (1999) is a C
alpha model derived from composite of 1BMF, 1A91 & 1AQT
Cytochrome C Oxidases aa.sub.3bovine heart mitochondria. 2.8 .ANG.
Tsukihara et al. (1996) aa.sub.3 Paracoccus denitrificans. 2.8
.ANG. Iwata et al. (1995) ba.sub.3 from T. thermophilus. 2.4 .ANG.
Soulimane et al. (2000) Cytochrome bc.sub.1 Complexes Bovine Heart
Mitochondria (5 subunits). 2.9 .ANG. Xia et al. (1997) Chicken
Heart Mitochondria. 3.16 .ANG. Zhang et al. (1998) Bovine Heart
Mitochondria (11 subunits). 2.8-3.0 .ANG.. Iwata et al. (1998) S.
cerevisiae (yeast, 9 subunits). 2.3 .ANG. Hunte et al. (2000)
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[0828] XVIII. Biosensors and Environmental Applications
[0829] XVIII.A. Minicell-Based Biosensors
[0830] The present invention is directed to a device that comprises
a sensor adapted to detect one or more specific health and/or
nutrition markers in a subject or in the environment. The device
may also signal the caretaker, the subject, or an actuator of the
occurrence. The sensor comprises a biosensor. As used herein, the
term "biosensor" is defined as a component comprising one or more
binding moities being adapted to detect a ligand found in one or
more target pathogenic microorganisms or related biomolecules.
[0831] Generally, biosensors function by providing a means of
specifically binding, and therefore detecting, a target
biologically active analyte. In this way, the biosensor is highly
selective, even when presented with a mixture of many chemical and
biological entities. Often the target biological analyte is a minor
component of a complex mixture comprising a multiplicity of
biological and other components. Thus, in many biosensor
applications, detection of target analytes occurs in the
parts-per-billion, parts-per-trillion, or even lower ranges
levels.
[0832] XVIII.A.1. Minicell-Based Biosensor Design
[0833] The biosensor of the present invention may comprise a
bio-recognition element, or molecular recognition element, that
provides the highly specific binding or detection selectivity for a
particular analyte. In a biosensor of the invention, the
bio-recognition element, or system, is a minicell displaying an
enzyme or sequence of enzymes; an antibody or antibody derivative;
a membrane receptor protein; or the like, and generally functions
to interact specifically with a target biological analyte. The
bio-recognition element is responsible for the selective
recognition of the analyte and the physico-chemical signal that
provides the basis for the output signal. The expressed protein or
molecule does not need to be a naturally occurring membrane bound
protein but could be a soluble protein or small molecule tethered
to the minicell by, for example, a transmembrane domain of another
protein such as the EGFR or ToxR.
[0834] Biosensors may include biocatalytic biosensors, and
bioaffinity biosensors. In biocatalytic biosensor embodiments, the
bio-recognition element minicell is "biocatalytic," e.g., displays
an enzyme. In biocatalytic biosensors, the selective binding sites
"turn over" (i.e., can be used again during the detection process),
resulting in a significant amplification of the input signal.
Biocatalytic sensors such as these are generally useful for
real-time, continuous sensing.
[0835] Bioaffinity sensors are generally applicable to bacteria,
viruses, toxins and other undesirable compounds and include
chemoreceptor-based biosensors and/or immunological sensors (i.e.,
immunosensors). Chemoreceptors are complex biomolecular
macroassemblies responsible, in part, for a viable organism's
ability to sense chemicals in its environment with high
selectivity. Chemoreceptor-based biosensors comprise one or more
natural or synthetic chemoreceptors associated with a means to
provide a signal (visual, electrical, etc.) of the presence or
concentration of a target biological analyte. In certain
embodiments, the chemoreceptor may be associated with an electrode
(i.e., an electrical transducer) so as to provide a detectable
electrical signal. In the biosensors of the invention, minicells
displaying a receptor are used in place of chemoreceptors. The
minicell has many desired features of a viable cell, and performs
similar functions, but is more durable.
[0836] On the other hand, the bio-recognition elements of
immunosensors are generally antibodies or antibody derivatives. In
any case, bioaffinity biosensors are generally irreversible because
the receptor sites of the biosensor become saturated when exposed
to the target biological analyte. In a biosensor of the invention,
an immunosensor may be a minicell displaying an antibody or
antibody fragment.
[0837] Biocatalytic and bioaffinity biosensor systems are described
in more detail in Journal of Chromatography, 510 (1990) 347-354 and
in the Kirk-Othmer Encyclopedia of Chemical Technology, 4.sup.th
ed. (1992), John Wiley & Sons, NY, the disclosure of which is
incorporated by reference herein.
[0838] The biosensors of the present invention may detect
biologically active analytes related to impending (i.e., future
presentation of symptoms is likely) or current human systemic
disease states, including, but not limited to, pathogenic bacteria,
parasites (e.g., any stage of the life cycle, including eggs or
portions thereof, cysts, or mature organisms), viruses, fungi such
as Candida albicans, antibodies to pathogens, and/or microbially
produced toxins. Additionally, the biosensor may target
biologically active analytes related to impending or current
localized health issues, such as stress proteins (e.g., cytokines)
and interleukin 1-alpha that may precede the clinical presentation
of skin irritation or inflammation. In preferred embodiments, the
biosensor functions as a proactive sensor, detecting and signaling
the subject, a caretaker or medical personnel of the impending
condition prior to the presentation of clinical symptoms. This
allows time to administer prophylactic or remedial treatments to
the subject which can significantly reduce, if not prevent, the
severity and duration of the symptoms. Further, the sensor, by
detecting the presence of a target biological analyte in a sample
from the subject, may detect residual contamination on a surface,
such as skin or environmental surface, in contact with the
biosensor, and provide and appropriate signal.
[0839] The physico-chemical signal generated by the bio-recognition
element or elements may be communicated visually to the caretaker
or medical personnel (i.e., via a color change visible to the human
eye). Other embodiments may produce optical signals, which may
require other instrumentation to enhance the signal. These include
flourescence, bioluminesence, total internal reflectance resonance,
surface plasmon resonance, Raman methods and other laser-based
methods, such as LED or laser diode sensors. For example, exemplary
surface plasmon resonance biosensors are available as IBIS I and
IBIS II from XanTec Analysensysteme of Muenster, Germany, which may
comprise bioconjugate surfaces as bio-recognition elements.
Alternatively, the signal may be processed via an associated
transducer which, for example, may produce an electrical signal
(e.g., current, potential, inductance, or impedance) that may be
displayed (e.g., on a readout such as an LED or LCD display) or
which triggers an audible or tactile (e.g., vibration) signal or
which may trigger an actuator, as described herein. The signal may
be qualitative (e.g., indicating the presence of the target
biological analyte) or quantitative (i.e., a measurement of the
amount or concentration of the target biological analyte). In such
embodiments, the transducer may optionally produce an optical,
thermal or acoustic signal.
[0840] In any case, the signal may also be durable (i.e., stable
and readable over a length of time typically at least of the same
magnitude as the usage life of the device) or transient (i.e.,
registering a real-time measurement). Additionally, the signal may
be transmitted to a remote indicator site (e.g., via a wire, or
transmitter, such as an infrared or rf transmitter) including other
locations within or on the device or remote devices. Further, the
sensor, or any of its components, may be adapted to detect and/or
signal only concentrations of the target biological analyte above a
predefined threshold level (e.g., in cases wherein the target
biological analyte is normally present in the bodily waste or when
the concentration of the analyte is below a known "danger"
level).
[0841] The target analytes that the biosensors of the present
invention are adapted to detect may also be viruses. These may
include diarrhea-inducing viruses such as rotavirus, or other
viruses such as rhinovirus and human immunodeficiency virus (HIV).
An exemplary biosensor adapted to detect HIV is described in U.S.
Pat. Nos. 5,830,341 and 5,795,453, referenced above. The disclosure
of each of these patents is incorporated by reference herein.
Biosensors are adopted to use in different tissues; see, e.g., U.S.
Pat. No. 6,342,037; Roe et al. Jan. 29, 2002; Device having fecal
component sensor; and using different binding molecules, see, e.g.,
U.S. Pat. No. 6,329,160; Schneider et al. Dec. 11, 2001;
Biosensors.
[0842] When minicells are incorporated into a biosensor, they may
be immobilized in the biosensor by techniques known in the art such
as entrapment, adsorption, crosslinking, encapsulation, covalent
attachment, any combination thereof, or the like. Further, the
immobilization can be carried out on many different substrates such
as known the art. In certain preferred embodiments, the
immobilization substrate may be selected from the group of
polymer-based materials, hydrogels, tissues, nonwoven materials or
woven materials.
[0843] In certain embodiments, biosensor embodiments, may comprise,
be disposed on, or be operatively associated with a microchip, such
as a silicon chip, MEMs (i.e., micro electromechanical system)
device, or an integrated circuit. Microchip-based biosensors may be
known as "biochips". Regardless of the type of sensor, the
microchip may comprise a multiplicity of sensor components having
similar or different sensitivities, kinetics, and/or target
analytes (i.e., markers) in an array adapted to detect differing
levels or combinations of the analyte(s). Further, each sensor in
such an array may provide a different type of signal, including
those types disclosed herein, and may be associated with different
actuators and/or controllers. Also, each sensor in an array may
operate independently or in association with (e.g., in parallel,
combination, or series) any number of other sensors in the
array.
[0844] A minicell of a biosensor of the invention may comprise a
detectable compound that produces a signal once ligands have bound
to the minicell. By way of non-limiting example, a minicell may
display a receptor for a ligand and contain a fluorescent compound.
The binding and internalization of the ligand into the minicell
results in FRET, shifting the wavelength of the signal. See, by way
of non-limiting example, Billinton et al., Development of a green
fluorescent protein reporter for a yeast genotoxicity biosensor,
Biosensors & Bioelectronics 13:831-838, 1998. A biosensor
according to the invention may use microbalance sensor systems
(Hengerer et al., Determination of phage antibody affinities to
antigen by a microbalance sensor system, BioTechniques 26:956-964,
1999).
[0845] XVIII.A.2. Surface Plasmon Resonance
[0846] Kd is measured using surface plasmon resonance on a chip,
for example, with a BIAcore.RTM. chip coated with immobilized
binding components, or similar systems such as the IAsys from
Thermo Labsystems, Affinity Sensors Division (Cambridge, U.K.) or
the BIOS-1 system from Artificial Sensing, Inc. (Zurich,
Switzerland). See Fitzgerald, Coupling optical biosensor technology
with micropreparative HPLC: Part 1, Am Biotech Lab November 2000,
p. 10 and 12; Fitzgerald, Coupling optical biosensor technology
with micropreparative HPLC: Part 2, Am Biotech Lab February 2001,
14, 16 and 18; and Leatherbarrow et al., Analysis of molecular
recognition using optical sensors, Current Opinion in Chem Biol
3:544-547, 1999).
[0847] Surface plasmon resonance is used to characterize the
microscopic association and dissociation constants of reaction
between an antibody or antibody fragment and its ligand. Such
methods are generally described in the following references that
are incorporated herein by reference. (Vely F. et al., BIAcore
analysis to test phosphopeptide-SH2 domain interactions, Methods in
Molecular Biology. 121:313-21, 2000; Liparoto et al., Biosensor
analysis of the interleukin-2 receptor complex, Journal of
Molecular Recognition. 12:316-21, 1999; Lipschultz et al.,
Experimental design for analysis of complex kinetics using surface
plasmon resonance, Methods. 20):310-8, 2000; Malmqvist., BIACORE:
an affinity biosensor system for characterization of biomolecular
interactions, Biochemical Society Transactions 27:335-40, 1999;
Alfthan, Surface plasmon resonance biosensors as a tool in antibody
engineering, Biosensors & Bioelectronics. 13:653-63, 1998;
Fivash et al., BIAcore for macromolecular interaction, Current
Opinion in Biotechnology. 9:97-101, 1998; Price et al.; Summary
report on the ISOBM TD-4 Workshop: analysis of 56 monoclonal
antibodies against the MUC1 mucin. Tumour Biology 19 Suppl 1:1-20,
1998; Malmqvist et al, Biomolecular interaction analysis: affinity
biosensor technologies for functional analysis of proteins, Current
Opinion in Chemical Biology. 1:378-83, 1997; O'Shannessy et al.,
Interpretation of deviations from pseudo-first-order kinetic
behavior in the characterization of ligand binding by biosensor
technology, Analytical Biochemistry. 236:275-83, 1996; Malmborg et
al., BIAcore as a tool in antibody engineering, Journal of
Immunological Methods. 183:7-13, 1995; Van Regenmortel, Use of
biosensors to characterize recombinant proteins, Developments in
Biological Standardization. 83:143-51, 1994; O'Shannessy,
Determination of kinetic rate and equilibrium binding constants for
macromolecular interactions: a critique of the surface plasmon
resonance literature, Current Opinions in Biotechnology. 5:65-71,
1994).
[0848] BIAcore.RTM. uses the optical properties of surface plasmon
resonance (SPR) to detect alterations in protein concentration
bound within to a dextran matrix lying on the surface of a
gold/glass sensor chip interface, a dextran biosensor matrix. In
brief, proteins are covalently bound to the dextran matrix at a
known concentration and a ligand for the protein (e.g., antibody)
is injected through the dextran matrix. Near infra red light,
directed onto the opposite side of the sensor chip surface is
reflected and also induces an evanescent wave in the gold film,
which in turn, causes an intensity dip in the reflected light at a
particular angle known as the resonance angle. If the refractive
index of the sensor chip surface is altered (e.g., by ligand
binding to the bound protein) a shift occurs in the resonance
angle. This angle shift can be measured and is expressed as
resonance units (RUs) such that 1000 RUs is equivalent to a change
in surface protein concentration of 1 ng/mm2. These changes are
displayed with respect to time along the y-axis of a sensorgram,
which depicts the association and dissociation of any biological
reaction.
[0849] Additional details may be found in Jonsson et al.,
Introducing a biosensor based technology for real-time biospecific
interaction analysis, (1993) Ann. Biol. Clin. 51:19-26; Jonsson et
al., Real-time biospecific interaction analysis using surface
plasmon resonance and a sensor chip technology, (1991)
Biotechniques 11:620-627; Johnsson et al., Comparison of methods
for immobilization to carboxymethyl dextran sensor surfaces by
analysis of the specific activity of monoclonal antibodies, (1995)
J. Mol. Recognit. 8:125-131; and Johnsson, Immobilization of
proteins to a carboxymethyldextran-modified gold surface for
biospecific interaction analysis in surface plasmon resonance
sensors (1991) Anal. Biochem. 198:268-277, Karlsson et. al.,
Kinetic analysis of monoclonal antibody-antigen interactions with a
new biosensor based analytical system J. Immunol. Meth., 145, 229,
1991; Weinberger et al., Recent trends in protein biochip
technology, Pharmacogenomics 2000 November; 1(4):395-416;
Lipschultz et al., Experimental design for analysis of complex
kinetics using surface plasmon resonance, Methods 2000 March;
20(3):310-8.
[0850] XVIII.B. Toxicological Sampling
[0851] Minicells are ideally suited for in vitro diagnostic
toxicological applications in which toxins, poisons, infectious
agents or pathogens, heavy metals, pollutants, caustic agents,
allergens, organic molecules, radionuclides, or other environmental
contaminants present either in air, water, soil samples and/or
fluid and/or tissue samples of organisms can be assessed. An
embodiment of this invention, minicells expressing proteins or
other molecules could be used in variety of diagnostic detection
platforms, including microwell formats, lateral flow devices,
molecular switches, biosensors, badges and other sensing devices.
Without being limited to the following examples, such devices could
be used for early warning of chemical and/or bioweapon attack,
illegal drug detection, explosives detection, biohazard detection,
pollution assessment, pesticide contamination, allergen detection
and detection of toxic or hazardous gasses. In a related
application, minicells could be used to eliminate, modify or
inactivate the agents.
[0852] In one non-limiting example of protein expression on
minicells for toxicological detection, olfactory receptors could be
expressed by minicells. The olfactory system possesses the ability
to recognize and differentiate between a wide range of odorants
based on odor molecules interacting with specific receptor proteins
in the ciliary membrane of olfactory neurons (Lancet, D., 1986.
Vertebrate olfactory reception. Ann. Rev. Neurosci. 9:329-355;
Shepherd, G. M., 1994. Discrimination of molecular signals by the
olfactory receptor neuron. Neuron 13:771-790). These receptors were
found to be 7-transmembrane-domain members of the G protein-coupled
receptor family (Buck, L. and R. Alex. 1991. A novel multigene
family may encode odorant receptors: A molecular basis for odor
recognition. Cell 65:175-187). Using a murine receptor library,
olfactory receptors were functionally expressed in HEK-293 cells
(Krautwurst, D., et al., 1998. Identification of ligands for
olfactory receptors by functional expression of a receptor library.
Cell. 95:917-926). By coexpressing the cloned receptors with
G.quadrature.15,16 subunits, the modified receptor system upon
activation leads to an increase in intracellular Ca.sup.2.+-..
Calcium levels were measured employing the dye FURA-2 and
ratiofluormetric imaging. This system demonstrated ligand
specificity and structure-function relationships for identified
olfactory receptors. Employing similar techniques, OR17-40, a human
olfactory receptor protein, was expressed in human embryonic kidney
293 cells and Xenopus Laevis oocytes (Wetzel, H., et al. 1999.
Specificity and sensitivity of a human olfactory receptor
functionally expressed in Human Embryonic Kidney 293 Cells and
Xenopus Laevis Oocytes. J. Neurosciences. 19:7426-7433). The
receptor was functionally expressed in a manner designed to assess
the specificity of its binding to the ligand, helional.
[0853] In one non-limiting example of target protein
identification, primers from homologous areas in transmembrane II
and transmembrane VII of olfactory GPCRs will be used to identify
unique receptor sequences. These sequences are inserted into
expression vectors. Minicell producing bacteria are transformed
with these vectors and cultured. Minicells are isolated from the
culture as previously described and subsequently induced. Using HTS
previously described, the functional receptor/minicells which
generate signal for binding of an odiferous toxin to the receptor
are identified. Large scaleLarge-scale production of the minicells
is carried out and the minicells are covalently coupled to the
surface of a microarraymicro array chip. The chip is supported in
an air sampler, which feeds atmosphere over surface of the chip on
a continuous basis. If the toxic agent is present in the air, the
binding to the receptor activates a series of events ending in the
generation of a signal identifying the presence of the agent in the
air.
[0854] By way of non-limiting example, standard molecular
biological techniques can be used as follows: cDNA for GFP is
ligated to the 3' end of cDNA sequence for the receptor described
above. The resulting sequence is inserted into an expression
vector. Minicell producing bacteria are transformed with these
vectors and cultured. Minicells are isolated from the culture as
previously described and subsequently induced. The minicells now
contain the receptor to the ligand on the surface of the minicell
with a GFP tag on the C-terminus of the protein in the cytosol.
These minicells are packed into filters. Air is passed through the
filter. If the ligand is present, it will bind to the receptor. The
filter packing is suspended on applied to a diagnostic device.
Antibody to the ligand/receptor binding site complex is fixed on
the capture zone. When the sample is applied to the device, the
receptor/ligand complex is captured. The capture zone is screened
for signal resulting from the presence of GFP. This can be
extrapolated to have multiple unique receptor/minicell moieties in
the same sampling device. Each receptor would have a unique
fluorescing protein tag such that different emissions identify
specific agents in the air.
[0855] Other methods for quantification associated take advantage
of the composition of the minicell. Loading of the minicell by
transiently permeabilizing the membrane to allow for migration of
molecules into to the cytosol. These molecules include but are not
limited to radiolabeled molecules (i.e., nucleotides), stains or
dyes (DAPI or other DNA staining, heavy metals, fluorophores. The
molecules could also be synthesized within the minicell (i.e. GFP).
The association of a specific ligand with the minicell could cause
a redox shift that induce a color change in the solution or could
shift the energy potential in the reaction are generating an
electrical current. Each of this examples are associated with well
know methods for measuring each of the resulting changes. These
include but are not limited to radioactivity or fluorescence
generated or the color shift by spectrophotometry.
[0856] A multigene family of gustatory G protein-coupled receptors
expressed in the lingual epithelia has been identified with
structural similarities to olfactory receptors (Abe, K., et al.
1993. Multiple genes for G protein-coupled receptors and their
expression in lingual epithelia. FEBS. 316:253-256; Abe, K., et al.
1993. Primary structure and cell-type specific expression of a
gustatory G protein-coupled receptor related to olfactory
receptors. J. Bio. Chem.). This provides an addition example of
receptors which can be isolated, expressed in minicells and then be
used for identification of specific substances in various matrices
in similar manners as identified for olfactory receptor
minicells.
[0857] As a non-limiting example of minicell use in
toxicological/environmental detection, arrays could be constructed
in which each well contains a distinct minicell subtype displaying
membrane-bound proteins or other molecules for each of several
potential toxins or agents in the environment. For example,
minicells in such a format could be used to determine which agents
are present in the environment as a consequence of a chemical
and/or biological weapons attack. Non-limiting examples of
biosensors that have been used toxicological/environmental
detection include those described by Sticher et al., Development
and characterization of a whole-cell bioluminescent sensor for
bioavailable middle-chain alkanes in contaminated groundwater
samples, Appl. Envir. Microbiol. 63:4053-4060, 1997; Willardson et
al., Development and Testing of a Bacterial Biosensor for
Toluene-Based Environmental Contaminants, Appl. Envir. Microbiol.
64:1006-1012, 1998; Lars et al., Detection of Oxytetracycline
Production by Streptomyces rimosus in Soil Microcosms by Combining
Whole-Cell Biosensors and Flow Cytometry, Appl. Envir. Microbiol.
67:239-244, 2001; Hojberg et al., Oxygen-Sensing Reporter Strain of
Pseudomonas fluorescens for Monitoring the Distribution of
Low-Oxygen Habitats in Soil, Appl. Envir. Microbiol. 1999 65:
4085-4093, 1999; R. P. Hollis et al., Design and Application of a
Biosensor for Monitoring Toxicity of Compounds to Eukaryotes, Appl.
Envir. Microbiol. 66: 1676-1679, 2000; Heitzer et al., Optical
biosensor for environmental on-line monitoring of naphthalene and
salicylate bioavailability with an immobilized bioluminescent
catabolic reporter bacterium, Appl. Envir. Microbiol. 60:1487-1494,
1994; Selifonova et al., Bioluminescent sensors for detection of
bioavailable Hg(II) in the environment, Appl. Envir. Microbiol. 59:
3083-3090, 1993; Jaeger et al., Mapping of Sugar and Amino Acid
Availability in Soil around Roots with Bacterial Sensors of Sucrose
and Tryptophan, Appl. Envir. Microbiol. 65: 2685-2690, 1999; and
Larsen et al., A Microsensor for Nitrate Based on Immobilized
Denitrifying Bacteria, Appl. Envir. Microbiol. 62: 1248-1251,
1996.
[0858] XVIII.C. Toxin Elimination
[0859] In another embodiment of the invention, minicells displaying
a receptor for a particular toxic agent could be used for the
elimination of the agent from the environment. In a non-limiting
example of this technology, minicells could be placed in a
filtering apparatus to eliminate the toxic agent from the
environment (e.g., air, water soil). In the example of atmospheric
contamination, the air would be circulated through a forced air
system containing in-line filters composed of a housing, support
matrix and receptor/minicells. As air passes over the minicells,
the toxin is bound to the receptor. The purified air passed out of
the system and into the atmosphere. A similar method for water
purification would follow a similar protocol replacing the receptor
for the toxin with the receptor or other protein binding a unique
epitope on contaminant wishing to be removed. Examples include but
are not limited to removing toxins, parasites or microbes from the
matrix such as water or air. This represent non-limiting example of
minicell-based technology for expression of functional receptors or
binding moieties of receptors on the minicell's surface for the
specific purpose of selectively capturing, identifying, quantifying
and/or removing molecules of interest for environmental
compartments to include but not limited to air water, soil, other
gas phases or liquid solutions.
[0860] Representative toxins include, but are not limited to, those
associated with "red tides"; eubacterial toxins, such as those
toxins produced by Corynebacterium diphtheriae (diphtheria),
Bordetella pertussis (whooping cough), Vibrio cholerae (cholera),
Bacillus anthracis (anthrax), Clostridium botulinum (botulism),
Clostridium tetani (tetanus), and enterohemorrhagic Escherichia
coli (bloody diarrhea and hemolytic uremic syndrome); and fungal
toxins (e.g., aflatoxin, gliotoxin, cyclopeptides, orellanine,
gyrometrin, coprine, muscarine, ibotenic acid, psilocybin, psilocin
and baeocystin).
[0861] The treatment of "red tides" with minicells exemplifies this
aspect of the invention. A red tide occurs as a result of a
higher-than-normal concentration of an algae or dinoflagellate
which, when present in dense concentrations as a result of a
"bloom," form colored patches on the surface of water. The colored
patches are pink, violet, orange, yellow, blue, green, brown, or
red, with red being the most common color. The organisms that cause
red tides often produce toxins that have negative impacts on other
organisms, including humans.
[0862] For example, Karenia brevis (formerly Gymnodinium breve)
produces a toxin (domoic acid) that affects the central nervous
system of fish, shellfish and other organisms, resulting in a state
of paralysis. Alexandrium species (e.g., A. tamarense, A.
fundyense), Dinophysis and Gonyaulax species; and Pseudo-nitzschia
multiseries, which cause, respectively, paralytic, diarrhetic and
amnesic shellfish poisoning. Because shellfish containing the toxin
taste and appear the same as shellfish that do not, and cooking
does not destroy the toxin, human ingestion of the former can cause
disease in humans and other organisms. For example, one form of
paralytic shellfish poisoning, which can be fatal to humans,
results from saxitoxin, which is produced by Gonyaulax tamarenis,
Protogonyaulax catanella, and other species. Other algae that can
result in red tides include Gonyaulax catenella, and Ptychdiscus
breve.
[0863] Minicells that comprise a binding moiety of an organism that
produces a red tide, or of the toxin produced thereby, can be used
for remediation. For example, a minicell having a binding moiety
directed to a red tide-producing organism can be used to deliver an
antibiotic thereto, and a minicell with a binding moiety directed
to a toxin can be used to bind and/or internalize the toxin. As is
explained in more detail elsewhere herein, a minicell with a
binding moiety directed to a toxin can also be used for therapeutic
purposes.
[0864] XVIII.D. Bioremediation
[0865] In another non-limiting example of the potential use of
minicells in a toxicological context is their use in
bioremediation, the process by which living organisms act to
degrade or transform hazardous organic contaminants. As used
herein, "bioremediation" is the process of using biological or
biologically derived compositions that alter the chemical structure
and/or bind, an undesirable substance in order to reduce the
effective concentration of the undesirable substance, thereby
reducing or eliminating the effect(s) of the undesirable compound
on the environment. Undesirable substances include, but are not
limited to, pollutants (e.g., heavy metals, pesticides, herbicides,
petroleum products); biological toxins (e.g., such as those
produced by "red tides", e.g., domoic acid, saxitoxin); pathogens
(e.g., viruses, eubacteria); organisms that produce toxins;
biological waste products (e.g., sewage, guano), and undesirable
organisms therewithin (e.g., pathogenic eubacteria).
[0866] The term "bioremediation" encompasess both biodegradation,
the breakdown of organic substances by microorganisms, and
biotransformation, the alteration of the structure of a compound by
a living organism or enzyme. The minicells of the invention may be
incorporated into biofilters, i.e., devices in which gases,
liquids, powders and the like are passed through media containing
biodegrading minicells, including but not limited to devices that
biodegrade volatile organic compounds in air by passing the air
therethrough.
[0867] Bioremediation can be used to process undesirable substances
in a composition prior to or after the release of the composition
into the environment. For example, bioremediation can be applied in
sewage treatment plants to process sewage prior to its release, or
to sewage that has been accidentally or otherwise released into the
environment.
[0868] Environmental microbiologists have sought to identify and
use specific bacteria that degrade pollutants and other
environmental containments. See, for example, Chakrabarty,
Microbial Degradation of Toxic Chemicals: Evolutionary Insights and
Practical Considerations, Am. Soc. Micro. Biol. News 62:130-137,
1996; and U.S. Pat. Nos. 4,511,657; 4,493,895; 4,871,673; and
4,535,061. In instances where a live organism is placed into the
environment to process undesirable substances, there is a concern
that the organism might have undesirable effects that would be made
more deleterious due to the ability of the live organism to
replicate (Sayler G S, Ripp S. Field applications of genetically
engineered microorganisms for bioremediation processes. Curr Opin
Biotechnol. 2000 June; 11(3):286-9; and Diaz E, Ferrandez A, Prieto
M A, Garcia J L. Biodegradation of aromatic compounds by
Escherichia coli. Microbiol Mol Biol Rev. 2001 December;
65(4):523-69). For example, when it has been proposed to use
genetically altered eubacteria to process oil spills, the concern
has been raised that the eubacteria might spread beyond the oil
spill and into supplies of petroleum products that are used to
produce energy, where they would process and render useless the
stored petroleum products. However, because they lack the ability
to replicate, such a scenario will not occur when minicells are use
for bioremediation.
[0869] By way of non-limiting example, octane enhances such as
methyl t-butyl ether or aromatic hydrocarbons contaminate the
aquifer and soil. These agents negatively impact the many microbes
in the effected area thus limiting capability of the microbial
community rectify the environmental insult. Bioaugmentation, the
addition to the environment of microorganisms that can metabolize
and grow on specific organic compounds, to facilitate degradation
may prove useful, but concerns exist relative to the regulation of
newly introduced bacteria. The minicell provides a vehicle to
accomplish biodegradation without bacterial overgrowth.
[0870] Diphenyl ethers and cyclic ethers such as dioxane and furan
have shown to be metabolized by soil bacteria. Using classic
isolation and screening techniques identified above, genes encoding
for the oxygenases or hydroylases are isolated. The enzyme sequence
is inserted into an expression vector using standard molecular
biology techniques. Minicell producing bacteria are transformed
with these vectors and cultured. Minicells are isolated from the
culture as previously described and subsequently induced. The
minicells are applied to the area contaminated with aromatic
hydrocarbons. These compounds are transported either actively or
passively in to the minicell and subsequently degraded by the
oxygenase or hydroylase. One advantage of this focused degradation
is the minimizing of feedback inhibition because the only machinery
of consequence in the minicell is that related to the degradation
of the ether compounds.
[0871] Similarly, beginning with genetic material from Dehalobacter
enzymes responsible for the biodegrading of tetrachloroethane could
be isolated as described above. The sequence for the enzyme is
inserted into the expression vector and used to transform
minicell-producing bacteria. The bacteria are cultured, minicells
isolated from the culture and the minicells induced as previously
described. Minicell preps are lyophilized using standard
lyophilization techniques. The resulting material is transported to
the site of tetrachloroethene contamination and reconstituted and
applied. As the tetracholoethene was assimilated, it is be degraded
by the enzyme system.
[0872] These are non-limiting examples scope of
bioremediation/biotransformation using minicell technology. The
scope of the invention includes taking advantage of metabolic
pathways organism in general to include but not limited to
eukaryotes, prokaryotes, fungi, animals or plants.
[0873] XVIII.E. Fermentation
[0874] Delivery of specific enzymes in an untargeted fashion by the
minicell allows for packaged delivery without the increased biomass
and complex metabolic products associated with processes using live
organisms. This aspect can be taken advantage of in fermentation,
where the addition of minicells into which unique enzymes have been
added are used to modulate the composition of the environment to
include but not limited to the alcohol, sugar and acid levels.
[0875] XVIII.F. Pesticides
[0876] Bacillus thurigenesis produces a toxin that kills plant
chewing insect larvae as well as mosquito larvae. The toxin,
Cry1Ac, binds to aminopeptidase N receptor on the endothelium of
the midgut. Minicell technology is allows for delivery of the
toxin. The toxin sequence is modified by ligation of a sequence
coding for a transmembrane domain as previously described. The
sequence for this fusion protein inserted into an expression vector
using standard molecular biology techniques. To facilitate the
consumption of the toxin/minicell plasmids containing sequences
incorporating the sequence for pheromones coupled at the C-terminus
to the sequence for a transmembrane domain is generated using
standard molecular biological techniques. This fusion protein
sequence is inserted into the expression containing coding region
for the toxin fusion protein or inserted into a unique expression
vector. Minicell producing bacteria are transformed with these
vectors and cultured. Minicells are isolated from the culture as
previously described and subsequently induced. The minicells are
distributed (e.g crop dusting) to the area of infestation. The
toxin/minicells are ingested by the larvae and kill the larvae as
the minicells passes through the gut.
[0877] XIX. Pharmaceutical Compositions
[0878] Another aspect of the invention is drawn to compositions,
including but not limited to pharmaceutical compositions. According
to the invention, a "composition" refers to a mixture comprising at
least one carrier, preferably a physiologically acceptable carrier,
and one or more minicell compositions. The term "carrier" defines a
chemical compound that does not inhibit or prevent the
incorporation of the biologically active peptide(s) into cells or
tissues. A carrier typically is an inert substance that allows an
active ingredient to be formulated or compounded into a suitable
dosage form (e.g., a pill, a capsule, a gel, a film, a tablet, a
microparticle (e.g., a microsphere), a solution; an ointment; a
paste, an aerosol, a droplet, a colloid or an emulsion etc.). A
"physiologically acceptable carrier" is a carrier suitable for use
under physiological conditions that does not abrogate (reduce,
inhibit, or prevent) the biological activity and properties of the
compound. For example, dimethyl sulfoxide (DMSO) is a carrier as it
facilitates the uptake of many organic compounds into the cells or
tissues of an organism.
[0879] Preferably, the carrier is a physiologically acceptable
carrier, preferably a pharmaceutically or veterinarily acceptable
carrier, in which the minicell composition is disposed.
[0880] A "pharmaceutical composition" refers to a composition
wherein the carrier is a pharmaceutically acceptable carrier, while
a "veterinary composition" is one wherein the carrier is a
veterinarily acceptable carrier. The term "pharmaceutically
acceptable carrier" or "veterinarily acceptable carrier" includes
any medium or material that is not biologically or otherwise
undesirable, i.e., the carrier may be administered to an organism
along with a minicell composition without causing any undesirable
biological effects or interacting in a deleterious manner with the
complex or any of its components or the organism. Examples of
pharmaceutically acceptable reagents are provided in The United
States Pharmacopeia, The National Formulary, United States
Pharmacopeial Convention, Inc., Rockville, Md. 1990, hereby
incorporated by reference herein into the present application. The
terms "therapeutically effective amount" or "pharmaceutically
effective amount" mean an amount sufficient to induce or effectuate
a measurable response in the target cell, tissue, or body of an
organism. What constitutes a therapeutically effective amount will
depend on a variety of factors, which the knowledgeable
practitioner will take into account in arriving at the desired
dosage regimen.
[0881] The compositions of the invention can further comprise other
chemical components, such as diluents and excipients. A "diluent"
is a chemical compound diluted in a solvent, preferably an aqueous
solvent, that facilitates dissolution of the composition in the
solvent, and it may also serve to stabilize the biologically active
form of the composition or one or more of its components. Salts
dissolved in buffered solutions are utilized as diluents in the
art. For example, preferred diluents are buffered solutions
containing one or more different salts. A preferred buffered
solution is phosphate buffered saline (particularly in conjunction
with compositions intended for pharmaceutical administration), as
it mimics the salt conditions of human blood. Since buffer salts
can control the pH of a solution at low concentrations, a buffered
diluent rarely modifies the biological activity of a biologically
active peptide.
[0882] An "excipient" is any more or less inert substance that can
be added to a composition in order to confer a suitable property,
for example, a suitable consistency or to form a drug. Suitable
excipients and carriers include, in particular, fillers such as
sugars, including lactose, sucrose, mannitol, or sorbitol cellulose
preparations such as, for example, maize starch, wheat starch, rice
starch, agar, pectin, xanthan gum, guar gum, locust bean gum,
hyaluronic acid, casein potato starch, gelatin, gum tragacanth,
methyl cellulose, hydroxypropylmethyl-cellulose, polyacrylate,
sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
If desired, disintegrating agents can also be included, such as
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate. Other suitable excipients and
carriers include hydrogels, gellable hydrocolloids, and chitosan.
Chitosan microspheres and microcapsules can be used as carriers.
See WO 98/52547 (which describes microsphere formulations for
targeting compounds to the stomach, the formulations comprising an
inner core (optionally including a gelled hydrocolloid) containing
one or more active ingredients, a membrane comprised of a water
insoluble polymer (e.g., ethylcellulose) to control the release
rate of the active ingredient(s), and an outer layer comprised of a
bioadhesive cationic polymer, for example, a cationic
polysaccharide, a cationic protein, and/or a synthetic cationic
polymer; U.S. Pat. No. 4,895,724. Typically, chitosan is
cross-linked using a suitable agent, for example, glutaraldehyde,
glyoxal, epichlorohydrin, and succinaldehyde. Compositions
employing chitosan as a carrier can be formulated into a variety of
dosage forms, including pills, tablets, microparticles, and
microspheres, including those providing for controlled release of
the active ingredient(s). Other suitable bioadhesive cationic
polymers include acidic gelatin, polygalactosamine, polyamino acids
such as polylysine, polyhistidine, polyornithine, polyquaternary
compounds, prolamine, polyimine, diethylaminoethyldextran (DEAE),
DEAE-imine, DEAE-methacrylate, DEAE-acrylamide, DEAE-dextran,
DEAE-cellulose, poly-p-aminostyrene, polyoxethane,
copolymethacrylates, polyamidoamines, cationic starches,
polyvinylpyridine, and polythiodiethylaminomethylethylene.
[0883] The compositions of the invention can be formulated in any
suitable manner. Minicell compositions may be uniformly
(homogeneously) or non-uniformly (heterogenously) dispersed in the
carrier. Suitable formulations include dry and liquid formulations.
Dry formulations include freeze dried and lyophilized powders,
which are particularly well suited for aerosol delivery to the
sinuses or lung, or for long term storage followed by
reconstitution in a suitable diluent prior to administration. Other
preferred dry formulations include those wherein a composition
according to the invention is compressed into tablet or pill form
suitable for oral administration or compounded into a sustained
release formulation. When the composition is intended for oral
administration but is to be delivered to epithelium in the
intestines, it is preferred that the formulation be encapsulated
with an enteric coating to protect the formulation and prevent
premature release of the minicell compositions included therein. As
those in the art will appreciate, the compositions of the invention
can be placed into any suitable dosage form. Pills and tablets
represent some of such dosage forms. The compositions can also be
encapsulated into any suitable capsule or other coating material,
for example, by compression, dipping, pan coating, spray drying,
etc. Suitable capsules include those made from gelatin and starch.
In turn, such capsules can be coated with one or more additional
materials, for example, and enteric coating, if desired. Liquid
formulations include aqueous formulations, gels, and emulsions.
[0884] Some preferred embodiments concern compositions that
comprise a bioadhesive, preferably a mucoadhesive, coating. A
"bioadhesive coating" is a coating that allows a substance (e.g., a
minicellcomposition) to adhere to a biological surface or substance
better than occurs absent the coating. A "mucoadhesive coating" is
a preferred bioadhesive coating that allows a substance, for
example, a composition according to the invention, to adhere better
to mucosa occurs absent the coating. For example, micronized
particles (e.g., particles having a mean diameter of about 5, 10,
25, 50, or 100 .mu.m) can be coated with a mucoadhesive. The coated
particles can then be assembled into a dosage form suitable for
delivery to an organism. Preferably, and depending upon the
location where the cell surface transport moiety to be targeted is
expressed, the dosage form is then coated with another coating to
protect the formulation until it reaches the desired location,
where the mucoadhesive enables the formulation to be retained while
the composition interacts with the target cell surface transport
moiety.
[0885] The compositions of the invention may be administered to any
organism, preferably an animal, preferably a mammal, bird, fish,
insect, or arachnid. Preferred mammals include bovine, canine,
equine, feline, ovine, and porcine animals, and non-human primates.
Humans are particularly preferred. Multiple techniques of
administering or delivering a compound exist in the art including,
but not limited to, oral, rectal (e.g. an enema or suppository)
aerosol (e.g., for nasal or pulmonary delivery), parenteral, and
topical administration. Preferably, sufficient quantities of the
biologically active peptide are delivered to achieve the intended
effect. The particular amount of composition to be delivered will
depend on many factors, including the effect to be achieved, the
type of organism to which the composition is delivered, delivery
route, dosage regimen, and the age, health, and sex of the
organism. As such, the particular dosage of a composition
incorporated into a given formulation is left to the ordinarily
skilled artisan's discretion.
[0886] Those skilled in the art will appreciate that when the
compositions of the present invention are administered as agents to
achieve a particular desired biological result, which may include a
therapeutic or protective effect(s) (including vaccination), it may
be necessary to combine the fusion proteins of the invention with a
suitable pharmaceutical carrier. The choice of pharmaceutical
carrier and the preparation of the fusion protein as a therapeutic
or protective agent will depend on the intended use and mode of
administration. Suitable formulations and methods of administration
of therapeutic agents include those for oral, pulmonary, nasal,
buccal, ocular, dermal, rectal, or vaginal delivery.
[0887] Depending on the mode of delivery employed, the
context-dependent functional entity can be delivered in a variety
of pharmaceutically acceptable forms. For example, the
context-dependent functional entity can be delivered in the form of
a solid, solution, emulsion, dispersion, micelle, liposome, and the
like, incorporated into a pill, capsule, tablet, suppository,
areosol, droplet, or spray. Pills, tablets, suppositories,
areosols, powders, droplets, and sprays may have complex,
multilayer structures and have a large range of sizes. Aerosols,
powders, droplets, and sprays may range from small (1 micron) to
large (200 micron) in size.
[0888] Pharmaceutical compositions of the present invention can be
used in the form of a solid, a lyophilized powder, a solution, an
emulsion, a dispersion, a micelle, a liposome, and the like,
wherein the resulting composition contains one or more of the
compounds of the present invention, as an active ingredient, in
admixture with an organic or inorganic carrier or excipient
suitable for enteral or parenteral applications. The active
ingredient may be compounded, for example, with the usual
non-toxic, pharmaceutically acceptable carriers for tablets,
pellets, capsules, suppositories, solutions, emulsions,
suspensions, and any other form suitable for use. The carriers
which can be used include glucose, lactose, mannose, gum acacia,
gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn
starch, keratin, colloidal silica, potato starch, urea, medium
chain length triglycerides, dextrans, and other carriers suitable
for use in manufacturing preparations, in solid, semisolid, or
liquid form. In addition auxiliary, stabilizing, thickening and
coloring agents and perfumes may be used. Examples of a stabilizing
dry agent includes triulose, preferably at concentrations of 0.1%
or greater (See, e.g., U.S. Pat. No. 5,314,695). The active
compound is included in the pharmaceutical composition in an amount
sufficient to produce the desired effect upon the process or
condition of diseases.
[0889] XX. Small Molecules
[0890] The term "small molecule" includes any chemical or other
moiety that can act to affect biological processes. Small molecules
can include any number of therapeutic agents presently known and
used, or can be small molecules synthesized in a library of such
molecules for the purpose of screening for biological function(s).
Small molecules are distinguished from macromolecules by size. The
small molecules of this invention usually have molecular weight
less than about 5,000 daltons (Da), preferably less than about
2,500 Da, more preferably less than 1,000 Da, most preferably less
than about 500 Da.
[0891] Small molecules include without limitation organic
compounds, peptidomimetics and conjugates thereof. As used herein,
the term "organic compound" refers to any carbon-based compound
other than macromolecules such nucleic acids and polypeptides. In
addition to carbon, organic compounds may contain calcium,
chlorine, fluorine, copper, hydrogen, iron, potassium, nitrogen,
oxygen, sulfur and other elements. An organic compound may be in an
aromatic or aliphatic form. Non-limiting examples of organic
compounds include acetones, alcohols, anilines, carbohydrates,
monosaccharides, oligosaccharides, polysaccharides, amino acids,
nucleosides, nucleotides, lipids, retinoids, steroids,
proteoglycans, ketones, aldehydes, saturated, unsaturated and
polyunsaturated fats, oils and waxes, alkenes, esters, ethers,
thiols, sulfides, cyclic compounds, heterocylcic compounds,
imidizoles and phenols. An organic compound as used herein also
includes nitrated organic compounds and halogenated (e.g.,
chlorinated) organic compounds. Methods for preparing
peptidomimetics are described below. Collections of small
molecules, and small molecules identified according to the
invention are characterized by techniques such as accelerator mass
spectrometry (AMS; see Turteltaub et al., Curr Pharm Des 2000
6(10):991-1007, Bioanalytical applications of accelerator mass
spectrometry for pharmaceutical research; and Enjalbal et al., Mass
Spectrom Rev 2000 19(3):139-61, Mass spectrometry in combinatorial
chemistry.)
[0892] Preferred small molecules are relatively easier and less
expensively manufactured, formulated or otherwise prepared.
Preferred small molecules are stable under a variety of storage
conditions. Preferred small molecules may be placed in tight
association with macromolecules to form molecules that are
biologically active and that have improved pharmaceutical
properties. Improved pharmaceutical properties include changes in
circulation time, distribution, metabolism, modification,
excretion, secretion, elimination, and stability that are favorable
to the desired biological activity. Improved pharmaceutical
properties include changes in the toxicological and efficacy
characteristics of the chemical entity.
[0893] XXI. Polypeptides and Derivatives
[0894] XXI.A. Polypeptides
[0895] As used herein, the term "polypeptide" includes proteins,
fusion proteins, oligopeptides and polypeptide derivatives, with
the exception that peptidomimetics are considered to be small
molecules herein. Although they are polypeptides, antibodies and
their derivatives are described in a separate section. Antibodies
and antibody derivatives are described in a separate section, but
antibodies and antibody derivatives are, for purposes of the
invention, treated as a subclass of the polypeptides and
derivatives.
[0896] A "protein" is a molecule having a sequence of amino acids
that are linked to each other in a linear molecule by peptide
bonds. The term protein refers to a polypeptide that is isolated
from a natural source, or produced from an isolated cDNA using
recombinant DNA technology; and has a sequence of amino acids
having a length of at least about 200 amino acids.
[0897] A "fusion protein" is a type of recombinant protein that has
an amino acid sequence that results from the linkage of the amino
acid sequences of two or more normally separate polypeptides.
[0898] A "protein fragment" is a proteolytic fragment of a larger
polypeptide, which may be a protein or a fusion protein. A
proteolytic fragment may be prepared by in vivo or in vitro
proteolytic cleavage of a larger polypeptide, and is generally too
large to be prepared by chemical synthesis. Proteolytic fragments
have amino acid sequences having a length from about 200 to about
1,000 amino acids.
[0899] An "oligopeptide" is a polypeptide having a short amino acid
sequence (i.e., 2 to about 200 amino acids). An oligopeptide is
generally prepared by chemical synthesis.
[0900] Although oligopeptides and protein fragments may be
otherwise prepared, it is possible to use recombinant DNA
technology and/or in vitro biochemical manipulations. For example,
a nucleic acid encoding an amino acid sequence may be prepared and
used as a template for in vitro transcription/translation
reactions. In such reactions, an exogenous nucleic acid encoding a
preselected polypeptide is introduced into a mixture that is
essentially depleted of exogenous nucleic acids that contains all
of the cellular components required for transcription and
translation. One or more radiolabeled amino acids are added before
or with the exogenous DNA, and transcription and translation are
allowed to proceed. Because the only nucleic acid present in the
reaction mix is the exogenous nucleic acid added to the reaction,
only polypeptides encoded thereby are produced, and incorporate the
radiolabelled amino acid(s). In this manner, polypeptides encoded
by a preselected exogenous nucleic acid are radiolabeled. Although
other proteins are present in the reaction mix, the preselected
polypeptide is the only one that is produced in the presence of the
radiolabeled amino acids and is thus uniquely labeled.
[0901] As is explained in detail below, "polypeptide derivatives"
include without limitation mutant polypeptides, chemically modified
polypeptides, and peptidomimetics.
[0902] The polypeptides of this invention, including the analogs
and other modified variants, may generally be prepared following
known techniques. Preferably, synthetic production of the
polypeptide of the invention may be according to the solid phase
synthetic method. For example, the solid phase synthesis is well
understood and is a common method for preparation of polypeptides,
as are a variety of modifications of that technique [Merrifield
(1964), J. Am. Chem. Soc., 85: 2149; Stewart and Young (1984),
Solid Phase polypeptide Synthesis, Pierce Chemical Company,
Rockford, Ill.; Bodansky and Bodanszky (1984), The Practice of
polypeptide Synthesis, Springer-Verlag, New York; Atherton and
Sheppard (1989), Solid Phase polypeptide Synthesis: A Practical
Approach, IRL Press, New York]. See, also, the specific method
described in Example 1 below.
[0903] Alternatively, polypeptides of this invention may be
prepared in recombinant systems using polynucleotide sequences
encoding the polypeptides. For example, fusion proteins are
typically prepared using recombinant DNA technology.
[0904] XXI.B. Polypeptide Derivatives
[0905] A "derivative" of a polypeptide is a compound that is not,
by definition, a polypeptide, i.e., it contains at least one
chemical linkage that is not a peptide bond. Thus, polypeptide
derivatives include without limitation proteins that naturally
undergo post-translational modifications such as, e.g.,
glycosylation. It is understood that a polypeptide of the invention
may contain more than one of the following modifications within the
same polypeptide. Preferred polypeptide derivatives retain a
desirable attribute, which may be biological activity; more
preferably, a polypeptide derivative is enhanced with regard to one
or more desirable attributes, or has one or more desirable
attributes not found in the parent polypeptide. Although they are
described in this section, peptidomimetics are taken as small
molecules in the present disclosure.
[0906] XXI.C. Mutant Polypeptide Derivatives
[0907] A polypeptide having an amino acid sequence identical to
that found in a protein prepared from a natural source is a
"wildtype" polypeptide. Mutant oligopeptides can be prepared by
chemical synthesis, including without limitation combinatorial
synthesis.
[0908] Mutant polypeptides larger than oligopeptides can be
prepared using recombinant DNA technology by altering the
nucleotide sequence of a nucleic acid encoding a polypeptide.
Although some alterations in the nucleotide sequence will not alter
the amino acid sequence of the polypeptide encoded thereby
("silent" mutations), many will result in a polypeptide having an
altered amino acid sequence that is altered relative to the parent
sequence. Such altered amino acid sequences may comprise
substitutions, deletions and additions of amino acids, with the
proviso that such amino acids are naturally occurring amino
acids.
[0909] Thus, subjecting a nucleic acid that encodes a polypeptide
to mutagenesis is one technique that can be used to prepare mutant
polypeptides, particularly ones having substitutions of amino acids
but no deletions or insertions thereof. A variety of mutagenic
techniques are known that can be used in vitro or in vivo including
without limitation chemical mutagenesis and PCR-mediated
mutagenesis. Such mutagenesis may be randomly targeted (i.e.,
mutations may occur anywhere within the nucleic acid) or directed
to a section of the nucleic acid that encodes a stretch of amino
acids of particular interest. Using such techniques, it is possible
to prepare randomized, combinatorial or focused compound libraries,
pools and mixtures.
[0910] Polypeptides having deletions or insertions of naturally
occurring amino acids may be synthetic oligopeptides that result
from the chemical synthesis of amino acid sequences that are based
on the amino acid sequence of a parent polypeptide but which have
one or more amino acids inserted or deleted relative to the
sequence of the parent polypeptide. Insertions and deletions of
amino acid residues in polypeptides having longer amino acid
sequences may be prepared by directed mutagenesis.
[0911] XXI.D. Chemically Modified Polypeptides
[0912] As contemplated by this invention, the term "polypeptide"
includes those having one or more chemical modification relative to
another polypeptide, i.e., chemically modified polypeptides. The
polypeptide from which a chemically modified polypeptide is derived
may be a wildtype protein, a mutant protein or a mutant
polypeptide, or polypeptide fragments thereof; an antibody or other
polypeptide ligand according to the invention including without
limitation single-chain antibodies, bacterial proteins and
polypeptide derivatives thereof; or polypeptide ligands prepared
according to the disclosure. Preferably, the chemical
modification(s) confer(s) or improve(s) desirable attributes of the
polypeptide but does not substantially alter or compromise the
biological activity thereof. Desirable attributes include but are
limited to increased shelf-life; enhanced serum or other in vivo
stability; resistance to proteases; and the like. Such
modifications include by way of non-limiting example N-terminal
acetylation, glycosylation, and biotinylation.
[0913] XXI.D.1. Polypeptides with N-Terminal or C-Terminal Chemical
Groups
[0914] An effective approach to confer resistance to peptidases
acting on the N-terminal or C-terminal residues of a polypeptide is
to add chemical groups at the polypeptide termini, such that the
modified polypeptide is no longer a substrate for the peptidase.
One such chemical modification is glycosylation of the polypeptides
at either or both termini. Certain chemical modifications, in
particular N-terminal glycosylation, have been shown to increase
the stability of polypeptides in human serum (Powell et al. (1993),
Pharma. Res. 10: 1268-1273). Other chemical modifications which
enhance serum stability include, but are not limited to, the
addition of an N-terminal alkyl group, consisting of a lower alkyl
of from 1 to 20 carbons, such as an acetyl group, and/or the
addition of a C-terminal amide or substituted amide group.
[0915] XXI.D.2. Polypeptides with a Terminal D-Amino Acid
[0916] The presence of an N-terminal D-amino acid increases the
serum stability of a polypeptide that otherwise contains L-amino
acids, because exopeptidases acting on the N-terminal residue
cannot utilize a D-amino acid as a substrate. Similarly, the
presence of a C-terminal D-amino acid also stabilizes a
polypeptide, because serum exopeptidases acting on the C-terminal
residue cannot utilize a D-amino acid as a substrate. With the
exception of these terminal modifications, the amino acid sequences
of polypeptides with N-terminal and/or C-terminal D-amino acids are
usually identical to the sequences of the parent L-amino acid
polypeptide.
[0917] XXI.D.3. Polypeptides with Substitution of Natural Amino
Acids by Unnatural Amino Acids
[0918] Substitution of unnatural amino acids for natural amino
acids in a subsequence of a polypeptide can confer or enhance
desirable attributes including biological activity. Such a
substitution can, for example, confer resistance to proteolysis by
exopeptidases acting on the N-terminus. The synthesis of
polypeptides with unnatural amino acids is routine and known in the
art (see, for example, Coller, et al. (1993), cited above).
[0919] XXI.D.4. Post-Translational Chemical Modifications
[0920] Different host cells will contain different
post-translational modification mechanisms that may provide
particular types of post-translational modification of a fusion
protein if the amino acid sequences required for such modifications
is present in the fusion protein. A large number (.about.100) of
post-translational modifications have been described, a few of
which are discussed herein. One skilled in the art will be able to
choose appropriate host cells, and design chimeric genes that
encode protein members comprising the amino acid sequence needed
for a particular type of modification.
[0921] Glycosylation is one type of post-translational chemical
modification that occurs in many eukaryotic systems, and may
influence the activity, stability, pharmacogenetics, immunogenicity
and/or antigenicity of proteins. However, specific amino acids must
be present at such sites to recruit the appropriate glycosylation
machinery, and not all host cells have the appropriate molecular
machinery. Saccharomyces cerevisieae and Pichia pastoris provide
for the production of glycosylated proteins, as do expression
systems that utilize insect cells, although the pattern of
glyscoylation may vary depending on which host cells are used to
produce the fusion protein.
[0922] Another type of post-translation modification is the
phosphorylation of a free hydroxyl group of the side chain of one
or more Ser, Thr or Tyr residues. Protein kinases catalyze such
reactions. Phosphorylation is often reversible due to the action of
a protein phosphatase, an enzyme that catalyzes the
dephosphorylation of amino acid residues.
[0923] Differences in the chemical structure of amino terminal
residues result from different host cells, each of which may have a
different chemical version of the methionine residue encoded by a
start codon, and these will result in amino termini with different
chemical modifications.
[0924] For example, many or most bacterial proteins are synthesized
with an amino terminal amino acid that is a modified form of
methionine, i.e., N-formyl-methionine (fMet). Although the
statement is often made that all bacterial proteins are synthesized
with an fMet initiator amino acid; although this may be true for E.
coli, recent studies have shown that it is not true in the case of
other bacteria such as Pseudomonas aeruginosa (Newton et al., J.
Biol. Chem. 274:22143-22146, 1999). In any event, in E. coli, the
formyl group of fMet is usually enzymatically removed after
translation to yield an amino terminal methionine residue, although
the entire fMet residue is sometimes removed (see Hershey, Chapter
40, "Protein Synthesis" in: Escherichia Coli and Salmonella
Typhimurium: Cellular and Molecular Biology, Neidhardt, Frederick
C., Editor in Chief, American Society for Microbiology, Washington,
D.C., 1987, Volume 1, pages 613-647, and references cited therein.)
E. coli mutants that lack the enzymes (such as, e.g., formylase)
that catalyze such post-translational modifications will produce
proteins having an amino terminal fMet residue (Guillon et al., J.
Bacteriol. 174:4294-4301, 1992).
[0925] In eukaryotes, acetylation of the initiator methionine
residue, or the penultimate residue if the initiator methionine has
been removed, typically occurs co- or post-translationally. The
acetylation reactions are catalyzed by N-terminal
acetyltransferases (NATs, a.k.a. N-alpha-acetyltransferases),
whereas removal of the initiator methionine residue is catalyzed by
methionine aminopeptidases (for reviews, see Bradshaw et al.,
Trends Biochem. Sci. 23:263-267, 1998; and Driessen et al., CRC
Crit. Rev. Biochem. 18:281-325, 1985). Amino terminally acetylated
proteins are said to be "N-acetylated," "N alpha acetylated" or
simply "acetylated."
[0926] Another post-translational process that occurs in eukaryotes
is the alpha-amidation of the carboxy terminus. For reviews, see
Eipper et al. Annu. Rev. Physiol. 50:333-344, 1988, and Bradbury et
al. Lung Cancer 14:239-251, 1996. About 50% of known endocrine and
neuroendocrine peptide hormones are alpha-amidated (Treston et al.,
Cell Growth Differ. 4:911-920, 1993). In most cases, carboxy
alpha-amidation is required to activate these peptide hormones.
[0927] XXI.E. Peptidomimetics
[0928] In general, a polypeptide mimetic ("peptidomimetic") is a
molecule that mimics the biological activity of a polypeptide but
is no longer peptidic in chemical nature. By strict definition, a
peptidomimetic is a molecule that contains no peptide bonds (that
is, amide bonds between amino acids). However, the term
peptidomimetic is sometimes used to describe molecules that are no
longer completely peptidic in nature, such as pseudo-peptides,
semi-peptides and peptoids. Examples of some peptidomimetics by the
broader definition (where part of a polypeptide is replaced by a
structure lacking peptide bonds) are described below. Whether
completely or partially non-peptide, peptidomimetics according to
this invention provide a spatial arrangement of reactive chemical
moieties that closely resembles the three-dimensional arrangement
of active groups in the polypeptide on which the peptidomimetic is
based. As a result of this similar active-site geometry, the
peptidomimetic has effects on biological systems that are similar
to the biological activity of the polypeptide.
[0929] There are several potential advantages for using a mimetic
of a given polypeptide rather than the polypeptide itself. For
example, polypeptides may exhibit two undesirable attributes, i.e.,
poor bioavailability and short duration of action. Peptidomimetics
are often small enough to be both orally active and to have a long
duration of action. There are also problems associated with
stability, storage and immunoreactivity for polypeptides that are
not experienced with peptidomimetics.
[0930] Candidate, lead and other polypeptides having a desired
biological activity can be used in the development of
peptidomimetics with similar biological activities. Techniques of
developing peptidomimetics from polypeptides are known. Peptide
bonds can be replaced by non-peptide bonds that allow the
peptidomimetic to adopt a similar structure, and therefore
biological activity, to the original polypeptide. Further
modifications can also be made by replacing chemical groups of the
amino acids with other chemical groups of similar structure. The
development of peptidomimetics can be aided by determining the
tertiary structure of the original polypeptide, either free or
bound to a ligand, by NMR spectroscopy, crystallography and/or
computer-aided molecular modeling. These techniques aid in the
development of novel compositions of higher potency and/or greater
bioavailability and/or greater stability than the original
polypeptide (Dean (1994), BioEssays, 16: 683-687; Cohen and
Shatzmiller (1993), J. Mol. Graph., 11: 166-173; Wiley and Rich
(1993), Med. Res. Rev., 13: 327-384; Moore (1994), Trends
Pharmacol. Sci., 15: 124-129; Hruby (1993), Biopolymers, 33:
1073-1082; Bugg et al. (1993), Sci. Am., 269: 92-98, all
incorporated herein by reference].
[0931] Thus, through use of the methods described above, the
present invention provides compounds exhibiting enhanced
therapeutic activity in comparison to the polypeptides described
above. The peptidomimetic compounds obtained by the above methods,
having the biological activity of the above named polypeptides and
similar three-dimensional structure, are encompassed by this
invention. It will be readily apparent to one skilled in the art
that a peptidomimetic can be generated from any of the modified
polypeptides described in the previous section or from a
polypeptide bearing more than one of the modifications described
from the previous section. It will furthermore be apparent that the
peptidomimetics of this invention can be further used for the
development of even more potent non-peptidic compounds, in addition
to their utility as therapeutic compounds.
[0932] Specific examples of peptidomimetics derived from the
polypeptides described in the previous section are presented below.
These examples are illustrative and not limiting in terms of the
other or additional modifications.
[0933] XXI.E.1. Peptides with a Reduced Isostere Pseudopeptide
Bond
[0934] Proteases act on peptide bonds. It therefore follows that
substitution of peptide bonds by pseudopeptide bonds confers
resistance to proteolysis. A number of pseudopeptide bonds have
been described that in general do not affect polypeptide structure
and biological activity. The reduced isostere pseudopeptide bond is
a suitable pseudopeptide bond that is known to enhance stability to
enzymatic cleavage with no or little loss of biological activity
(Couder, et al. (1993), Int. J. Polypeptide Protein Res.
41:181-184, incorporated herein by reference). Thus, the amino acid
sequences of these compounds may be identical to the sequences of
their parent L-amino acid polypeptides, except that one or more of
the peptide bonds are replaced by an isostere pseudopeptide bond.
Preferably the most N-terminal peptide bond is substituted, since
such a substitution would confer resistance to proteolysis by
exopeptidases acting on the N-terminus.
[0935] XXI.E.2. Peptides With A Retro-Inverso Pseudopeptide
Bond
[0936] To confer resistance to proteolysis, peptide bonds may also
be substituted by retro-inverso pseudopeptide bonds (Dalpozzo, et
al. (1993), Int. J. Polypeptide Protein Res. 41:561-566,
incorporated herein by reference). According to this modification,
the amino acid sequences of the compounds may be identical to the
sequences of their L-amino acid parent polypeptides, except that
one or more of the peptide bonds are replaced by a retro-inverso
pseudopeptide bond. Preferably the most N-terminal peptide bond is
substituted, since such a substitution will confer resistance to
proteolysis by exopeptidases acting on the N-terminus.
[0937] XXI.E.3. Peptoid Derivatives
[0938] Peptoid derivatives of polypeptides represent another form
of modified polypeptides that retain the important structural
determinants for biological activity, yet eliminate the peptide
bonds, thereby conferring resistance to proteolysis (Simon, et al.,
1992, Proc. Natl. Acad. Sci. USA, 89:9367-9371 and incorporated
herein by reference). Peptoids are oligomers of N-substituted
glycines. A number of N-alkyl groups have been described, each
corresponding to the side chain of a natural amino acid.
[0939] XXII. KITS
[0940] The invention provides for diagnostic and therapeutic kits
related useful for therapeutic, diagnostic, and research
applications. Exemplary kits are disclosed in U.S. Pat. Nos.
5,773,024; 6,017,721; and 6,232,127 B1. The kits of the invention
incorporate minicells, and/or include methods of using minicells
described herein.
[0941] XXII.A. Diagnostic and Research Use Kit Components
[0942] In one embodiment, the invention relates to kits for
determining the diagnosis or prognosis of a patient. These kits
preferably comprise devices and reagents for measuring one or more
marker levels in a test sample from a patient, and instructions for
performing the assay. Optionally, the kits may contain one or more
means for converting marker level(s) to a prognosis. Such kits
preferably contain sufficient reagents to perform one or more such
determinations.
[0943] More specifically, a diagnostic kit of the invention
comprises any of the following reagents and/or components in any
combination.
[0944] (1) A detectable or detectably labeled first reagent that
binds a ligand of interest. The binding reagent can, but need not,
be an antibody or an antibody derivative comprising a detectable
moiety. The sphingolipid-binding reagent is stored in an openable
container in the kit, or is bound to a surface of a substrate such
that it is accessible to other reagents. Examples of the latter
include test strips.
[0945] (2) If the first reagent in neither detectable nor
detectably labeled, the kit may comprise a detectable or detectably
labeled second reagent that binds to the first reagent (e.g., a
secondary antibody) or which produces a detectable signal when in
close proximity to the first reagent (e.g., as results from
fluorescent resonance energy transfer FRET). In either case, the
signal produced from the second reagent correlates with the amount
of ligand in the sample.
[0946] (3) One or more positive control reagents. Typically, these
reagents comprise a compound that is known to produce a signal in
the assay. In one embodiment, the positive control reagents are
standards, i.e., comprise a known amount of a detectable or
detectably labeled compound, the signal from which may be compared
to the signal from a test sample. In addition to serving as
positive control reagents, they may be used to develop calibration
curves that relate the amount of signal to the known concentration
of a detectable or detectably labeled compound. The signal from a
test sample is compared to the calibration curve in order to
determine what concentration of the detectable or detectably
labeled compound corresponds to the signal from the test sample. In
this embodiment, the kit provides quantitative measurements of the
amount of a ligand in a test sample.
[0947] (4) One or more negative control reagents. Typically, these
control reagents may comprise buffer or another solution that does
not contain any of the detectable or detectably labeled first or
second reagents and should thus not produce any detectable signal.
Any signal that is detected reflects the background level of
"noise" in the assay. Another type of negative control reagent
contains most of the components necessary for the signal of the
assay to be produced, but lacks at least one such component and
therefor should not produce a signal. Yet another type of negative
control reagent contains all of the components necessary for the
signal of the assay to be produced, but also contains an inhibitor
of the process that produced the signal.
[0948] (5) One or more auxiliary reagents for use in the diagnostic
assays of the kit, e.g., buffers, alcohols, acid solutions, etc.
These reagents are generally available in medical facilities and
thus are optional components of the kit. However, these reagents
preferably are included in the kit to ensure that reagents of
sufficient purity and sterility are used, since the resulting
protein conjugates are to be administered to mammals, including
humans, for medical purposes, and to provide kits that can be used
in situations where medical facilities are not readily available,
e.g., when hiking in places located far from medical facilities, or
in situations where the presence of these auxiliary reagents allows
for the immediate treatment of a patient outside of a medical
facility as opposed to treatment that arrives at some later
time).
[0949] (6) Instructions to a person using a kit for its use. The
instructions can be present on one or more of the kit components,
the kit packaging and/or a kit package insert.
[0950] XXII.B. Therapeutic Kit Components
[0951] A therapeutic kit of the invention comprises any of the
following reagents and/or components in any combination.
[0952] (1) One or more therapeutic agents.
[0953] (2) If the therapeutic agent(s) are not formulated for
delivery via the alimentary canal, which includes but is not
limited to sublingual delivery, a device capable of delivering the
therapeutic agent through some other routes. One type of device for
parenteral delivery is a syringe that is used to inject the
therapeutic agent into the body of an animal in need of the
therapeutic agent. Inhalation devices may also be used.
[0954] (3) Separate containers, each of which comprises one or more
reagents of the kit. In a preferred embodiment, the containers are
vials contain sterile, lyophilized formulations of a therapeutic
composition that are suitable for reconstitution. Other containers
include, but are not limited to, a pouch, tray, box, tube, or the
like. Kit components may be packaged and maintained sterilely
within the containers.
[0955] (4) Instructions to a person using a kit for its use. The
instructions can be present on one or more of the kit components,
the kit packaging and/or a kit package insert. Such instructions
include, by way of non-limiting example, instructions for use of
the kit and its reagents, for reconstituting lyophilized reagents
or otherwise preparing reagents.
[0956] A preferred kit of the present invention comprises the
elements useful for performing an immunoassay. A kit of the present
invention can comprise one or more experimental samples (i.e.,
formulations of the present invention) and one or more control
samples bound to at least one pre-packed dipstick or ELISA plate,
and the necessary means for detecting immunocomplex formation
(e.g., labelled secondary antibodies or other binding compounds and
any necessary solutions needed to resolve such labels, as described
in detail above) between antibodies contained in the bodily fluid
of the animal being tested and the proteins bound to the dipstick
or ELISA plate. It is within the scope of the invention that the
kit can comprise simply a formulation of the present invention and
that the detecting means can be provided in another way.
[0957] An alternative preferred kit of the present invention
comprises elements useful for performing a skin test. A kit of the
present invention can comprise at least one pre-packed syringe and
needle apparatus containing one or more experimental samples and/or
one or more control samples. A kit according to the invention may
be designed for both diagnostic and therapeutiuc applications. Any
combination of the above elements XX.A.(1)-(6) and XX.B.(1)-(4) may
be used in a kit, optionally with additional reagents, standards,
sample containers, an the like.
[0958] XXIII. Immunogenic Minicells
[0959] XXIII.A. In General
[0960] Minicells are used to immunize subjects. An organism is said
to be "immunized" when, after contact with an immunogen, the
organism produces antibodies directed to the immunogen, or has
increased proliferation or activity of cytotoxic and/or helper T
cells, or both. Increased proliferation or activity of T cells may
be particularly desirable in the case of parasites that cause a
decrease in T cell proliferation.
[0961] The use of minicells to present antigens has several
potential advantages. An intact membrane protein can be presented
in its native form on the surface of an immunogenic minicell,
rather than as a denatured protein or as oligopeptides derived from
the amino acid sequence of a membrane protein, which allows for
antibodies to be developed that are directed to epitopes which, due
to protein folding, occur only in the native protein. The minicell
surface may naturally be, or may be modified to be, an adjuvant.
Moreover, pharmacokinetic properties of minicells, as discussed
elsewhere herein, may be improved relative to other forms of
administration.
[0962] The applications of immunogenic minicells include, but are
not limited to, research, prophylactic, diagnostic and therapeutic
applications.
[0963] In research applications, immunogenic minicells are used to
generate antibodies to an antigen displayed on a minicell. Such
antibodies are used to detect an antigen, which may be a chemical
moiety, molecule, virus, organelle, cell, tissue, organ, or
organism that one wishes to study. Classically, such antibodies
have been prepared by immunizing an animal, often a rat or a
rabbit, and collecting antisera therefrom. Molecular biology
techniques can be used to prepare antibodies and antibody
fragments, as is described elsewhere herein. Single-chain antibody
fragments (scFv) may also be identified, purified, and
characterized using minicells displaying a membrane protein or
membrane bound chimeric soluble protein.
[0964] In prophylactic applications, immunogenic minicells are used
to stimulate a subject to produce antibodies and/or activate T
cells, so that the subject is "pre-immunized" before contact with a
pathogen or hyperproliferative cell. Thus, in the case of a
pathogens, the subject is protected by antibodies and/or T cells
that are specifically directed to the pathogen before
infection.
[0965] In therapeutic applications, immunogenic minicells are used
in immunotherapy.
[0966] Certain aspects of the invention involve active
immunotherapy, in which treatment relies on the in vivo stimulation
of the endogenous host immune system to react against pathogens or
tumors due to the administration of agents that cause, enhance or
modulate an immune response. Such agents include, but are not
limited to, immunogens, adjuvants, cytokines and chemokines.
[0967] Other therapeutic applications involve passive
immunotherapy, in which treatment involves the delivery of agents
(such as antibodies or effector cells) that are specifically
directed to an immunogen of a pathogen or a hyperproliferative
cell, and does not necessarily depend on an intact host immune
system. Examples of effector cells include T cells; T lymphocytes,
such as CD8+ cytotoxic T lymphocytes and CD4+ T-helper
tumor-infiltrating lymphocytes; killer cells, such as Natural
Killer (NK) cells and lymphokine-activated killer cells.
[0968] XXIII.B. Hyperproliferative Disorders
[0969] The immunogenic minicells of the invention can be used to
treat hyperproliferative disorders by inducing an immune response
to an antigen associated therewith. The term "hyperproliferative
disorder" refers to disorders characterized by an abnormal or
pathological proliferation of cells, for example, cancer,
psoriasis, hyperplasia and the like.
[0970] For reviews of immunotherapy as applied to
hyperproliferative disorders, see Armstrong et al., Cellular
immunotherapy for cancer, BMJ 323:1289-1293, 2001; Evans, Vaccine
therapy for cancer--fact or fiction?, Proc R Cell Physicians Edinb
31:9-16, 2001; Ravindranath and Morton, "Active Specific
Immunotherapy with Vaccines," Chapter 61 in: Holland-Frei Cancer
Medicine, Fifth Edition, Bast, Robert C., et al., editors, B. C.
Decker, Inc., Hamilton, 2000, pages 800-814.
[0971] Types of cancers include without limitation fibrosarcoma,
myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,
chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,
sweat gland carcinoma, sebaceous gland carcinoma, papillary
carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, meningioma, melanoma, neuroblastoma,
retinoblastoma, leukemia, lymphoma, multiple myeloma, Waldenstrom's
macroglobulinemia, and heavy chain disease.
[0972] Tumor specific antigens (TSAs), tumor-associated
differentiation antigens (TADAs) and other antigens associated with
cancers and other hyperproliferative disorders include, but are not
limited to, C1 IAC, a human cancer associated protein (Osther, U.S.
Pat. No. 4,132,769); the CA125 antigen, an antigen associated with
cystadenocarcinoma of the ovary, (Hanisch et al., Carbohydr. Res.
178:29-47, 1988; O'Brien, U.S. Pat. No. 4,921,790); CEA, an antigen
present on many adenocarcinomas (Honig et al., Strategies for
cancer therapy using carcinembryonic antigen vaccines, Expert
Reviews in Molecular Medicine, http://www-ermm.cbcu.cam.ac.uk: 1,
2000); CORA (carcinoma or orosomucoid-related antigen) described by
Toth et al. (U.S. Pat. No. 4,914,021); DF3 antigen from human
breast carcinoma (Kufe, in U.S. Pat. Nos. 4,963,484 and 5,053,489);
DU-PAN-2, a pancreatic carcinoma antigen (Lan et al., Cancer Res.
45:305-310, 1985); HCA, a human carcinoma antigen (Codington et
al., U.S. Pat. No. 5,693,763); Her2, a breast cancer antigen
(Fendly et al., The Extracellular Domain of HER2/neu Is a Potential
Immunogen for Active Specific Immunotherapy of Breast Cancer,
Journal of Biological Response Modifiers 9:449-455, 1990); MSA, a
breast carcinoma glycoprotein (Tjandra et al., Br. J. Surg.
75:811-817, 1988); MFGM, a breast carcinoma antigen (Ishida et al.,
Tumor Biol. 10:12-22, 1989); PSA, prostrate specific antigen (Nadji
et al., Prostatic-specific-antigen, Cancer 48:1229-1232, 1981);
STEAP (six transmembrane epithelial antigens of the prostate)
proteins (Afar et al., U.S. Pat. No. 6,329,503); TAG-72, a breast
carcinoma glycoprotein (Kjeldsen et al., Cancer Res. 48:2214-2220,
1988); YH206, a lung carcinoma antigen (Hinoda et al., Cancer J.
42:653-658, 1988); the p97 antigen of human melanoma (Estin et al.,
Recombinant Vaccinia Virus Vaccine Against the Human Melanoma
Antigen p97 for Use in Immunotherapy, Proc. Natl. Acad. Sci. USA,
85:1052-1056, 1988); and the melanoma specific antigen described by
Pfreundschuh in U.S. Pat. No. 6,025,191);
[0973] XXIII.B. Intracellular Pathogens
[0974] In certain aspects of the invention, vaccines comprising
immunogenic minicells are used to prevent or treat diseases caused
by intracellular pathogens. Vaccines may be prepared that stimulate
cytotoxic T cell responses against cells infected with viruses
including, but not limited to, hepatitis type A, hepatitis type B,
hepatitis type C, influenza, varicella, adenovirus, herpes simplex
type I (HSV-I), herpes simplex type II (HSV-II), rinderpest,
rhinovirous, echovirus, rotavirus, respiratory syncytial virus,
papilloma virus, papova virus, cytomegalovirus, echinovirus,
arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus,
rubella virus, polio virus, human immunodeficiency virus type I
(HIV-I), and human immunodeficiency virus type II (HIV-II).
Vaccines also may be prepared that stimulate cytotoxic T cell
responses against cells infected with intracellular obligates,
including but not limited to Chlamydia, Mycobacteria and
Rickettsia. Vaccines also may be prepared that stimulate cytotoxic
T cell responses against cells infected with intracellular
protozoa, including, but not limited to, leishmania, kokzidioa, and
trypanosoma.
[0975] The causative agent of Lyme disease, the spirochete Borrelia
burgdorfei, is also of interest. The outer surface proteins (Osps)
A, B and C of B. burgdorfei are known antigens that are
lipoproteins that associate with membranes. Amino-terminal cysteine
residues in Osp proteins are the sites of triacyl lipid
modifications that serve as membrane-anchoring moities. The
N-terminal portions of the Osp proteins are highly conserved and
are preferred portions for display on immunogenic minicells.
[0976] XXIII.C. Eukaryotic Pathogens
[0977] In addition to intracellular pathogens, other eukaryotic
pathogens exist and may also be treated using immunogenic minicells
displayed antigens therefrom. A number of antigens have been used
to develop anti-parasitic vaccines, e.g. the recombinant 45w
protein of Taenia ovis; EG95 oncosphere proteins of Echinococcus
granulosis; cathepsin L antigen of the liver fluke, Fasciola
hepatica; and the H11 antigen of Haemonchus contortus (Dalton et
al., Parasite vaccines--a reality?, Vet Parasitol 98:149-167,
2001). Other eukaryotic pathogens include, but are not limited
to:
[0978] Protozoans, including but not limited to, Entamoeba
histolytica, a pathogenic amoeba that causes amoebic dysentery and
occasionally digests its way through the intestinal wall to invades
other organs, which may cause morbidity; Balantinium coli, a
ciliate that causes diarrhea in humans; Giardia lamblia, a
flagellate that causes diarrhea and abdominal pain, along with a
chronic fatigue syndrome that is otherwise asymptomatic and
difficult to diagnose; Trypanosoma brucei, a hemoflagellate causing
sleeping sickness; and Trypanosoma cruzi, the cause of Chagas
disease);
[0979] Plasmodia, sporozoan obligate intracellular parasites of
liver and red blood cells, including but not limited to P.
falciparum, the causative agent of malaria. Dozens of P. falciparum
antigens have been identified, e.g., CSP-1, STARP, SALSA, SSP-2,
LSA-1, EXP-1, LSA-3, RAP-1, RAP-2, SERA-1, MSP-1, MSP-2, MSP-3,
MSP-4, MSP-5, AMA-1, EBA-175, RESA, GLURP, EMP-1, Pfs25, Pfg27,
Pf35, Pf55, Pfs230, Pfg27, Pfs16, Pfs28 and Pfs45/48.
[0980] Helminthes including but not limited to Ascaris lumbricoides
(roundworm); Enterobius vermicularis (pinworm); Trichuris
trichiuria (whipworm); and Fasciola hepatica (liver fluke);
[0981] Taenia sp. (tapeworms and cestodes);
[0982] Schistosoma (trematodes), such as Schistosoma mansoni, which
comprises the Sm32 antigen (asparaginyl endopeptidase), which can
induce antibody formation in mice (Chlichlia et al., DNA
vaccination with asparaginyl endopeptidase (Sm32) from the parasite
Schistosoma mansoni: anti-fecundity effect induced in mice, Vaccine
20:439-447, 2001); and acetylcholinesterase (Arnon et al.,
Acetylcholinesterase of Schistosoma mansoni-Functional correlates,
Protein Science 8:2553-2561, 1999); and
[0983] Ticks and other invertebrates, including but not limited to
insects, arachnids, etc. For example, a description of a vaccine
against the cattle tick Boophilus microplus has been described
(Valle et al., The evaluation of yeast derivatives as adjuvants for
the immune response to the Bm86 antigen in cattle, BMC Biotechnol.
1:2, 2001)
[0984] XXIII.D. Formulation and Administration of Immunogenic
Minicells
[0985] Vaccine formulations of immunogenic minicells include a
suitable carrier. Because minicells may be destroyed by digestion,
or prevented from acting due to antibody secretion in the gvut,
they are preferably administered parenterally, including, for
example, administration that is subcutaneous, intramuscular,
intravenous, or intradermal. Formulations suitable for parenteral
administration include aqueous and non-aqueous sterile injection
solutions which may contain antioxidants, buffers, and solutes
which render the formulation isotonic with the bodily fluid,
preferably the blood, of the individual; and aqueous and
non-aqueous sterile suspensions which may include suspending agents
or thickening agents. The formulations may be presented in
unit-dose or multi-dose containers, for example, sealed ampules and
vials and may be stored in a freeze-dried condition requiring only
the addition of the sterile liquid carrier immediately prior to
use. The vaccine formulation may also include adjuvant systems for
enhancing the immunogenicity of the formulation. Adjuvants are
substances that can be used to augment a specific immune response.
Normally, the adjuvant and the composition are mixed prior to
presentation to the immune system, or presented separately, but
into the same site of the mammal being immunized. Examples of
materials suitable for use in vaccine compositions are provided in
Osol, A., ed., Remington's Pharmaceutical Sciences, Mack Publishing
Co, Easton, Pa. (1980), pp. 1324-1341, which reference is entirely
incorporated herein by reference.
[0986] Compositions comprising immunogenic minicells are injected
into a human or animal at a dosage of 1-1000 ug per kg body weight.
Antibody titers against growth factor are determined by ELISA,
using the recombinant protein and horseradish peroxidase-conjugated
goat anti-human or animal immunoglobulins or other serologic
techniques (e.g., sandwich ELISA). Booster injections are
administered as needed to achieve the desired levels of protective
antibodies and/or T cells.
[0987] Routes and frequency of administration, as well as dosage,
will vary from individual to individual. Between 1 and 10 doses may
be administered for a 52-week period. Preferably, 6 doses are
administered, at intervals of 1 month, and booster vaccinations may
be given periodically thereafter. Alternate protocols may be
appropriate for individual patients. In immunotherapy of
hyperproliferative disorders, a suitable dose is an amount of a
compound that, when administered as described above, is capable of
promoting an anti-tumor immune response. Such response can be
monitored by measuring the anti-tumor antibodies in a patient or by
vaccine-dependent generation of cytolytic effector cells capable of
killing the patient's tumor cells in vitro. Such vaccines should
also be capable of causing an immune response that leads to an
improved clinical outcome (e.g., more frequent remissions, complete
or partial or longer disease-free survival) in vaccinated patients
as compared to non-vaccinated patients.
[0988] The vaccine according to the invention may contain a single
species of immunogenic minicells according to the invention or a
variety of immunogenic minicells, each of which displays a
different immunogen. Additionally or alternatively, immunogenic
minicells may each display and/or express more than one
immunogen.
[0989] The summary of the invention described above is non-limiting
and other features and advantages of the invention will be apparent
from the following detailed description of the invention, and from
the claims.
EXAMPLES
Example 1
Creation of a Minicell-Producing Bacterial Cell Line (MC-T7) that
Expresses an Exogenous RNA Polymerase
[0990] In order to maximize the amount of RNA transcription from
episomal elements in minicells, a minicell-producing cell line that
expresses an RNA polymerase specific for certain episomal
expression elements was created. This E. coli strain, designated
MC-T7, was created as follows.
[0991] The P678-54 E. coli strain contains mutations that influence
cell division and induce the production of minicells (Adler et al.,
Proc. Natl. Acad. Sci. 57:321-326 (1967), Allen et al., Biochem.
Biophys. Res. Communi. 47:1074-1079 (1972), Hollenberg et al., Gene
1:33-47 (1976)). The P678-54 strain is resistant to Lambda phage
due to a mutation in the malT gene (Gottesman, Bacteriophage
Lambda: The Untold Story. J. Mol. Biol. 293:177-180, 1999;
Friedman, Interactions Between Bacteriophage Lambda and its
Escherichia Coli Host. Curr. Opin. Genet. Dev. 2:727-738, 1992).
Thus, as an initial step, the P678-54 strain was altered so as to
be sensitive to Lambda phage so that it could form lysogens of
Lambda-DE3 (see below). Wildtype MalT-encoding sequences were
restored via a HFR (high frequency recombination) conjugation
protocol using the G43 E. coli strain (CGSC stain 4928).
[0992] Recipient (P678-54) and donor (G43:BW6169) strains were
grown overnight in 10 mL of LB media (10 g NaCl, 10 g select
peptone 140, and 5 g yeast extract in one liter ddH.sub.20). The
samples were centrifuged and then concentrated in about 0.2 mL of
LB media. The concentrated samples were combined and incubated with
slow rotation for 30 minutes at 30.degree. C., and were then plated
on LB agar plates that contained streptomycin (50 .mu.g/mL) and
tetracycline (50 .mu.g/mL). (Ampicillin, streptomycin,
tetracycline, and all other chemicals were purchased from Sigma
Chemical (St. Louis, Mo.) unless otherwise indicated.) Recipient
cells were resistant to streptomycin and donor cells were resistant
to tetracycline; only conjugates, which contained both resistance
genes, were able to grow on the LB agar plates that contained
streptomycin (50 .mu.g/mL) and tetracycline (50 .mu.g/mL).
[0993] Putative conjugates were screened for Lambda phage
sensitivity using a cross streak technique, in which putative
colonies were cross-streaked on an LB agarose plate (streptomycin,
50 .mu.g/mL, and tetracycline, 50 .mu.g/mL) that had been streaked
with live Lambda phage. The streaked conjugate colonies were
streaked perpendicular to the Lambda phage streak; if a conjugate
was sensitive to Lambda phage infection then, upon contact with the
Lambda phage streak, there was cell lysis and thus less or no
bacterial growth. Thus, in the case of conjugates that were
sensitive to Lambda phage, there was deceased bacterial growth
"downstreak" from the phage streak.
[0994] The conjugate E. coli that were found to be sensitive to
Lambda phage infection were then used to create Lambda lysogens.
Lysogenization is a process during which Lambda phage incorporates
its genome, including exogenous genes added thereto, into a
specific site on the chromosome of its E. coli host cell.
[0995] The DE3 gene, which is present in the genome of the Lamda
phage used to create lysogens, encodes RNA polymerase from
bacteriophage T7. Lysogenation was carried out using the
DE3-Lysogenation kit (Novagen, Madison, Wis.) essentially according
to the manufacturer's instructions. A T7 polymerase dependent
tester phage was used to confirm the presence and expression of the
DE3 gene on the bacterial chromosome. The T7-dependent tester phage
can only form plaques on a bacterial known in the presence of T7
polymerase. The phage uses a T7 promoter for expression of its
essential genes. Therefore in a plaque-forming assay only cells
which express T7 polymerase can be lysed by the tester phage and
only these cells will allow for the formation of plaques. As is
described in more detail herein, episomal expression elements that
are used in minicells may be designed such that transcription and
translation of a cloned gene is driven by T7 RNA polymerase by
utilizing expression sequences specific for the T7 RNA
polymerase.
Example 2
Cloning of Rat Edg-1 into the pCAL-c Expression Vector
Materials
[0996] Taq Polymerase, PCR Buffers, and PCR reagents were purchased
from Roche Molecular Biochemicals (Indianapolis, Ind.). All
restriction enzymes were purchased from Gibco BRL (Grand Island,
N.Y.) and Stratagene (La Jolla, Calif.). QIAprep mini and maxi
kits, PCR purification Kits, RNeasy miniprep kits, and the One Step
RT-PCR Kit were purchased from QIAGEN (Valencia, Calif.). The
Geneclean Kit was purchased from BIO 101 (Carlsbad, Calif.). IPTG
(isopropy-beta-D-thiogalactopyranoside), T4 DNA Ligase, LB Media
components and agarose were purchased from Gibco BRL. The pCAL-c
prokaryote expression vector and competent cells were purchased
from Stratagene.
[0997] The pCAL-c expression vector has a structure in which an ORF
may be operably linked to a high-level (but T7 RNA polymerase
dependent) promoter, sequences that bind the E. coli Lac repressor,
and the strong T7 gene 10 ribosome-binding site (RBS). The Lad
repressor is also encoded by an expressed from to pCAL-c vector. As
long as it is bound to its recognition sequences in the pCAL-c
expression element, the lac repressor blocks transcription from the
T7 promoter. When an inducing agent, such as IPTG is added, the lac
repressor is released from its binding sites and transcription
proceeds from the T7 promoter, provided the T7 RNA polymerase is
present. After induction, the cloned and expressed protein may
constitute the majority of newly expressed cellular proteins due to
the efficient transcription and translation processes of the
system.
[0998] Amplification
[0999] The first step in cloning rat Edg-1 (rEDG-1) into an
expression vector was to design primers for amplification via PCR
(polymerase chain reaction). PCR primers were designed using the
rat Edg-1 sequence (Nakajima et al., Biophy, J. 78:319 A, 2000) in
such a manner that they contained either sites for NheI (GCTAGC) or
BamHI (GGATCC) on their five prime ends. The upstream primer had
the sequence of SEQ ID NO:31. The three prime downstream primer
(SEQ ID NO:32) also contained a stop codon, as the pCAL-c vector
contains a Calmodulin Binding Protein (CBP) "tag" at its carboxyl
terminus which was not intended to be incorporated into the rat
Edg-1 polypeptide in this expression construct. The primer and
resulting PCR products were designed so that the five prime end of
the rat Edg-1 ORF was in frame with the methionine start codon
found in the pCAL-c vector.
Oligonucleotide Primer Sequences for Cloning into pCAL-c:
TABLE-US-00007 Edg1/pCAL-c construct primers: Upstream primer (SEQ
ID NO: 31) 5'-AATTGCTAGCTCCACCAGCATCCCAGTGGTTA-3' Downstream primer
(SEQ ID NO: 32) 5'-AATTGGATCCTTAAGAAGAAGAATTGACGTTT-3' Edg1/CBP
fusion construct primers: Upstream primer (SEQ ID NO: 31)
5'-AATTGCTAGCTCCACCAGCATCCCAGTGGTTA-3' Downstream primer (SEQ ID
NO: 33) 5'-AATTGGATCCAGAAGAAGAATTGACGTTTCCA-3' Edg1/His6 construct
primers: Upstream primer (SEQ ID NO: 31)
5'-AATTGCTAGCTCCACCAGCATCCCAGTGGTTA-3' Downstream primer (SEQ ID
NO: 34) 5'-AATTGGATCCTTAATGATGATGATGATGATGAGAAGAAGAATTGAC GTTTCC-3'
Edg3/rtPCR primers: Upstream primer (SEQ ID NO: 35)
5'-TTATGGCAACCACGCACGCGCAGG-3' Downstream primer (SEQ ID NO: 36)
5'-AGACCGTCACTTGCAGAGGAC-3' Edg3/pCAL-c construct primers: Upstream
primer (SEQ ID NO: 37) 5'-AATTGCTAGCACGCACGCGCAGGGGCACCCGC-3'
Downstream primer (SEQ ID NO: 38)
5'-AATTGGTACCTCACTTGCAGAGGACCCCATTCTG-3' Edg3/His6 construct
primers: Upstream primer (SEQ ID NO: 39)
5'-AATTGCTAGCACGCACGCGCAGGGGCACCCGC-3' Downstream primer (SEQ ID
NO: 16) 5'-AATTGGTACCTCAATGATGATGATGATGATGCTTGCAGAGGACCCC ATTCTG-3'
GFP/pCAL-c construct primers: Upstream primer (SEQ ID NO: 40)
5'-GGTCGCCACCATGGTGAGCAA-3' Downstream primer (SEQ ID NO: 41)
5'-TTAAGGATCCTTACTTGTACAGCTCGTCCAT-3' GFP/CBP construct primers:
Upstream primer (SEQ ID NO: 42) 5'-GGTCGCCACCATGGTGAGCAA-3'
Downstream primer (SEQ ID NO: 43)
5'-TTAAGGATCCCTTGTACAGCTCGTCCATGCC-3' Notes: Restriction
endonuclease sites are underlined Stop codons are double
underlined
[1000] The primers were used to amplify the rEdg-1 DNA ORF using
the polymerase chain reaction (PCR). The template used for
amplification was mRNA isolated from rat muscle tissue using the
RNeasy Miniprep Kit (Qiagen) and was carried out essentially
according to the manufacturer's protocol. Both the rtPCR and PCR
amplification steps were carried out in a single reaction using the
One Step RT-PCR Kit (Qiagen). The resulting rat Edg-1 PCR fragment
was purified using the PCR Purification Kit (Qiagen). The amplified
double stranded rEdg-1 DNA sequence contained the NheI site at the
5-prime end and the BamHI site at the 3-prime end. This amplified
rEdg-1 fragment was used for cloning into the pCAL-c expression
vector.
[1001] The pCAL-c expression vector contains NcoI, NheI, and BamHI
restriction sites in its multiple cloning site. In order to insert
rEdg-1-encoding sequence into the expression vector, the rEdg-1 PCR
fragment and the pCAL-c expression vector were digested with NheI
and BamHI restriction enzymes for one hour at 37.degree. C. The
reaction mixture for the digestion step consisted of 1 .mu.g of
DNA, 1.times. restriction buffer, and 1 .mu.L of each enzyme. The
reaction mixture was brought to a final volume of 20 .mu.L with
ddH.sub.2O (dd, double distilled). After 45 minutes, 1 .mu.L of
Calf Intestine Alkaline Phosphatase (CIAP) was added to the pCAL-c
reaction mixture in order to remove the terminal phosphates from
the digested plasmid DNA. The reactions were incubated for an
additional 15 minutes at 37.degree. C. The digested DNA samples
were then run on a 1% TAE (Tris-acetate/EDTA electrophoresis
buffer) agarose gel at 130 volts for 45 minutes. The bands were
visualized with UV light after the gel was stained with ethidium
bromide.
[1002] The appropriate bands were cut out of the gel for
purification using the Geneclean Kit (BIO101). The Purified DNA
fragments were then quantified on a 1% TAE agarose gel. For the
ligation reaction, ratios of insert to vector of 6:1 and 3:1 were
used. A negative control comprising vector only was also included
in the ligation reactions. The reaction mixtures contained insert
and vector DNA, 4 .mu.L Ligase buffer, and 2 .mu.L Ligase. The
reaction was brought up to a final volume of 20 .mu.L with
ddH.sub.2O. The ligation was carried out at room temperature for
about 2 hours. Ten (10) .mu.L of the ligation reaction mixture was
used for subsequent transformation steps.
[1003] Ligated DNA was introduced into Epicurian Coli XL1-Blue
competent cells using the heat shock transformation technique as
follows. The ligation mixture was added to 100 .mu.L of competent
cells, placed on ice, and was incubated for about 30 minutes. The
cells were then heat shocked at 37.degree. C. for 1 minute and put
back on ice for 2 minutes. Following heat shock, 950 .mu.L of room
temperature LB media was added to the cells and the cells were
shaken at 37.degree. C. for 1 hour. Following the 1-hour agitation
the cells were pelleted for one minute at 12000 rpm in a Eppendorf
5417C microcentrifuge. The supernatant was carefully poured off so
that about 200 .mu.L remained. The cells were then resuspended in
the remaining LB media and spread on 100.times.15 mm LB agarose
plates containing 50 .mu.g/mL ampicillin. The plates were incubated
overnight at 37.degree. C. Colonies were counted the following day,
and the ratio of colonies between the negative control and the
ligated samples was determined. A high ratio of the number of
colonies when the ligation mixture was used to transform cells, as
contrasted to the number of negative control colonies indicated
that the cloning was successful. Transformed colonies were
identified, isolated, and grown overnight in LB media in the
presence of ampicillin. The resulting bacterial populations were
screened for the presence of the Edg-1-pCAL-c expression
construct.
[1004] Plasmid DNA was isolated from the cells using the QIAprep
Spin Miniprep Kit (Qiagen). Isolated Edg-1-pCAL-c constructs were
screened using the restriction enzyme ApaI, which digests the
Edg-1-pCAL-c construct at two different sites: one in the Edg-1
coding sequence and one in the pCAL-c vector itself. The plasmid
preparations were digested with ApaI electrophoresed on a 1% TAE
agarose gel and visualized using uv light and ethidium bromide
staining. The predicted sizes of the expected DNA fragments were
2065 bp and 4913 bp. As shown in FIG. 3, bands of the predicted
size were present on the gel. The entire Edg-1-pCAL-c construct was
sequenced in order to confirm its structure. This expression
construct, a pCAL-c derivative that contains the rat Edg-10RF
operably linked to a T7 promoter and lac repressor binding sites,
is designated "prEDG-1" herein.
Example 3
Construction of Rat Edg-1-CBP Fusion Protein
[1005] In order to detect rat Edg-1 protein expression, rEdg-1
coding sequences were cloned into the pCAL-c vector in frame with a
CBP fusion tag. The cloning strategy for the rEdg-1-CBP construct
was performed essentially as described for the Edg-1-pCAL-c
construct with the following differences. The PCR primers (SEQ ID
NOS:3 and 5) were as described for the Edg-1-pCAL-c cloning except
for the omission of the stop codon in the downstream primer (SEQ ID
NO:33). The removal of the stop codon is required for the
construction of the Edg-1-CBP fusion protein. The pCAL-c vector is
designed so that, when the BamHI site is used for insertional
cloning, and no stop codon is present in an ORF inserted into the
pCAL-c expression vector the cloned ORF will be in-frame with the
CBP fusion tag. Because the three prime downstream primer did not
contain a stop codon, a CBP fusion tag could be cloned in-frame
with the Edg-1 ORF. Other cloning steps were performed essentially
as described before. The resulting plasmid, a pCAL-c derivative
that comprises an ORF encoding a rat Edg-1-CBP fusion protein
operably linked to a T7 promoter and lac repressor binding sites,
is designated "prEDG-1-CBP" herein.
Example 4
Cloning of a His-Tagged Rat Edg-1 into pCAL-c Expression Vector
[1006] The rEdg-1 protein was manipulated to generate a fusion
protein having a 6.times.His tag at its carboxyl terminus. A
"6.times.His tag" or "His tag" is an amino acid sequence consisting
of six contiguous histidine residues that can be used as an epitope
for the binding of anti-6.times.His antibodies, or as ligand for
binding nickel atoms. The His-tagged rEdg-1 fusion protein is used
to detect rEdg-1 protein expression in the minicell expression
system environment.
[1007] The rEdg-1-6.times.His construct was cloned using the
strategy described above for the construction of the rEdg-1-pCAL-c
expression construct (prEDG-1), with the upstream primer having the
sequence of SEQ ID NO:3, but with the exception that the three
prime downstream primer (SEQ ID NO:34) was designed to contain six
histidine codons followed by a stop codon. The 18 base pair
6.times.His tag was incorporated into the carboxyl terminus of the
Edg-1 protein as expressed from the pCAL-c vector. Subsequent
cloning procedures (PCR, restriction digest, gel purification,
ligation, transformation, etc.) were performed as described
previously for the Edg-1-pCAL-c construct (prEDG-1). The resulting
plasmid, a pCAL-c derivative that comprises an ORF encoding a
carboxy-terminal His-tagged rat Edg-1-CBP fusion protein operably
linked to a T7 promoter and lac repressor binding sites, is
designated "prEDG-1-6.times.His" herein.
Example 5
Amplification and Cloning of Rat Edg-3 Sequences
[1008] The Edg-3 full length coding sequence was amplified via PCR
from rat skeletal muscle mRNA using primers (SEQ ID NOS:35 and 36)
designed from the known mouse sequence (Genbank accession
NM.sub.--010101). The mRNA used as a template for the amplification
reaction was isolated using the RNeasy Miniprep Kit (Qiagen). Both
the rtPCR and PCR amplification steps were carried out in a single
reaction using the One Step RT-PCR Kit (Qiagen). The rEdg-3 PCR
products were visualized with UV after electrophoresis in 1% TAE
agarose gels and ethidium bromide staining.
[1009] The predicted size of the amplified PCR products is 1145
base pairs. An appropriately-sized DNA band was isolated from the
TAE gel and purified using the Geneclean Kit (B10101). The purified
band was ligated to the pCR3.1 vector using the TA-cloning kit
(Invitrogen). Other cloning steps were carried out as described
previously for the cloning of the rEdg-1-pCAL-c construct (prEDG-1)
with the exception that the samples were screened using the EcoRI
restriction enzyme. The expected sizes of the digested bands were
1145 base pairs and 5060 base pairs. Positive clones were analyzed
by automated sequencing. The nucleotide sequences were analyzed
using BLAST searches from the NCBI web site
(www.ncbi.nlm.nih.gov/). The predicted full length rat Edg-3 amino
acid sequence was assembled from the nucleotide sequencing data
using in silico translation. The pCR3.1 vector comprising the rat
Edg-3 ORF is designated "pCR-rEDG-3" herein.
Example 6
Cloning of Rat Edg-3 Coding Sequences into the pCAL-c Expression
Vector
[1010] In order to express it in the minicell expression system,
the rat Edg-3 ORF was cloned into the pCAL-c expression vector. The
cloning strategy used was as described above for the cloning of the
rat Edg-1 gene into the pCAL-c vector with the following
exceptions. The primers used for PCR amplification were designed
from the rat Edg-3 sequence and contained sites for the restriction
enzymes NheI and KpnI (GGTACC). The NheI site was added to the five
prime upstream primer (SEQ ID NO:37) and the KpnI site was added to
the three prime downstream primer; SEQ ID NO:38). The NheI and KpnI
restriction enzymes were used for the digestion reaction. The
reaction mixture for the digestion step consisted of 1 .mu.g of
DNA, 1.times. restriction buffer (provided with the enzyme), and 1
.mu.L of each enzyme. Plasmid preparations were screened by
digestion with NheI and KpnI. The digested plasmid DNA was
electrophosesed on a TAE agarose gel and visualized by UV after
staining with ethidium bromide. The resultant band sizes were
predicted to be 1145 base pairs and 5782 base pairs. The positive
plasmid clones were analyzed with automated sequencing. The
resulting plasmid, a pCAL-c derivative that comprises an ORF
encoding a rat Edg-3 protein operably linked to a T7 promoter and
lac repressor binding sites, is designated "pEDG-3" herein.
Example 7
Cloning of a His-Tagged Rat Edg-3 into the pCAL-c Expression
Vector
[1011] In order to detect expression of the rat Edg-3 protein in
the minicell expression system, the rat Edg-3 coding sequence was
manipulated so as to contain a 6.times.His tag at the carboxyl
terminus of the protein. The cloning strategy used to create this
construct was essentially the same as described above for the
rEdg-3-pCAL-c (prEDG-3) construct cloning, with the upstream primer
having the sequence of SEQ ID NO:37, with the exception that the
three-prime downstream primer (SEQ ID NO:18) was designed to
contain a 6.times.His coding sequence followed by a stop codon,
which allowed for the incorporation of the 6.times.His amino acid
sequence onto the carboxyl terminus of the Edg-3 receptor protein.
Other cloning and screening steps were performed as described
above. The resulting plasmid, a pCAL-c derivative that comprises an
ORF encoding a carboxy-terminal His-tagged rat Edg-3 fusion protein
operably linked to a T7 promoter and lac repressor binding sites,
is designated "prEDG-3-6.times.His" herein.
Example 8
GFP Cloning into pCAL-c Expression Construct
[1012] Cloning of GFP-encoding nucleotide sequences into the pCAL-c
vector was performed in order to produce an expression construct
having a reporter gene that can be used to detect protein
expression (GFP, green fluorescent protein). The cloning strategy
used was essentially the same as the cloning strategy described
above with the following exceptions. The template used for PCR
amplification was the peGFP plasmid "construct" (GFP construct sold
by Clontech). The primers used for amplification were designed from
the GFP coding sequence and contained sites for the restriction
enzymes NcoI and BamHI. The NcoI site was added to the five prime
upstream primer (SEQ ID NO:40) and the BamHI site was added to the
three prime downstream primer; see SEQ ID NO:41) The NcoI and BamHI
restriction enzymes were used for the digestion reaction. The
reaction mixture for the digestion step consisted of 1 .mu.g of
DNA, 1.times. restriction buffer (provided with the enzyme), and 1
.mu.L of each enzyme. The screening of the plasmid preparations was
carried out using NcoI and BamHI. Digested plasmid preparations
were electrophoresed and visualized on TAE agarose gels with UV
after staining with ethidium bromide. Restriction products having
the predicted sizes of 797 and 5782 base pairs were seen. Positive
plasmid clones were sequenced using an automated sequencer. The
resulting plasmid, a pCAL-c derivative that comprises an ORF
encoding a rEdg-3-GFP fusion protein operably linked to a T7
promoter and lac repressor binding sites, is designated
"prEDG-3-GFP" herein.
Example 9
Design Construction of Control Expression Elements
[1013] Control expression elements used to detect and quantify
expression of proteins in minicells were preposed. These controls
direct the expression of detectable proteins. An expression element
used as positive control is pPTC12, which is supplied with the
pCAL-c expression vector from Stratagene. This construct contains
an ORF encoding a fusion protein comprising beta-galactosidase
linked to CBP. Induction of expression of pTC12 should result in
the production of a protein of about 120 kD, and this protein is
detected via its enzymatic activity or by using antibodies directed
to epitopes on the beta-galactosidase or CBP polypeptide.
[1014] A GFP fusion construct was created and used as a positive
control for the CBP detection kit. This construct was a positive
control for induction of protein expression in the minicell
expression system. The cloning strategy used to create the
construct was essentially the same as that used for the cloning of
the GFP into the pCAL-c expression vector, with the exception that
the three prime downstream primer did not contain a stop codon;
this allowed for the in frame incorporation of the CBP fusion tag
to the GFP protein. The upstream primer had the sequence of SEQ ID
NO:42, and the downstream primer had the sequence SEQ ID NO:43. The
nucleotide sequence of the expression element was confirmed using
an automated sequencer. The resulting plasmid, a pCAL-c derivative
that comprises an ORF encoding GFP operably linked to a T7 promoter
and lac repressor binding sites, is designated "pGFP-CBP"
herein.
Example 10
Introduction of pCAL-c Expression Constructs Into the MC-T7
Escherichia Coli Strain
[1015] The MC-T7 E. coli strain was made competent using the
CaCl.sub.2 technique. In brief, cells were grown in 40 mL LB medium
to an OD.sub.600 of 0.6 to 0.8, and then centrifuged at 8000 rpm
(7,700 g) for 5 min at 4.degree. C. The pellet was resuspended in
20 mL of cold CaCl.sub.2 and left on ice for five minutes. The
cells were then centrifuged at 8000 rpm (7,700 g) for 5 min at
4.degree. C. The cell pellet was resuspended in 1 mL of cold
CaCl.sub.2 and incubated on ice for 30 min. Following this
incubation 1 mL of 25% glycerol was added to the cells and they
were distributed and frozen in 200 .mu.L aliquots. Liquid nitrogen
was used to freeze the cells. These cells subsequently then used
for the transformation of expression constructs.
Example 11
Preparation of Minicells
[1016] To some degree, the preparation of minicells varied
according to the type of expression approach that is used. In
general, there are two such approaches, although it should be noted
from the outset that these approaches are neither limiting nor
mutually exclusive. One approach is designed to isolate minicells
that already contain an expressed therapeutic protein or nucleic
acid. Another approach is designed to isolate minicells that will
express the protein or nucleic acid in the minicell following
isolation.
[1017] E. coli are inoculated into bacterial growth media (e.g.,
Luria broth) and grown overnight. After this, the overall protocol
varies with regards to methods of induction of expression. The
minicell producing cultures used to express protein post isolation
are diluted and grown to the desired OD.sub.600 or OD450, typically
in the log growth phase of bacterial cultures. The cultures are
then induced with IPTG and then isolated. The IPTG concentration
and exposure depended on which construct was being used, but was
usually about 500 .mu.M final for a short time, typically about 4
hours. This treatment results in the production of the T7
polymerase, which is under control of the LacUVR5 promoter, which
is repressed by the Lad repressor protein. IPTG relieves the LacI
repression and thus induces expression from the LacUVR5 promoter
which controls expression of the T7 polymerase from the chromosome.
This promoter is "leaky" that is, there is always a basal level of
T7 polymerase which can be selected for or against so that the
induction before isolation is not required. (This induction step is
not required if a non-T7 expression system is used, as the reason
for this step is to express the T7 RNA polymerase in the
minicell-producing cells so that the polymerase and molecules
segregate with the minicell.)
[1018] The E. coli cultures that produce minicells containing a
therapeutic protein or nucleic acid have different induction
protocols. The overnight cultures are diluted as described above;
however, in the case of proteins that are not toxic to the parent
cells, this time the media used for dilution already contains IPTG.
The cultures are then grown to mid-log growth and minicells are
isolated. These cultures produce the therapeutic protein or nucleic
acid as they grow, and the minicells derived therefrom contain the
therapeutic protein or nucleic acid.
[1019] Alternatively or additionally, IPTG is added and expression
is induced after the isolation of minicells. In the case of
non-toxic proteins or nucleic acids that are expressed from
expression elements in minicells, this treatment enhances
production of the eposimally encoded gene product. In the case of
toxic gene products induction post-isolation is preferred.
Example 12
Minicell Isolation
[1020] Minicells were isolated from the minicell producing MC-T7
strain of E. coli using centrifugation techniques. The protocol
that was used is essentially that of Jannatipour et al.
(Translocation of Vibrio Harveyi N,N'-Diacetylchitobiase to the
Outer Membrane of Escherichia Coli, J. Bacteriol. 169: 3785-3791,
1987) and Matsumura et al. (Synthesis of Mot and Che Products of
Escherichia coli Programmed by Hybrid ColE1 Plasmids in Minicells,
J. Bacteriol. 132:996-1002, 1977).
[1021] In brief, MC-T7 cells were grown overnight at 37.degree. C.
in 2 to 3 mL of LB media containing ampicillin (50 .mu.g/mL),
streptomycin (50 .mu.g/mL), and tetracycline (50 .mu.g/mL)
(ampicillin was used only when growing MC-T7 cells containing a
pCAL-c expression construct). The cells were diluted 1:100 in a
total volume of 100 to 200 mL LB media with antibiotics, and grown
at 37.degree. C. until they reached an OD.sub.600 of 0.4 to 0.6,
which is roughly beginning of the log growth phase for the MC-T7 E.
coli. During this incubation the remainder of the overnight culture
was screened for the presence of the correct expression construct
using the techniques described above. When the cultures reached the
appropriate OD.sub.600 they were transferred to 250 mL GS3
centrifuge bottles and centrifuged (Beckman centrifuge) at 4500 rpm
(3,500 g) for 5 min. At this point the supernatant contains mostly
minicells, although a few relatively small whole cells may be
present.
[1022] The supernatant was transferred to a clean 250 mL GS3
centrifuge bottle and centrifuged at 8000 rpm (11,300 g) for 10
min. The pellet was resuspended in 2 mL of 1.times.BSG
(10.times.BSG: 85 g NaCl, 3 g KH.sub.2PO.sub.4, 6 g
Na.sub.2HPO.sub.4, and 1 g gelatin in 1 L ddH.sub.2O) and layered
onto a 32 mL 5 to 20% continuous sucrose gradient. The sucrose
gradient was made with sucrose dissolved in 1.times.BSG.
[1023] The sucrose gradient was then loaded in a Beckman SW24 rotor
and centrifuged in a Beckman Ultracentrifuge at 4500 rpm (9,000 g)
for 14 min. Following ultracentrifugation a single diffuse band of
minicells was present. The top two thirds of this band was
aspirated using a 10 mL pipette and transferred to a 30 mL Oakridge
tube containing 10 mL of 1.times.BSG. The sample was then
centrifuged at 13,000 rpm (20,400 g) for 8 min. Following
centrifugation, the pellet was resuspended in 2 mL 1.times.BSG, and
the resuspended cells were loaded onto another 5 to 20% sucrose
gradient. This sucrose gradient was centrifuged and the minicells
were collected as described above. The sucrose gradient procedure
was repeated a total of three times.
[1024] Following the final sucrose gradient step the entire
minicell band was collected from the sucrose gradient and added to
a 30 mL Oakridge tube that contained 10 mL of MMM buffer (200 mL
1.times.M9 salts, 2 mL 20% glucose, and 2.4 mL DIFCO Methionine
Assay Medium). This minicell solution was centrifuged at 13,000 rpm
(20,400 g) for 8 min. The pellet was resuspended in 1 mL of MMM
Buffer.
[1025] The concentration of minicells was determined using a
spectrophotometer. The OD.sub.450 was obtained by reading a sample
of minicells that was diluted 1:100.
Example 13
Other Methods to Prepare and Isolate Minicells
[1026] By way of non-limiting example, induction of E. coli
parental cells to form minicells may occur by overexpression of the
E. coli ftsZ gene. To accomplish this both plasmid-based and
chromosomal overexpression constructs were created that place the
ftsZ gene under the control of various regulatory elements (Table
6).
TABLE-US-00008 TABLE 6 REGULATORY CONSTRUCTS CONTROLLING FTSZ
EXPRESSION. Regulatory region inducer [inducer] SEQ ID NO.:
Para::ftsZ Arabinose 10 mM 1, 3 Prha::ftsZ Rhamnose 1 mM 2, 4
Ptac::ftsZ IPTG 30 .mu.M 5, Garrido et al..sup.a .sup.aGarrido, T.
et al. 1993. Transcription of ftsZ oscillates during the cell cycle
of Escherichia coli.
Oligonucleotide Names and PCR Reactions Use the Following
Format:
[1027] "gene-1" is N-terminal, 100% homology oligo for chromosomal
or cDNA amplification [1028] "gene-2" is C-terminal, 100% homology
oligo for chromosomal or cDNA amplification [1029] "gene-1-RE site"
is same sequence as gene-1 with additional residues for remainder
of sequence, RE sites, and/or chimeric fusions. [1030] "gene-2-RE
site" is same sequence as gene-1 with additional residues for
remainder of sequence, RE sites, and/or chimeric fusions. Use
"gene-1, 2" combo for chromosomal/cDNA amplification and "gene-1 RE
site, gene-2-RE site" to amplify the mature sequence from the
"gene-1, 2" gel-purified product.
TABLE-US-00009 [1030] TABLE 7 OLIGONUCLEOTIDE PRIMER SEQUENCES FOR
TABLE 6 CONSTRUCTS SEQ ID NO.: Primer name 5' to 3' sequence 44
FtsZ-1 CCAATGGAACTTACCAATGACGCGG 45 FtsZ-2 GCTTGCTTACGCAGGAATGCTGGG
46 FtsZ-1-PstI CGCGGCTGCAGATGTTTGAACCAAT GGAACTTACCAATGACGCGG 47
FtsZ-2-XbaI GCGCCTCTAGATTATTAATCAGCTT GCTTACGCAGGAATGCTGGG
Table 7 Oligonucleotide Sequences are for Use in Cloning FTSZ Into
SEQ ID NO.:1 and 2 (Insertions of FTSZ Behind the Arabinose
Promotor (SEQ ID NO.: 1) and the Rhamnose Promotor (SEQ ID NO.:
2).
TABLE-US-00010 [1031] TABLE 8 OLIGONUCLEOTIDE PRIMER SEQUENCES FOR
FTSZ CHROMOSOMAL DUPLICATION CONSTRUCTS SEQ ID Primer NO.: name 5'
to 3' sequence 48 Kan-1 GCTAGACTGGGCGGTTTTATGGACAGCAAGC 49 Kan-2
GCGTTAATAATTCAGAAGAACTCGTCAAGAAGGCG 50 Kan-1-X-
GCGCCTACTGACGTAGTTCGACCGTCGGACTAGCGA frt
AGTTCCTATACTTTCTAGAGAATAGGAACTTCGCTA GACTGGGCGGTTTTATGGACAGCAAGC 51
Kan-2- CAAGATGCTTTGCCTTTGTCTGAGTTGATACTGGCTT intD-frt
TGGGAAGTTCCTATTCTCTAGAAAGTATAGGAACTT
CGCGTTAATAATTCAGAAGAACTCGTCAAGAAGGCG 52 AraC-1
CGTTACCAATTATGACAACTTGACGG 53 RhaR-1 TTAATCTTTCTGCGAATTGAGATGACGCC
54 LacI.sup.q-1 GTGAGTCGATATTGTCTTTGTTGACCAG 55 Ara-1-
GCCTGCATTGCGGCGCTTCAGTCTCCGCTGCATACTG intD
TCCCGTTACCAATTATGACAACTTGACGG 56 RhaR-1-
GCCTGCATTGCGGCGCTTCAGTCTCCGCTGCATACTG intD
TCCTTAATCTTTCTGCGAATTGAGATGACGCC 57 LacI.sup.q-1-
GCCTGCATTGCGGCGCTTCAGTCTCCGCTGCATACTG intD
TCCTTAATAAAGTGAGTCGATATTGTCTTTGTTGACC AG 58 FtsZ-1-X
GCCTGCATTGCGGCGCTTCAGTCTCCGCTGCATACTG
TCCCGTTACCAATTATGACAACTTGACGG
[1032] In like fashion, the ftsZ gene was amplified from SEQ ID
NO.: 1, 2 and Ptac::ftsZ (Garrido, T. et al. 1993. Transcription of
ftsZ oscillates during the cell cycle of Escherichia coli. EMBO J.
12:3957-3965) plasmid and chromosomal constructs, respectively
using the following oligonucleotides:
For amplification of araC through ftsZ of SEQ ID NO.: 1 use
oligonucleotides:
AraC-1
FtsZ-2
[1033] For amplification of rhaR through ftsZ of SEQ ID NO.: 2 use
oligonucleotides:
RhaR-1
FtsZ-2
[1034] For amplification of lacI.sup.q through ftsZ of Ptac::ftsZ
(Garrido, T., et al.) use oligonucleotides: lacI.sup.q-1 ftsZ-2
[1035] The above amplified DNA regions were gel-purified and used
as template for the second round of PCR using oligonucleotides
containing homology with the E. coli chromosomal gene intD and on
the other end with random sequence termed "X". Oligonucleotides
used in this round of PCR are shown below:
For amplification of araC through ftsZ from SEQ ID NO.: 1 to
contain homology to intD and the random X use oligonucleotides:
AraC-1-intD
FtsZ-1-X
[1036] For amplification of rhaR through ftsZ from SEQ ID NO.: 2 to
contain homology to intD and the random X use oligonucleotides:
RhaR-1-intD
FtsZ-1-X
[1037] For amplification of lacIq through ftsZ from Ptac::ftsZ to
contain homology to intD and the random X use oligonucleotides:
LacIq-1-intD
FtsZ-1-X
[1038] The PCR products from these PCR reactions are as shown
below:
[1039] intD-araC-Ara promotor-ftsZ-"X"
[1040] intD-rhaRS-Rha promotor-ftsZ-"X"
[1041] intD-lacIq-Ptac promotor-ftsZ-"X"
To amplify the mature complexes, the following regions were mixed
and amplified with the coupled oligonucleotide sequence
primers:
##STR00005##
[1042] These expression constructs may be expressed from the
plasmid, placed in single copy, replacing the native ftsZ copy on
the E. coli chromosome (Garrido, T., et al. 1993. Transcription of
ftsZ oscillates during the cell cycle of Escherichia coli. EMBO J.
12:3957-3965), or in duplicate copy retaining the native ftsZ copy
while inserting one of the expression constructs in Table 6 into
the intD gene on the same chromosome. Chromosomal duplications were
constructed using the RED recombinase system (Katsenko, K. A., and
B. L. Wanner. One-Step Inactivation of Chromosomal Genes in
Escherichia coli K-12 Using PCR Products. Proc. Natl. Acad. Sci.
97:6640-6645. 2000) and are shown in SEQ ID NO 3-5. The later
constructs allow native replication during non-minicell producing
conditions, thus avoiding selective pressure during strain
construction and maintenance. Furthermore, these strains provide
defined points of minicell induction that improve minicell
purification while creating conditions that allow strain
manipulation prior to, during, and following minicell production.
By way of non-limiting example these manipulations may be protein
production that the cytoplasmic redox state, modify plasmid copy
number, and/or produce chaperone proteins.
[1043] For minicell production, a minicell producing strain
described in the previous section is grown overnight in Luria broth
(LB) supplemented with 0.1% dextrose, 100 .mu.g/ml ampicillin, and
when using the single-copy ftsZ construct, 15 .mu.M IPTG. All
incubations were performed at 37.degree. C. For minicell induction
only, overnight strains are subcultured 1/1000 into the same media.
If minicell induction is to be coupled with co-expression of other
proteins that are controlled by a catabolite repression-sensitive
regulator, dextrose was excluded. Minicell induction is sensitive
to aeration and mechanical forces. Therefore, flask size, media
volume and shake speed is critical for optimal yields. Likewise,
bioreactor conditions must be properly regulated to optimize these
production conditions.
[1044] In shake-flask cultures, strains are grown to early
exponential (log) phase as monitored by optical density (OD) at 600
nm (OD.sub.600 0.05-0.20). (Bioreactor conditions may differ
significantly depending on the application and yield desired). For
minicell induction alone, early log phase cultures are induced with
the appropriate inducer concentration shown in Table 6. For coupled
co-expression, these cultures are induced as shown in Table 6 for
the appropriate minicell regulator, while the coupled protein(s) is
induced with the inducer appropriate for the regulator controlling
the synthesis of that protein. Cultures are grown under the
appropriate conditions and harvested during late log (OD.sub.600
0.8-1.2). Depending on the application, minicell induced cultures
may be immediately chilled on ice prior to purification, or
maintained at room temperature during the harvesting process.
[1045] To separate minicells from viable, parental cells, cultures
are subjected to differential centrifugation (Voros, J., and R. N.
Goodman. 1965. Filamentous forms of Erwinia amylovora. Phytopathol.
55:876-879). Briefly, cultures are centrifuged at 4,500 rpm in a
GSA rotor for 5 min. Supernatants are removed to a fresh bottle and
centrifuged at 8,000 rpm for an additional 10 min to pellet
minicells. Pelleted minicells (containing contaminating parental
cells) are resuspended in 2 ml LB, LBD (LB supplemented with 0.1%
dextrose), Min (minimal M63 salt media) (Roozen, K. J., et al.
1971. Synthesis of ribonucleic acid and protein in
plasmid-containing minicells of Escherichia coli K-12. J.
Bacteriol. 107:21-23), supplemented with 0.5% casamino acids) or
MDT (minimal M63 salt media, supplemented with 0.5% casamino acids,
0.1% dextrose, and thiamine). Resuspended minicells are next
separated using linear density gradients. By way of non-limiting
example, these gradients may contain sucrose (Cohen A., et al.
1968. The properties of DNA transferred to minicells during
conjugation. Cold Spring Harb. Symp. Quant. Biol. 33:635-641),
ficol, or glycerol. For example, linear sucrose gradients range
from 5-20% and are poured in LB, LBD, Minor MDT. Using a SW28
swinging bucket rotor, gradients are centrifuged at 4,500 rpm for
14 min. Banded minicells are removed, mixed with LB, LBD, Minor
MDT, and using a JA-20 rotor are centrifuged at 13,000 rpm for 12
min. Following centrifugation, pellets are resuspended in 2 ml LB,
LBD, Minor MDT and subjected to a second density gradient.
Following the second density separation, banded minicells are
removed from the gradient, pelleted as described, and resuspended
in LB, LBD, Minor MDT for use and/or storage.
[1046] Purified minicells are quantitated using an OD.sub.600
measurement as compared to a standard curve incorporating LPS
quantity, minicell size, and minicell volume. Quantitated minicells
mixtures are analyzed for contaminating, viable parental cells by
plating on the appropriate growth media (Table 9).
TABLE-US-00011 TABLE 9 MINICELL PURIFICATION AND PARENTAL CELL
QUANTITATION Total MC/PC Fold- Purification Total cells parental
cells ratio purification Before 4.76 .times. 10.sup.11 3.14 .times.
10.sup.11 0.25/1 -- After 1.49 .times. 10.sup.11 6.01 .times.
10.sup.4 2.48 .times. 10.sup.6/1 5.23 .times. 10.sup.6
Example 14
Protoplast Formation
[1047] In order to allow a membrane receptor to be presented to the
outside environment (displayed), minicells are made into
protoplasts. In order to make the integral membrane protein
receptors in the inner membrane more accessible for ligand binding,
the outer membrane and cell wall were removed. The removal of the
outer membrane and cell wall from E. coli whole cells and minicells
to produce protoplasts was performed essentially according to
previously described protocols with a few modifications (Birdsell
et al., Production and Ultrastructure of Lysozyme and
Ethylenediaminetetraacetate-Lysozyme Spheroplasts of Escherichia
coli, J. Bacteriol. 93:427-437, 1967; Weiss et al., Protoplast
Formation in Escherichia Coli, J. Bacteriol. 128:668-670, 1976.
Both minicells and whole cells were processed the same way.
[1048] In brief, the cells were grown to mid-log phase and pelleted
at room temperature (minicells were isolated from cultures in
mid-log phase). The pellet was washed twice with 10 mM Tris.
Following the second wash protoplast production may be performed
using two approaches. In the first approach, following the second
wash, the cells were resuspended in 100 mM Tris (pH 8.0) that
contained 6-20% sucrose and put in a 37.degree. C. waterbath (the
Tris/sucrose buffer was pre-warmed to 37.degree. C.). The volume
used to resuspend the cells was determined by the following
equation: (volume of cells.times.OD.sub.450)/10=resuspension
volume. After a 1 minute incubation, 2 mg/mL lysozyme was added to
a final concentration of 5-100 .mu.g/mL. The samples were then
incubated for 12 minutes at 37.degree. C. while being gently mixed.
Next, 100 mM EDTA (pH 7) was slowly added over a period of 2.5
minutes (amount of EDTA added=1/00-1/10 volume of cells) followed
by a 10 min incubation at 37.degree. C. The protoplasts are also
diluted from 20% sucrose down to either 10% or 5% sucrose, which
facilitates the complete removal of the outer membrane and cell
wall. The protoplasts thus generated were separated from the outer
membrane and cell wall using a sucrose step gradient. A sucrose
step gradient does not have a gradual increase in sucrose
percentage; rather, it goes directly from one percent to the other.
For example, protoplasts generated from whole cells are loaded on a
step gradient that is made from 5% and 15% sucrose. The protoplasts
spin through the 15% sucrose but the debris generated when making
the protoplasts does not spin through the 15% sucrose. The
protoplasts are thus separated from the debris. The second method
to prepare protoplasts, following the second wash, 1.times.10.sup.9
cells were resuspended with 50 mM Tris, pH 8.0 containing 0.5-50 mM
EDTA and 6-20% sucrose. This mixture was incubated at 37.degree. C.
for 10 min. Following incubation, the mixture was centrifuged at
13,200 RPM in a microcentrifuge for 2 min. After centrifugation,
the pellet was resuspened in 50 mM Tris, pH 8.0 containing 5-100
.mu.g/ml lysozyme and 6-20% sucrose. This mixture was incubated at
37.degree. C. for 10 min. Following incubation, the mixture was
centrifuged at 13,200 RPM in a microcentrifuge for 2 min,
resuspended in 50 mM Tris pH 8.0 containing 6-20% sucrose for
use.
[1049] An alternative method to remove contaminating LPS is to use
affinity absorption with an anti-LPS antibody (Cortex). To
accomplish this, the anti-LPS antibody was coated on either an
activated agarose or sepharose matrix (Sigma) or epoxy-coated
magnetic M-450 beads (Dynal). The spheroplast/protoplast mixture
was subjected to the antibody coated matrix either in batch or
using column chromatographic techniques to remove contaminating
LPS. Following exposure, the unbound fraction(s) was collected and
re-exposed to fresh matrix. To monitor the efficiency of the
protoplasting reaction and LPS removal, three constructs were used
(Table 10).
TABLE-US-00012 TABLE 10 PROTOPLAST MONITORING CONSTRUCTS SEQ ID SEQ
ID Inducible Construct NO Plasmid NO protein Inducer PMPX-5 6
pMPX-32 7 .DELTA.phoA Rhamnose PMPX-5 6 pMPX-53 8 phoA Rhamnose
PMPX-5 6 pMPX-33 9 toxR-phoA Rhamnose
TABLE-US-00013 TABLE 11 OLIGONUCLEOTIDE PRIMER SEQUENCES FOR TABLE
10 CONSTRUCTS SEQ ID NO.: Primer name 5' to 3' sequence 59
.DELTA.phoA-1 GCCTGTTCTGGAAAACCGGGCTGCTCAGGG 60 .DELTA.phoA-2
GCGGCTTTCATGGTGTAGAAGAGATCGG 61 .DELTA.phoA-1-PstI
CCGCGCTGCAGATGCCTGTTCTGGAAAACCGGGCTGCT CAGGG 62 .DELTA.phoA-2-XbaI
GCGCCTCTAGATTATTATTTCAGCCCCAGAGCGGCTTTC ATGGTGTAGAAGAGATCGG 63
PhoA-1 GTCACGGCCGAGACTTATAGTCGC 64 PhoA-2
GCGGCTTTCATGGTGTAGAAGAGATCGG 65 PhoA-1-PstI
CCGCGCTGCAGATGTCACGGCCGAGACTTATAGTCGC 66 PhoA-2-XbaI
GCGCCTCTAGATTATTATTTCAGCCCCAGAGCGGCTTTC ATGGTGTAGAAGAGATCGG 67
T-phoA-1-PstI CCGCGCTGCAGATGAACTTGGGGAATCGACTGTTTATTC
TGATAGCGGTCTTACTTCCCCTCGCAGTATTACTGCTCA
TGCCTGTTCTGGAAAACCGGGCTGCTCAGGG 68 T-phoA-2-XbaI
GCGCCTCTAGATTATTATTTCAGCCCCAGAGCGGCTTTC ATGGTGTAGAAGAGATCGG
[1050] Oligonucleotides SEQ ID NOS.:59, 60, 61 and 62 were used to
amplify phoA lacking a leader sequence (.DELTA.phoA) form the E.
coli chromosome. Once amplified, this region was inserted into SEQ
ID NO.: 6 using PstI and XbaI to create SEQ ID NO.: 7.
[1051] Oligonucleotides SEQ ID NOS.:63, 64, 65 and 66 were used to
amplify phoA containing a leader sequence (phoA) form the E. coli
chromosome. Once amplified, this region was inserted into SEQ ID
NO.: 6 using PstI and XbaI to create SEQ ID NO.: 8.
[1052] Oligonucleotides SEQ ID NOS.:59, 60, 67 and 68 were used to
amplify phoA lacking a leader sequence (.DELTA.phoA) form the E.
coli chromosome and form a translational fusion between the
transmembrane domain of toxR from Vibrio cholerae. Once amplified,
this region was inserted into SEQ ID NO.: 6 using PstI and XbaI to
create SEQ ID NO.: 9.
[1053] By co-expression of minicells and protein, minicells were
prepared that contained cytoplasmic PhoA (pMPX-32 expresses phoA
lacking a leader sequence [.DELTA.phoA]), periplasmic PhoA (pMPX-53
expresses native phoA that exports to the periplasmic space), or
inner membrane-bound PhoA (pMPX-33 expresses phoA lacking a leader
sequence fused to the transmembrane domain (TMD) of the toxR gene
product from Vibrio cholerae). Using these expressed proteins, the
efficiency of minicell protoplasting was monitored (Table 12).
TABLE-US-00014 TABLE 12 EFFICIENCY OF MINICELL PROTOPLAST
PREPARATION AND PURIFICATION Step Location .sup.a .DELTA.PhoA PhoA
T-PhoA LPS total .sup.b Minicell Pellet 100 100 100 100
EDTA/lysozyme Whole 100 100 100 100 1.sup.st Anti-LPS Pellet 80 0
80 30 2.sup.nd Anti-LPS Pellet 60 0 60 0 .sup.a Measuring the
location of protein being measured using an anti-BAP antibody
(Sigma). Pellet refers to the presence of the expressed protein in
the low-speed centrifugation pellet. These pellets contain only
intact cellular bodies. Whole refers to the reaction mixture prior
to low-speed centifugation. .sup.b Measured using a slot-blot
apparatus (Bio-Rad) using the anti-LPS antibody (Cortex)
[1054] The data suggests that periplasmic PhoA is lost during the
preparation, while both cytoplasmic and membrane-bound PhoA are
retained in a cellular body that lacks LPS. However, during this
process .about.40% of the total minicell content is lost.
Example 15
T7-Dependent Induction of Expression
[1055] Expression from the pCAL-c expression vector is driven from
a T7 bacteriophage promoter that is repressed by the LacI gene
product. Transcription of the DNA into mRNA, and subsequent
translation of mRNA into proteins, does not occur as long as the
Lad repressor is bound to the T7 promoter. However, in the presence
of IPTG, the Lad repressor does not bind the T7 promoter. Thus,
induction of expression from pCAL-c sequences is dependent on the
presence of IPTG. Slightly different protocols were used for the
induction of Escherichia coli whole and for the induction of
minicells. Slight differences are also present in the protocols for
induction of minicells for .sup.35S-methionine labeling of proteins
in contrast to those for the induction of minicells for Western
blot analysis. These induction protocols are described bellow.
[1056] For expression in E. coli whole cells, the cells were first
grown overnight in 3 mL of LB and antibiotics. The cultures were
screened for the presence of the desired expression element as
previously described. Cultures containing the desired expression
elements were diluted 1:100 and grown to an OD.sub.600 of between
0.4 to 0.6. The culture size varied depending on the intended use
of the cells. IPTG was then added to a final concentration of 200
.mu.g/mL, and the cells were shaken at 30.degree. C. for 4 hours.
Following the induction, cells were harvested for analysis.
[1057] The induction of minicells was carried out as follows. The
minicells were diluted in MMM buffer to 1 mL total volume according
to the concentration obtained from the isolation procedure
(OD.sub.450 of about 0.5). The cells were then treated with 50
.mu.g/mL of cycloserine for 30 minutes at 37.degree. C. to stop
whole cell growth. Following the cycloserine treatment the cells
were provided with an amino acid, methionine, which the MMM buffer
does not contain. For .sup.35S-labeled protein induction
.sup.35S-methionine was added to the minicell sample whereas, for
unlabeled protein induction unlabeled methionine was added. Fifteen
(15) .mu.Ci of .sup.35S-methionine (Amersham Pharmacia Biotech,
Piscataway, N.J.) was added to the samples for radiolabeling and 5
.mu.mol of methionine was added to the non-labeled minicell
samples. Two hundred (200) .mu.g/mL IPTG was also added to the
minicell samples, which were then shaken at 30.degree. C. for about
4 hours. Following induction, the minicells were harvested for
further preparation or analysis.
Example 16
Western Blot Analysis
[1058] The CBP detection kit was purchased from Stratagene. SDS
running buffer, 10% Tris-HCl ready gels, Kaleidoscope Pre-stained
Standards, and Laemmli Sample Buffer were purchased from BIO RAD
(Hercules, Calif.). GFP (FL) HRP antibodies were purchased from
Santa Cruz Biotechnology (Santa Cruz, Calif.). Edg-3CT antibody an
antibody directed to the carboxy terminus of was purchased from
Exalpha Biologicals (Boston, Mass.). Anti-6.times.His antibody,
positrope, and the WesternBreeze Kit were purchased from Invitrogen
(Carlsbad, Calif.). Protocols were carried out essentially
according to the manufacturer's instructions unless otherwise
indicated.
[1059] Three different Western blot protocols were used to detect
protein expression in both a minicell expression system and in a
whole cell expression system. For both systems, the SDS-PAGE gel
and the transfer protocols were essentially as follows. The samples
were denatured by diluting the samples 1:1 in Laemmli buffer
(BIORAD) and then sonicated for 10 min. The denatured samples were
loaded onto a 10% Tris-Glycine gel (BIORAD) and electrophoresed at
130 V for about 1.5 hours in 1.times.SDS running buffer (BIORAD).
The electrophoresed proteins were electrotransferred to
nitrocellulose membranes at 0.5 Amps for 1.5 hours in Transfer
Buffer (5.8 g Tris, 2.9 g glycine, 200 mL methanol, and 3.7 mL of
10% SDS). The nitrocellulose membranes comprising the transferred
proteins were used for Western blotting.
[1060] GFP Western blots were carried out as follows. The
nitrocellulose membrane was blocked for 2 hours with 5% milk in
PBST (PBS buffer with 0.05% Tween). Following the blocking step the
nitrocellulose membrane was washed twice with PBST. For the
detection of GFP protein, an anti-GFP-HRP conjugated antibody
(Santa Cruz Biotechnology) was used at a dilution of 1:3000 in PBST
(HRP, horse radish peroxidase). The nitrocellulose membrane was
incubated in the anti-GFP-HRP antibody solution for one hour and
then washed twice with PBST. GFP proteins on the nitrocellulose
membrane were detected and visualized using the ECL system
(Amersham).
[1061] The His-tagged Edg-1 and Edg-3 proteins were detected using
a mouse anti-6.times.His antibody from Invitrogen and the
WesternBreeze chemoluminecent Kit (Invitrogen). The antibody was
diluted 1:4000 in buffers provided by the WesternBreeze Kit. The
WesternBreeze immunoblot was carried out essentially according to
the manufacturer's protocol. The Edg-1-CBP and GFP-CBP fusion
proteins were detected using the CBP detection Kit (Stratagene).
All antibodies and substrates were provided in the Kit. FIG. 3 is a
photo of the Western hybridization results showing the presence of
Edg-1-6.times.His and Edg-3-6.times.His in minicells and parent
cells.
Example 17
Methods to Induce Expression
[1062] Expression in minicells may proceed following purification
of minicells and/or minicell protoplasts from parental cells and
LPS constituents, respectively. However, for some applications it
is suitable to co-express proteins of interest with minicell
induction. For these approaches, one may use the protocol described
in EXAMPLE 13 for expression of the phoA constructs. By way of
non-limiting example, either of these approaches may be
accomplished using one or more of the following expression
constructs (Table 13).
TABLE-US-00015 TABLE 13 EXPRESSION CONSTRUCTS Regulatory SEQ ID
Plasmid element(s) inducer Plasmid NO.: pMPX-5 rhaRS Rhamnose
pUC-18 6 pMPX-7 uidR .beta.-glucuronate pUC-18 10 pMPX-8 melR
Melibiose pUC-18 11 pMPX-18 araC Arabinose pUC-18 12 pMPX-6 araC
Arabinose pUC-18 13
TABLE-US-00016 TABLE 14 OLIGONUCLEOTIDE PRIMER SEQUENCES FOR TABLE
13 CONSTRUCTS SEQ ID NO.: Primer name 5' to 3' sequence 69 Rha-1
GCGAATTGAGATGACGCCACTGGC 70 Rha-2 CCTGCTGAATTTCATTAACGACCAG 71
Rha-1-HindIII CGGCGAAGCTTAATTAATCTTTCTGCGAATTGAGATGA CGCCACTGGC 72
Rha-2-PstI CGCCGTAATCGCCGCTGCAGAATGTGATCCTGCTGAAT TTCATTAACGACCAG
73 Uid-1 CGCAGCGCTGTTCCTTTGCTCG 74 Uid-2 CCTCATTAAGATAATAATACTGG 75
Uid-1-HindIII GCCGCAAGCTTCGCAGCGCTGTTCCTTTGCTCG 76 Uid-2-PstI
CCAATGCATTGGTTCTGCAGGACTCCTCATTAAGATAA TAATACTGG 77 Mel-1
CGTCTTTAGCCGGGAAACG 78 Mel-2 GCAGATCTCCTGGCTTGC 79 Mel-1-HindIII
GCCGCAAGCTTCGTCTTTAGCCGGGAAACG 80 Mel-2-SalI
CGGTCGACGCAGATCTCCTGGCTTGC 81 Ara-1 CAAGCCGTCAATTGTCTGATTCG 82
Ara-2 GGTGAATTCCTCCTGCTAGCCC 83 Ara-1-HindIII
GCGCCAAGCTTCAAGCCGTCAATTGTCTGATTCG 84 Ara-2-PstI
CTGCAGGGTGAATTCCTCCTGCTAGCCC 85 Ara-1-XhoI
GCTTAACTCGAGCTTAATAACAAGCCGTCAATTGTCTG ATTC 86 Ara-2-SstI
GCTTAACCGCGGGCCAAGCTTGCATGCCTGCTCC
[1063] Oligonucleotides SEQ ID NOS.:69, 70, 71 and 72 were used to
amplify the rhaRS genes and their divergent control region from the
E. coli chromosome. Once amplified, this region was inserted into
pUC18 using HindIII and PstI to create SEQ ID NO.: 6.
[1064] Oligonucleotides SEQ ID NOS.:73, 74, 75 and 76 were used to
amplify the uidR control region, the uidR gene and the control
region for expression from the E. coli chromosome. Once amplified,
this region was inserted into pUC18 using HindIII and PstI to
create SEQ ID NO.: 10.
[1065] Oligonucleotides SEQ ID NOS.:77, 78, 79 and 80 were used to
amplify the melR gene and its divergent control region from the E.
coli chromosome. Once amplified, this region was inserted into
pUC18 using HindIII and SalI to create SEQ ID NO.: 11.
[1066] Oligonucleotides SEQ ID NOS.:81, 82, 83 and 84 were used to
amplify the araC gene and its divergent control region from the E.
coli chromosome. Once amplified, this region was inserted into
pUC18 using HindIII and PstI to create SEQ ID NO.: 12.
[1067] Oligonucleotides SEQ ID NOS.:81, 82, 85 and 86 were used to
amplify the araC gene and its divergent control region was PCR
amplified from pBAD-24. Once amplified, this region was inserted
into pEGFP (Clontech) using XhoI and SstI to create SEQ ID NO.:
13.
[1068] Except of pMPX-6, these expression constructs contain the
same multiple cloning site. Therefore, any protein of interested
may be inserted in each modular expression construct for simple
expression screening and optimization.
[1069] By way of non-limiting example, other proteins that may be
expressed are listed in Table 15.
TABLE-US-00017 TABLE 15 OTHER EXPRESSED PROTEINS SEQ ID Protein
Origin Construct Purpose NO.: Edg3 Rat native GPCR 14 .beta.2AR
Human native GPCR 15 TNFR-1a Human residues Receptor 18 (human)
29-455 TNFR-1b Human residues Receptor 17 (human) 41-455 TNF
(human) Human native Gene transfer 19 T-EGF Human chimera Gene
transfer 20 T-Invasin Y. chimera Gene transfer 21
pseudotuberculosis
TABLE-US-00018 TABLE 16 OLIGONUCLEOTIDE PRIMER SEQUENCES FOR TABLE
15 SEQ ID Primer NO.: name 5' to 3' sequence 87 Edg-1
GGCAACCACGCACGCGCAGGGCCACC 88 Edg-2 CAATGGTGATGGTGATGATGACCGG 89
Edg-1-SalI CGCGGTCGACATGGCAACCACGCACGCGCAGGGC CACC 90 Edg-2-KpnI
GCGCCGGTACCTTATCAATGGTGATGGTGATGAT GACCGG 91 .beta.2AR-1
GGGGCAACCCGGGAACGGCAGCGCC 92 .beta.2AR-2
GCAGTGAGTCATTTGTACTACAATTCCTCC 93 .beta.2AR-1-
CGCGGTCGACATGGGGCAACCCGGGAACGGCAGC SalI GCC 94 .beta.2AR-2-
GCGCCGGATCCTTATTATAGCAGTGAGTCATTTG BamHI TACTACAATTCCTCC 95
TNFR(29)-1 GGACTGGTCCCTCACCTAGGGGACAGGG 96 TNFR(29)-2
CTGAGAAGACTGGGCGCGGGCGGGAGG 97 TNFR(29)-1-
CGCGGGTCGACATGGGACTGGTCCCTCACCTAGG SalI GGACAGGG 98 TNFR(29)-2-
GCGCCGGTACCTTATTACTGAGAAGACTGGGCGC KpnI GGGCGGGAGG 99 TNFR(41)-1
GATAGTGTGTGTCCCC 100 TNFR(41)-2 CTGAGAAGACTGGGCGC 101 TNFR(41)-1-
GGGAGACCATGGATAGTGTGTGTCCCC NcoI 102 TNFR(41)-2-
GCCTCATCTAGATTACTGAGAAGACTGGGCGC XbaI 103 TNF-1
GAGCACTGAAAGCATGATCCGGGACG 104 TNF-2 CAGGGCAATGATCCCAAAGTAGACCTGC
105 TNF-1- CCGCGGAATTCATGAGCACTGAAAGCATGATCCG EcoRI GGACG 106
TNF-2- GGCGCAAGCTTATCACAGGGCAATGATCCCAAAG HindIII TAGACCTGC 107
T-EGF-1 TCTGATAGCGGTCTTACTTCCCCTCGCAGTATTA
CTGCTCAATAGTGACTCTGAATGTCCCCTGTCCC
ACGATGGGTACTGCCTCCATGATGGTGTGTGCAT GTATATTG 108 T-EGF-2
AGGTCTCGGTACTGACATCGCTCCCCGATGTAGC
CAACAACACAGTTGCATGCATACTTGTCCAATGC
TTCAATATACATGCACACACCATCATGGAGGCA 109 T-EGF-3
CCGCGGGTACCATGAACTTGGGGAATCGACTGTT TATTCTGATAGCGGTCTTACTTCCCCTCG
110 T-EGF-4 GCGCCAAGCTTATTAGCGCAGTTCCCACCACTTC
AGGTCTCGGTACTGACATCGCTCCCCG 111 Inv-1 TCATTCACATTGAGCGTCACCG 112
Inv-2 TTATATTGACAGCGCACAGAGCGG 113 Inv-1-
GCAAGAATTCACCATGAACTTGGGGAATCGACTG ToxR-EcoRI
TTTATTCTGATAGCGGTCTTACTTCCCCTCGCAG
TATTACTGCTCTCATTCACATTGAGCGTCACCG 114 Inv-2-PstI
CGCGGTTACGTAAGCAACTGCAGTTATATTGACA GCGCACAGAGCGG
[1070] Oligonucleotides SEQ ID NOS.:87, 88, 89 and 90 were used to
amplify rat Edg3 from rat cDNA. Once amplified, this region was
inserted into SEQ ID NO.: 6 (pMPX-5) using SalI and KpnI to create
SEQ ID NO.:14.
[1071] Oligonucleotides SEQ ID NOS.:91, 92, 93 and 94 were used to
amplify human .beta.2 adrenergic receptor (.beta.2AR) from human
heart cDNA. Once amplified, this region was inserted into SEQ ID
NO.: 6 (pMPX-5) using SalI and BamHI to create SEQ ID NO.:15.
[1072] Oligonucleotides SEQ ID NOS.:95, 96, 97 and 98 were used to
amplify human tumor necrosis factor receptor (TNFR residues 29-455)
from human Jurkat CL71 cDNA. Once amplified, this region was
inserted into SEQ ID NO.: 12 (pMPX-18) using SalI and KpnI to
create SEQ ID NO.:18.
[1073] Oligonucleotides SEQ ID NOS.:99, 100, 101 and 102 were used
to amplify human tumor necrosis factor receptor (TNFR residues
41-455) from human Jurkat CL71 cDNA. Once amplified, this region
was inserted into pBAD24 using NcoI and XbaI to create SEQ ID
NO.:17.
[1074] Oligonucleotides SEQ ID NOS.:103, 104, 105 and 106 were used
to amplify human tumor necrosis factor (TNF) from human Jurkat CL71
cDNA. Once amplified, this region was inserted into SEQ ID NO.: 13
(pMPX-6) using EcoRI and HindIII to create SEQ ID NO.:19.
TABLE-US-00019 TABLE 17 PROGRAM TO ANNEAL GRADIENT PCR WITH PFX
POLYMERASE Step Temp (.degree. C.) Time (min) 1 95 2.0 2 95 0.5 3
64 0.5 4 68 2.5 5 Goto 2, 2X 6 95 0.5 7 62 0.5 8 68 2.5 9 Goto 6,
4X 10 95 0.5 11 60 0.5 12 68 2.5 13 Goto 10, 6X 14 95 0.5 15 58 0.5
16 68 2.5 17 Goto 14, 24X 18 4 hold 19 end
[1075] Oligonucleotides SEQ ID NOS.:107, 108, 109 and 110 were
mixed and PCR amplified using anneal gradient PCR (Table 17) to
form mature human epidermal growth factor (EGF) (residues 971-1023)
translationally fused to the transmembrane domain of toxR from
Vibrio cholerae. Once amplified, this region was inserted into SEQ
ID NO.: 13 (pMPX-6) using KpnI and HindIII to create SEQ ID
NO.:20.
[1076] Using PFX polymerase (Invitrogen) oligonucleotide SEQ ID
NO.:111, 112, 113 and 114 were used to amplify invasin residues
490-986 (inv) from Yersinia pseudotuberculosis chromosomal DNA and
form a translational fusion between the transmembrane domain of
toxR from Vibrio cholerae. Once amplified, this region was inserted
into SEQ ID NO.:13 (pMPX-6) using EcoRI and PstI to create SEQ ID
NO.:21.
[1077] These proteins were proof-of-principle constructs used to
evaluate the minicell platform. For purposes of this initial
evaluation, all proteins except TNF, T-EGF and T-Invasin were
cloned into pMPX-5, with these later proteins cloned into pMPX-6
for gene transfer experiments.
[1078] Whether the approach for protein expression is co-expression
with minicell induction or expression following minicell and/or
protoplast isolation, the procedure to transform the expression
constructs is the same. To accomplish this, protein constructs were
initially cloned into E. coli MG1655 and then into the minicell
producing strain of interest. Transformation events were selected
prior to minicell induction. For co-induction of protein and
minicells, see the protocol for phoA expression above. For
post-minicell and/or protoplast purification induction experiments,
following minicell purification and/or protoplast preparation and
purification, these cellular bodies were induced for protein
production in either LBD or MDT at a minicell or protoplast/volume
ratio of 1.times.10.sup.9 minicells or protoplasts/1 ml media.
Media was supplemented with the appropriate inducer concentration
(see Table 6). Protein induction is sensitive to a variety of
factors including, but not limited to aeration and temperature,
thus reaction volume to surface area ratio is important, as is the
method of shaking and temperature of induction. Therefore, each
protein must be treated as required to optimize expression. In
addition to expression parameters, protoplasted minicells are
sensitive to osmotic and mechanical forces. Therefore, protoplast
protein induction reactions must also contain 10% sucrose with
greater volume to surface area ratios than required for intact
minicells to achieve similar aeration at lower revolutions.
[1079] Using the T-PhoA as a non-limiting example, protein
expression was performed during and following minicell isolation.
To accomplish this task, t-phoA co-expressed with minicell
induction was compared to t-phoA expressed after minicell
isolation. In both cases, overnight minicell-producing parental
strains containing pMPX-5::t-phoA were subcultured into LBD
supplemented with the appropriate antibiotic. Cultures were grown
to OD.sub.600 0.1 and induced for minicell production alone or for
both minicell and protein production. Both cultures were harvested
at OD.sub.600 1.0 and minicells produced were harvested as
described above. Minicells to be induced for T-phoA production
following purification were induced by introducing 1.times.10.sup.9
purified minicells into a 15 ml culture tube containing 1 ml MDT
with 1 mM L-rhamnose. Minicell protein induction was allowed to
proceed for up to 14 hours and compared to protein production
obtained using the co-expression approach. For each approach,
minicells were fractionated and analyzed for membrane association,
total protein, and membrane association-dependent enzymatic
activity. These observations were compared to post-induction,
pre-isolation parental cell/minicell (PC/MC) mixtures from the
co-expressed reactions. The first observation was that
co-expression of minicell and protein induction was superior to
post-minicell purification induction (Table 18). However, although
the kinetics are slower for the post-minicell purification
induction protocol, the end result is equivalent.
TABLE-US-00020 TABLE 18 COMPARATIVE EXPRESSION: CO-EXPRESSION
VERSUS POST MINICELL PURIFICATION INDUCTION Time of Purified
minicell Co-expression induction induction .sup.a induction .sup.a
1.0 8.0 -- 2.0 -- 812.2 4.0 70.0 -- 14.0 445.0 -- .sup.a Nanogram
expressed T-PhoA per 1 .times. 10.sup.9 minicells.
[1080] Using the co-expression induction procedure, the amount of
membrane-associated T-PhoA was measured and compared for both
parental cells and minicells. Briefly, following co-expression
induction of T-PhoA and minicells, minicells were purified and
their membranes isolated. For membrane isolation, minicells
containing expressed T-PhoA were subjected to three rounds of
freeze-thaw lysis in the presence of 10 .mu.g/ml lysozyme.
Following freeze-thaw cycling, the reaction was subjected to
sonication. Sonicated material was centrifuged at 6,000 rpm in a
microcentrifuge for 5 min at room temperature. Supernatants were
transferred to a fresh 1.5 ml Eppendorf tube and centrifuged at
70,000 rpm using a TLA-100 rotor. Following centrifugation, the
pellet was resuspended in buffer and analyzed for total T-PhoA
protein (Table 19) and T-PhoA enzyme activity (Table 20).
TABLE-US-00021 TABLE 19 MEMBRANE ASSOCIATED T-PHOA: PARENTAL CELLS
VERSUS MINICELLS Protein T-PhoA mem- mem- T-PhoA T- T- brane brane
% membrane Protein PhoA PhoA asso- asso- protein Cell type .sup.a
total .sup.a total .sup.b % total ciated .sup.a ciated .sup.b total
Parental 107.5 5.3 4.9 10.7 3.1 29.0 cells Minicells 4.6 0.8 17.5
1.0 0.5 50.0 Minicells 25.2 4.4 -- 5.5 2.7 -- EQ .sup.b .sup.a
Total protein as determined by BCA assay (Pierce) .sup.b Microgram
expressed T-PhoA per 1 .times. 10.sup.9 minicells as determined via
Western using an anti-PhoA antibody (Sigma) versus a PhoA standard
curve (BCA determined). .sup.c Equivalent membrane lipid to
parental cell
TABLE-US-00022 TABLE 20 PHOA ENZYMATIC ACTIVITY.sup.a (RELATIVE
UNITS): PARENTAL CELLS VERSUS MINICELLS. Cell type .sup.b Unlysed
Lysed, total Lysed, membrane Parent cell -- 358 240 Minicell 275
265 211 Minicell EQ .sup.c 1,504 1,447 1,154 .sup.aActivity
determined colorimetrically using PNPP measuring optical density at
405 nm .sup.b Based on 1 .times. 10.sup.9 parental cells or
minicells per reaction .sup.c Equivalent membrane lipid to parental
cell
[1081] These results suggest that co-expression induction of T-PhoA
and minicells together results in minicells containing an
equivalent amount of T-PhoA produced in both parental cells and
minicells. However, the percent of T-PhoA compared to total protein
is 3.5.times. greater in minicells than in parental cells.
Furthermore, of the protein made, T-PhoA constitutes 50% of the
total membrane protein in minicells, whereas it is only 29% in
parental cells. It should be noted that the T-PhoA protein
associated with the membrane can be easily removed by treatment
with mild, non-ionic detergent suggesting that the T-PhoA present
in the membrane pellet is indeed associated with the membrane and
not an insoluble, co-sedimenting precipitate (data not shown).
Finally, PhoA is a periplasmic enzyme that requires export to the
periplasmic space for proper folding and disulfide bond formation.
Both of which are required for enzymatic activity. In the time
course of this experiment, expression of .DELTA.PhoA lacking a
leader sequence does not demonstrate enzymatic activity.
Furthermore, there is no difference between unlysed and lysed
minicells containing expressed T-PhoA (Table 20) also demonstrating
that the PhoA enzyme domain of the T-PhoA chimera must be present
in the periplasmic space. Therefore, the T-PhoA construct must
membrane associate and the PhoA domain must orient into the
periplasmic space for enzymatic activity. Thus, when comparing
equivalent amounts of membrane lipid between parental cells and
minicells in Table 20, membrane association-dependent T-PhoA
activity is almost 5.times. greater than in parental cells. Taking
into account the data in Table 19 where 50% of T-PhoA is in the
membrane compared to 29% in parental cells, the difference in
T-PhoA membrane association is not sufficient to explain the almost
5.times. increase in minicell activity. These observations suggest
that minicells contain a capacity to support more expressed
membrane protein than parental cells and that the protein that
associates with the membrane is more active. This activity may be
simply result from minicells allowing greater efficiency of folding
and disulfide bond formation for this particular protein. However,
do to the fact that minicells do not contain chromosome, it is also
possible that the overexpression of this protein is readily finding
membrane-binding sites in the absence of chromosomally produced
competitors present in parental cells. Furthermore, overexpression
of proteins often leads to increased protease expression. Because
minicells do not contain chromosome, these otherwise degraded
surplus T-PhoA is allowed the continued opportunity to insert and
properly fold in the membrane, an attribute that could lend favor
to overexpression of more complex membrane proteins.
Example 18
Exemplary Methods to Induce and Study Complex Membrane Proteins
[1082] Expression of non-native (exogenous) complex membrane
proteins in bacterial systems can be difficult. Using the minicell
system, we are able to eliminate toxicity issues. However, issues
still remain with proper translation, compartmentalization at the
membrane, insertion in the membrane and proper folding for native
activity. To account for these potential problems we have
constructed a modular chimeric system that incorporates leader
sequences and chaperone-recognized soluble domains that are native
to our bacterial minicell system. In addition, we created modular
constructs that overexpress the native chaperones groESL and
trigger factor (tig). Finally, we have constructed
minicell-producing strains that contain mutations that effect
protein export and disulfide bond formation. For non-limiting
examples of these constructs see Table 21.
TABLE-US-00023 TABLE 21 NON-LIMITING TOOLS FOR EXOGENOUS COMPLEX
PROTEIN SYNTHESIS AND FUNCTION Residues of SEQ ID Tool Reference
sequence Purpose NO pMPX-5::phoA leader -- 1-48 Membrane 22
targeting pMPX-5::phoA leader -- 1-494 Membrane 23 targeting
pMPX-5::malE leader 1 1-28 Membrane 24 targeting pMPX-5::malE
leader 1 1-370 Membrane 25 targeting pMPX-17 (groESL, tig) -- --
Chaperone 26 pMPX-5::trxA::FLAG 2 2-109 .sup.a Solubility 27 .sup.a
Residues do not include FLAG sequence. References to Table 21. 1.
Grisshammer, R., et al. 1993. Expression of a rat neurotensin
receptor in Escherichia coli. Biochem. J. 295:571-576. 2. Tucker,
J., and R. Grisshammer. 1996. Purification of a rat neurotensin
receptor expressed in Escherichia coli. Biochem. J.
317:891-899.
TABLE-US-00024 TABLE 22 OLIGONUCLEOTIDE PRIMER SEQUENCES FOR TABLE
21 CONSTRUCTS SEQ ID NO.: Primer name 5' to 3' sequence 115 PhoA
lead-1 GTCACGGCCGAGACTTATAGTCGC 116 PhoA lead-2
GGTGTCCGGGCTTTTGTCACAGG 117 PhoA lead-1-PstI
CGCGGCTGCAGATGTCACGGCCGAGACTTATAG TCGC 118 PhoA lead-2-XbaI
CGCGGTCTAGATTCTGGTGTCCGGGCTTTTGTCA CAGG 119 PhoA complete
CAGCCCCAGAGCGGCTTTCATGG 120 PhoA complete-2-XbaI
CGCGGTCTAGATTTCAGCCCCAGAGCGGCTTTCA TGG 121 MalE lead-1
CGCGGCTGCAGATGAAAATAAAAACAGGTGCAC
GCATCCTCGCATTATCCGCATTAACGACGATGAT
GTTTTCCGCCTCGGCTCTCGCCAAAATCTCTAGA CGCGG 122 MalE lead-2
CCGCGTCTAGAGATTTTGGCGAGAGCCGAGGCG GAAAACATCATCGTCGTTAATGCGGATAATGCG
AGGATGCGTGCACCTGTTTTTATTTTCATCTGCA GCCGCG 123 MalE-1
GGTGCACGCATCCTCGCATTATCCGC 124 MalE-2 CGGCATACCAGAAAGCGGACATCTGC
125 MalE-1-PstI CGCGGCTGCAGATGAAAATAAAAACAGGTGCAC
GCATCCTCGCATTATCCGC 126 MalE-2-XbaI
CGCGGTCTAGAACGCACGGCATACCAGAAAGCG GACATCTGC 127 Tig-1
CGCGACAGCGCGCAATAACCGTTCTCG 128 Tig-2 GCTGGTTCATCAGCTCGTTGAAAGTGG
129 Tig-1-NarI GCGCCGGCGCCATACGCGACAGCGCGCAATAAC CGTTCTCG 130
Tig-2-XbaI GGCGCTCTAGATTATTATTACGCCTGCTGGTTCA TCAGCTCGTTGAAAGTGG
131 Gro-1 GGTAGCACAATCAGATTCGCTTATGACGG 132 Gro-2
GCCGCCCATGCCACCCATGCCGCCC 133 Gro-1-XbaI
GCGTCTAGAGGTAGCACAATCAGATTCGCTTAT GACGG 134 Gro-2-HindIII
GGCGCAAGCTTATTATTACATCATGCCGCCCATG CCACCCATGCCGCCC 135 TrxA-1
GCGATAAAATTATTCACCTGACTGACG 136 TrxA-2 GCGTCGAGGAACTCTTTCAACTGACC
137 TrxA-1-Fxa-PstI CGCGGCTGCAGATGATCGAAGCCCGCTCTAGAC
TCGAGAGCGATAAAATTATTCACCTGACTGACG 138 TrxA-2-FLAG-BamHI
CCGCGGGATCCTTATTAATCATCATGATCTTTAT
AATCGCCATCATGATCTTTATAATCCTCGAGCGC
CAGGTTAGCGTCGAGGAACTCTTTCAACTGACC
[1083] Oligonucleotides SEQ ID NOS.:115, 116, 117 and 118 were used
to amplify the phoA leader (residues 1-49) from E. coli chromosomal
DNA. Once amplified, this region was inserted into SEQ ID NO.: 6
(pMPX-5) using PstI and XbaI to create SEQ ID NO.:22.
[1084] Oligonucleotides SEQ ID NOS.:115, 117, 119 and 120 were used
to amplify the complete phoA gene from E. coli chromosomal DNA.
Once amplified, this region was inserted into SEQ ID NO.: 6
(pMPX-5) using PstI and XbaI to create SEQ ID NO.23.
[1085] Oligonucleotides SEQ ID NOS.:121 and 122 were used to
construct the malE leader (residues 1-28) sequence. Once annealed,
this construct was inserted into SEQ ID NO.: 6 (pMPX-5) using PstI
and XbaI to create SEQ ID NO.:24.
[1086] Oligonucleotides SEQ ID NOS.:123, 124, 125 and 126 were used
to amplify the malE expanded leader (residues 1-370) from E. coli
chromosomal DNA. Once amplified, this region was inserted into SEQ
ID NO.: 6 (pMPX-5) using PstI and XbaI to create SEQ ID NO.:25.
[1087] Oligonucleotides SEQ ID NOS.:127, 128, 129 and 130 were used
to amplify the tig control and gene region from E. coli chromosomal
DNA. Once amplified, this region was ligated to the groESL
amplified region below using XbaI prior to insertion into SEQ ID
NO.: 6 (pMPX-5) using NarI (from the tig region) and HindIII (from
the groESL region) to create SEQ ID NO.:26.
[1088] Oligonucleotides SEQ ID NOS.:131, 132, 133 and 134 were used
to amplify the groESL control and gene region from E. coli
chromosomal DNA. Once amplified, this region was ligated to the tig
amplified region above using XbaI prior to insertion into SEQ ID
NO.: 6 (pMPX-5) using Nan (from the tig region) and HindIII (from
the groESL region) to create SEQ ID NO.:26.
[1089] Oligonucleotides SEQ ID NOS.:135, 136, 137 and 138 were used
to amplify trxA (residues 2-109) from E. coli chromosomal DNA and
insert FLAG and Factor Xa sequences. Once amplified, this region
was inserted into SEQ ID NO.: 6 (pMPX-5) using PstI and BamHI to
create SEQ ID NO.:27.
[1090] By way of non-limiting example, the pMPX-5::phoA leader
(residues 1-48), pMPX-5::phoA leader (residues 1-494), pMPX-5::malE
leader (residues 1-28), and pMPX-5::malE leader (residues 1-370)
constructs are designed to direct expressed exogenous membrane
proteins to the minicell cytoplasmic membrane. In addition to these
constructs, By way of non-limiting example, mutations in E. coli
genes secA and secY, specifically mutation prlA4 (Strader, J., et
al. 1986. Kinetic analysis of lamB mutants suggests the signal
sequence plays multiple roles in protein export. J. Biol. Chem.
261:15075-15080), permit promiscuous targeting to the membrane.
These mutations, like the above constructs are integrated into the
minicell expression system. To complement these mutations, the
chaperone complex groESL and trigger factor have also been
incorporated into the expression system. By way of non-limiting
example, pMPX-5::trxA::FLAG will be used to create a
carboxy-terminal fusion to the protein of interest to increase the
membrane insertion efficiency of the membrane protein of interest
(Tucker, J., and R. Grisshammer. 1996. Purification of a rat
neurotensin receptor expressed in Escherichia coli. Biochem. J.
317:891-899). Also By way of non-limiting example,
pMPX-5::FLAG::toxR and pMPX-5::FLAG::.lamda.cI constructs will be
prepared to create a carboxy-terminal fusion to the protein of
interest for use in a reporter-based assay for protein-protein
interactions. By way of non-limiting example, the protein of
interest for this system is a GPCR. Also By way of non-limiting
example, this GPCR may be the neurotensin receptor from rat
(Grisshammer, R., et al. 1993. Expression of a rat neurotensin
receptor in Escherichia coli. Biochem. J. 295:571-576.), or the
.beta.2 adrenergic receptor from humans (Freissmuth, M., et al.
1991. Expression of two .beta.-adrenergic receptors in Escherichia
coli: functional interaction with two forms of the stimulatory G
protein. Proc. Natl. Acad. Sci. 88:8548-8552). Insertion of a GPCR
into one of these reporter constructs creates a carboxy-terminal
fusion between the GPCR of interest and the DNA-binding regulatory
domain of the ToxR positive activator, the .lamda.cI repressor, or
the AraC positive activator. To complete this reporter system, By
way of non-limiting example pMPX-5::(X)::toxR or
pMPX-5::(X)::.lamda.cI will be used to create a carboxy-terminal
fusion to the protein of interest for use in a reporter-based assay
for protein-protein interactions, where (X) may be any protein or
molecule involved in an intermolecular or intramolecular
interaction. By way of non-limiting example, this molecule of
interest may be a G-protein. This G-protein may be the
G.alpha..sub.i1-protein from rat (Grisshammer, R., and E. Hermans.
2001. Functional coupling with G.alpha.q and G.alpha.i1 protein
subunits promotes high-affinity agonist binding to the neurotensin
receptor NTS-1 expressed in Escherichia coli. FEBS Lett.
493:101-105), or the G.sub.s.alpha.-protein from human (Freissmuth,
M., et al. 1991. Expression of two .beta.-adrenergic receptors in
Escherichia coli: functional interaction with two forms of the
stimulatory G protein. Proc. Natl. Acad. Sci. 88:8548-8552) Like
the GPCR, insertion of a G-protein into one of these reporter
constructs creates a carboxy-terminal fusion between the G-protein
of interest and the DNA-binding regulatory domain of the ToxR
positive activator, the .lamda.cI repressor, or other regulatory
protein. Finally, these plasmid constructs contain the DNA-binding
domain of each regulator; the ctx regulatory region from Vibrio
cholerae (Russ, W. P., and D. M. Engelman. 1999. TOXCAT: a measure
of transmembrane helix association in a biological membrane.
96:863-868), or the P.sub.R1O.sub.R1 region of bacteriophage lambda
(Hu, J. C., et al. 1990. Sequence requirements for coiled-coils:
analysis with lambda repressor-GCN4 leucine zipper fusions.
Science. 250:1400-1403), respectively. By way of non-limiting
example, each binding domain is coupled to a reporter sequence
encoding luciferase (Dunlap, P. V., and E. P. Greenberg. 1988.
Control of Vibrio fischeri lux gene transcription by a cyclic AMP
receptor protein-luxR protein regulatory circuit. J. Bacteriol.
170:4040-4046), green fluorescent protein (GFP) (Yang, T. T., et
al. 1996. Dual color microscopic imagery of cells expressing the
green fluorescent protein and a red-shifted variant. Gene.
173:19-23; Matthys se, A. G., et al. 1996. Construction of GFP
vectors for use in gram-negative bacteria other than Escherichia
coli. FEMS Microbiol. Lett. 145:87-94), or other reporter.
Co-expression of these GPCR and G-protein chimeras will create a
system measuring the interaction between a GPCR and G-protein
within an intact minicell. This system is designed to be used as a
positive or negative read-out assay and may be used to detect loss
or gain of GPCR function. Although the GPCR-G-protein interaction
is provided as an example, this modular system may be employed with
any soluble or membrane protein system measuring protein-protein or
other intermolecular interaction.
Example 19
Exemplary Methods for Gene Transfer Using Minicells or Minicell
Protoplasts
[1091] Included in the design of the invention is the use of
minicells to transfer genetic information to a recipient cell. By
way of non-limiting example, this gene transfer may occur between a
minicell and a mammalian cell in vitro, or in vivo, and this gene
transfer may occur through cell-specific interactions, through
general interactions, or a combination of each. To accomplish this
task three basic constructs were created. Each of these constructs
is created in pMPX-6 which contains a CMV promotor controlling the
synthesis of GFP. The plasmid pMPX-6 was constructed by cloning the
araC through the multiple cloning site of pBAD24 into pEGFP
(Clontech). This construct provided a bacterial regulator as well
as a method to monitor the success of gene transfer using GFP
expression form the CMV promotor. In design, the protein expressed
using the bacterial promotor will drive the cell-cell interaction,
while the successful transfer of DNA from the minicell to the
recipient cell will initiate the production of GFP. By way of
non-limiting example, proteins that will drive the cell-cell
interaction may be the invasin protein from Yersinia
pseudotuberculosis, which stimulates 131 integrin-dependent
endocytic events. To properly display the invasin protein on the
surface of minicells, the domain of invasin that stimulates these
events (residues 490-986) (Dersch, P., and R. R. Isberg. 1999. A
region of the Yersinia pseudotuberculosis invasin protein enhances
integrin-mediated uptake into mammalian cells and promotes
self-association. EMBO J. 18:1199-1213) was fused to the
transmembrane domain of ToxR. Expression of this construct from
pMPX-6 will display T-Inv on the surface of the minicell and
stimulate endocytosis with any cell displaying a .beta.1 integrin.
Thus, T-Inv display will provide a general mechanism of gene
transfer from minicells. To provide specificity, By way of
non-limiting example, the ligand portion of epidermal growth factor
(EGF) may be fused to the transmembrane domain of ToxR, thus
creating a protein that will interact with cells displaying the EGF
receptor (EGFR). Likewise, tumor nucrosis factor (TNF) may also
serve this purpose by stimulating cell-cell interactions between
minicells displaying TNF and cells displaying TNF receptor (TNFR).
Although EGF-EGFR and TNF-TNFR interactions may stimulate cell-cell
fusion between minicells and recipient cells, or minicell uptake,
this alone may not be sufficient to efficiently transfer genetic
information from minicells. Therefore, a genetic approach to
increasing the cell-cell genetic transfer may be the development of
a genetic switch that senses the specificity interaction, e.g.
EGF-EGFR interaction, and turns on the production of a second gene
product, e.g. invasin, that stimulates the endocytic event. By way
of non-limiting example, this genetic switch may be similar to the
GPCR-G-protein interaction reporter system above, in that an
extracellular event stimulates the dimerization of a
transcriptional active regulator, thus turning on the production of
invasin or invasin-like protein. In either approach, the display
system to stimulate transfer of genetic information from minicells
to recipient cells may also be applicable to the transfer of
substances other than genetic information, e.g. pre-synthesized
therapeutic drugs.
[1092] To test this targeting methodology, different pMPX-6
constructs containing each of these general or specific cell-cell
interaction proteins will be transformed into a minicell producing
strain and either by co-expression induction of minicells, by
post-minicell purification induction, or by post-protoplasting
induction, minicells displaying the targeting protein of interest
will be produced. When using the co-expression induction and
post-minicell purification induction of the targeting protein
approaches, it is necessary to protoplast the purified minicells
after protein induction. Once the targeting protein has been
displayed on the surface of a minicell protoplast, these
protoplasts are ready to be exposed to target cells. For
preliminary experiments these interactions will be monitored using
cell culture of Cos cells in comparison to lipofectamine
(Invitrogen), electroporation, and other transfection techniques.
Initial experiments will expose protoplasts displaying T-Inv to Cos
cells and compare the transfection efficiency to protoplast
containing pMPX-6::t-inv in the absence of t-inv expression, naked
pMPX-6::t-inv alone, and naked pMPX-6::t-inv with lipofectamine.
Each of these events will be monitored using fluorescent microscopy
and/or flow cytometry. From these results the specific targeting
apparatus proteins will be tested. Using A-431 (display EGFR) and
K-562 (no EGFR) cell lines, the pMPX-6::t-egf constructs will be
tested. Using the same approaches as for the t-inv study, the level
of transfection between A-431 and K-562 cell lines will be measured
and compared to those achieved using lipofectamine. Similarly, the
ability of TNF to stimulate gene transfer will be studied using
L-929 cells. In all cases, the ability of these general and
specific targeting protein constructs will be compared to standard
transfection techniques. Upon positive results, these methodologies
will be tested on difficult to transfect cell lines, e.g. adult
cardiomyocytes. The basis of these results will create a foundation
for which applications into in vivo gene transfer may occur.
Example 20
Additional and Optimized Methods for Genetic Expression
[1093] Expression in minicells may occur following purification of
minicells and/or minicell protoplasts from parental cells and LPS
constituents, respectively. However, for some applications it is
preferred to co-express proteins of interest with minicell
induction. For these approaches, one may use the protocol described
in Example 13 for expression of the phoA constructs. Either of
these approaches may be accomplished using one or more of the
following expression constructs (Table 23) and/or optimized
expression constructs (Table 25).
[1094] Expression plasmid pCGV1 contains a temperature sensitive
lambda cI repressor (cI857) and both lambda PR and PL promoters
(Guzman, C. A., et al. 1994. A novel Escherichia coli
expression-export vector containing alkaline phosphatase as an
insertional inactivation screening system. Gene. 148:171-172) with
an atpE initiation region (Schauder, B., et al. 1987. Inducible
expression vectors incorporating the Escherichia coli atpE
translational initiation region. Gene. 52:279-283). Included in the
design of the invention is the modification of this expression
vector to best align the required Shine-Delgarno ribosomal binding
site with cloning sites. In addition, the pCGVI expression vector
was modified to incorporate a stem-loop structure at the 3-prime
end of the transcript in order to provide a strong transcriptional
stop sequence (Table 23).
[1095] Expression plasmid pCL478 contains a temperature sensitive
lambda cI repressor (cI857) and both lambda PR and PL promoters
(Love, C. A., et al. 1996. Stable high-copy bacteriophage promoter
vectors for overproduction of proteins in Escherichia coli. Gene.
176:49-53). Included in the design of the invention is the
modification of this expression vector to best align the required
Shine-Delgarno ribosomal binding site with cloning sites. In
addition, the pCL478 expression vector was modified to incorporate
a stem-loop structure at the 3-prime end of the transcript in order
to provide a strong transcriptional stop sequence (Table 23).
TABLE-US-00025 TABLE 23 LAMBDA CI857 EXPRESSION VECTOR
MODIFICATIONS New Parent Region Plasmid plasmid removed Region
added .sup.a SEQ ID NO pMPX-84 pCGV1 NdeI - BamHI NdeI, SD - PstI,
XbaI, KpnI, Stem-loop, BamHI 139 pMPX-85 pCGV1 NdeI - BamHI NdeI,
SD - SalI, XbaI, KpnI, Stem-loop, BamHI 140 pMPX-86 pCL478 BamHI -
XhoI BamHI, SD - PstI, XbaI, KpnI, Stem-loop, XhoI 141 pMPX-87
pCL478 BamHI - XhoI BamHI, SD - SalI, XbaI, KpnI, Stem-loop, XhoI
142 .sup.a "SD" refers to a Shine-Delgarno ribosome-binding
sequence; "Stem-loop" refers to a stem-loop structure that
functions as a transcriptional stop site.
TABLE-US-00026 TABLE 24 OLIGONUCLEOTIDE PRIMER SEQUENCES FOR TABLE
23 SEQ ID NO Primer name 5' to 3' sequence 143 CGV1-1-SalI
TATGTAAGGAGGTTGTCGACCGGCTCAGTCTAGAGGTACCCGCCCTCA TCCGAAAGGGCGTATTG
144 CGV1-2-SalI GATCCAATACGCCCTTTCGGATGAGGGCGGGTACCTCTAGACTGAGCC
GGTCGACAACCTCCTTACA 145 CGV1-1-PstI
TATGTAAGGAGGTTCTGCAGCGGCTCAGTCTAGAGGTACCCGCCCTCA TCCGAAAGGGCGTATTG
146 CGV1-2-PstI GATCCAATACGCCCTTTCGGATGAGGGCGGGTACCTCTAGACTGAGCC
GCTGCAGAACCTCCTTACA 147 CL478-1-SalI
GATCCTAAGGAGGTTGTCGACCGGCTCAGTCTAGAGGTACCCGCCCTC ATCCGAAAGGGCGTATTC
148 CL478-2-SalI TCGAGAATACGCCCTTTCGGATGAGGGCGGGTACCTCTAGACTGAGCC
GGTCGACAACCTCCTTAG 149 CL478-1-PstI
GATCCTAAGGAGGTTCTGCAGCGGCTCAGTCTAGAGGTACCCGCCCTC ATCCGAAAGGGCGTATTC
150 CL478-2-PstI TCGAGAATACGCCCTTTCGGATGAGGGCGGGTACCTCTAGACTGAGCC
GCTGCAGAACCTCCTTAG
[1096] Oligonucleoides SEQ ID NOS.: 143 and 144 were annealed to
each other to generate a DNA molecule with a 5' overhang at both
ends. The overhangs are designed so that the DNA can be directly
cloned into pCGVI cut with NdeI (5' overhang is TA) and BamHI (5'
overhang is GATC). Insertion of the annealed DNA into pCGVI creates
SEQ ID NO.: 139, pMPX-84.
[1097] Oligonucleoides SEQ ID NOS.: 145 and 146 were annealed to
each other to generate a DNA molecule with a 5' overhang at both
ends. The overhangs are designed so that the DNA can be directly
cloned into pCGVI cut with NdeI (5' overhang is TA) and BamHI (5'
overhang is GATC). Insertion of the annealed DNA into pCGVI creates
SEQ ID NO.: 140, pMPX-85.
[1098] Oligonucleoides SEQ ID NOS.: 147 and 148 were annealed to
each other to generate a DNA molecule with a 5' overhang at both
ends. The overhangs are designed so that the DNA can be directly
cloned into pCL478 cut with BamHI (5' overlap is GATC) and XhoI
(overhang is TCGA). Insertion of the annealed DNA into pCL578 cut
with BamHI and XhoI creates SEQ ID NO.: 141, pMPX-86.
[1099] Oligonucleoides SEQ ID NOS.: 149 and 150 were annealed to
were annealed to each other to generate a DNA molecule with a 5'
overhang at both ends. The overhangs are designed so that the DNA
can be directly cloned into pCL578 cut with BamHI (5' overlap is
GATC) and XhoI (overhang is TCGA). Insertion of the annealed DNA
into pCL478 cut with BamHI and XhoI creates SEQ ID NO.: 142,
pMPX-87.
[1100] The optimized expression constructs in Table 25 were created
from SEQ ID NOS.: 6, 11, and 12 (see Table 13). Modifications were
made to optimize the alignment of the SalI or PstI cloning sites
with the Shine-Delgarno ribosome-binding site. In addition,
stem-loop transcriptional termination sequences were added on the
3' end of the cloning region.
TABLE-US-00027 TABLE 25 EXPRESSION CONSTRUCTS Regulatory SEQ ID
Plasmid element(s) inducer Plasmid NO.: pMPX-67 RhaRS Rhamnose
PUC-18 151 pMPX-72 RhaRS Rhamnose PUC-18 152 pMPX-66 AraC Arabinose
PUC-18 153 pMPX-71 AraC Arabinose PUC-18 154 pMPX-68 MelR Melibiose
PUC-18 155
TABLE-US-00028 TABLE 26 OLIGONUCLEOTIDE PRIMER SEQUENCES FOR TABLE
25 CONSTRUCTS SEQ ID NO.: Primer name 5' to 3' sequence 69 Rha-1
GCGAATTGAGATGACGCCACTGGC 156 Rha-SD GCAGAACCTCCTGAATTTCATTACGACC 71
Rha-1-HindIII CGGCGAAGCTTAATTAATCTTTCTGCGAATTGAGATGA CGCCACTGGC 157
Rha-SD SalI CCGCGGGTACCAATACGCCCTTTCGGATGAGGGCGCG KpnI
GGGATCCTCTAGAGTCGACGTCGACAACCTCCTGAATT TCATTACGACC 158 Rha-SD KpnI
CCGCGGGTACCAATACGCCCTTTCGGATGAGGGCGCG KpnI
GGGATCCTCTAGAGTCGACCTGCAGAACCTCCTGAATT TCATTACGACC 81 Ara-1
CAAGCCGTCAATTGTCTGATTCG 159 Ara-SD
CTGCAGGGCCTCCTGCTAGCCCAAAAAAACGGGTATGG 83 Ara-1-HindIII
GCGCCAAGCTTCAAGCCGTCAATTGTCTGATTCG 160 Ara-SD SalI
CCGCGGGTACCAATACGCCCTTTCGGATGAGGGCGCG KpnI
GGGATCCTCTAGAGTCGACGTCGACGGCCTCCTGCTAG CCCAAAAAAACGGGTATGG 161
Ara-SD PstI CCGCGGGTACCAATACGCCCTTTCGGATGAGGGCGCG KpnI
GGGATCCTCTAGAGTCGACCTGCAGGGCCTCCTGCTAG CCCAAAAAAACGGGTATGG 77 Mel-1
CGTCTTTAGCCGGGAAACG 162 Mel-SD CCTCCTGGCTTGCTTGAATAACTTCATCATGG 79
Mel-1-HindIII GCCGCAAGCTTCGTCTTTAGCCGGGAAACG 163 Mel-SD-SalI
CCGCGGGTACCAATACGCCCTTTCGGATGAGGGCGCG KpnI
GGGATCCTCTAGAGTCGACCCCCTCCTGGCTTGCTTGA ATAACTTCATCATGGC
[1101] Oligonucleotides SEQ ID NOS.: 69, 156, 72, and 157 were used
to amplify the rhaRS genes and their divergent control region from
the E. coli chromosome and insertion of an optimized
SalI-Shine-Delgarno ribosome-binding alignment and a stem-loop
transcriptional termination sequence. Once amplified, this region
was inserted into pUC18 using HindIII and KpnI to create pMPX67,
SEQ ID NO.: 151.
[1102] Oligonucleotides SEQ ID NOS.: 69, 156, 72, and 158 were used
to amplify the rhaRS genes and their divergent control region from
the E. coli chromosome and insertion of an optimized
PstI-Shine-Delgarno ribosome-binding alignment and a stem-loop
transcriptional termination sequence. Once amplified, this region
was inserted into pUC18 using HindIII and KpnI to create, pMPX-72,
SEQ ID NO.: 152.
[1103] Oligonucleotides SEQ ID NOS.: 81, 159, 81, 160 were used to
amplify the araC genes and their divergent control region from the
E. coli chromosome and insertion of an optimized
SalI-Shine-Delgarno ribosome-binding alignment and a stem-loop
transcriptional termination sequence. Once amplified, this region
was inserted into pUC18 using HindIII and KpnI to create, pMPX-66,
SEQ ID NO.: 153.
[1104] Oligonucleotides SEQ ID NOS.: 81, 159, 81, 161 were used to
amplify the araC genes and their divergent control region from the
E. coli chromosome and insertion of an optimized
PstI-Shine-Delgarno ribosome-binding alignment and a stem-loop
transcriptional termination sequence. Once amplified, this region
was inserted into pUC18 using HindIII and KpnI to createm pMPX-71,
SEQ ID NO.: 154.
[1105] Oligonucleotides SEQ ID NOS.: 77, 162, 79, 163 were used to
amplify the melR genes and their divergent control region from the
E. coli chromosome and insertion of an optimized
SalI-Shine-Delgarno ribosome-binding alignment and a stem-loop
transcriptional termination sequence. Once amplified, this region
was inserted into pUC18 using HindIII and KpnI to create, pMPX-68,
SEQ ID NO.: 155.
Example 21
Optimization of Rat Neurotensin Receptor(NTR) Expression
[1106] Expression of specific GPCR proteins in minicells may
require chimeric domain fusions to stabilize the expressed protein
and/or direct the synthesized protein to the membrane. The NTR
protein from rat was cloned into several chimeric combinations to
assist in NTR expression and membrane association (Grisshammer, R.,
et al. 1993. Expression of a rat neurotensin receptor in
Escherichia coli. Biochem. J. 295:571-576; Tucker, J., and
Grisshammer, R. 1996. Purification of a rat neurotensin receptor
expressed in Escherichia coli. Biochem. J. 317:891-899). Methods
for construction are shown the Tables below.
TABLE-US-00029 TABLE 27 NEUROTENSIN RECEPTOR EXPRESSION
FACILITATING CONSTRUCTS SEQ ID Protein .sup.a Construct .sup.b NO
MalE(L) SalI-MalE (1-370)-Factor Xa-NTR homology 164 NTR Factor
Xa-NTR (43-424)-NotI-FLAG-KpnI 165 MalE(L)- SalI-MalE(1-370)-Factor
Xa-NTR(43-424)- 166 NTR NotI-FLAG-KpnI MalE(S)-
SalI-MalE(1-28)-Factor Xa-NTR(43-424)- 167 NTR NotI-FLAG-KpnI TrxA
NotI-TrxA(2-109)-NotI 168 MalE(L)- SalI-MalE(1-370)-Factor
Xa-NTR(43-424)- 169 NTR-TrxA NotI-TrxA(2-109)-FLAG-KpnI MalE(S )-
SalI-MalE(1-28)-Factor Xa-NTR(43-424)- 170 NTR-TrxA
NotI-TrxA(2-109)-FLAG-KpnI .sup.a (L) refers to MalE residues
1-370, and (S) refers to MalE residues 1-28. .sup.b All mature
constructs were cloned into SalI and KpnI sites of SEQ ID NOS.:
140, 142, 151 and 153.
TABLE-US-00030 TABLE 28 OLIGONUCLEOTIDE PRIMER SEQUENCES FOR TABLE
27 SEQ ID NO.: Primer name 5' to 3' sequence 171 MalE-1
GGTGCACGCATCCTCGCATTATCCGC 172 MalE-2
CGCACGGCATACCAGAAAGCGGACATCTGCG 173 MalE-1-SalI
CCGCGGTCGACATGAAAATAAAAACAGGTGCACGCATCCTCGC 174 MalE-2-XaNTR
GCCGTGTCGGATTCCGAGGTGCGGCCTTCGATACGCACGGCAT ACCAAGAAAGCGGGATGTTCGGC
175 NTR-1 CCTCGGAATCCGACACGGCAGGGC 176 NTR-2
GTACAGGGTCTCCCGGGTGGCGCTGG 177 NTR-1-Xa
CCGCGATCGAAGGCCGCACCTCGGAATCCGACACGGCAGGGCC 178 NTR-2-Flag
GGCGCGGTACCTTTGTCATCGTCATCTTTATAATCTGCGGCCGC
GTACAGGGTCTCCCGGGTGGCGCTGGTGG 179 NTR-2-Stop
GCGGCGGTACCTTATTATTTGTCATCGTCATCTTTATAATCTGC KpnI GGCCGCG 180
NTR-1-Xa Lead CCGCATTAACGACGATGATGTTTTCCGCCTCGGCTCTCGCCAAA
ATCATCGAAGGCCGCACCTCGGAATCCGACACGGC 181 NTR-2-Lead2
CCGCGGTCGACATGAAAATAAAAACAGGTGCACGCATCCTCGC SalI
ATTATCCGCATTAACGACGATGATGTTTTCCGCCTCGGC 182 TrxA-1
CCGCGAGCGATAAAATTATTCACCTGACTGACG 183 TrxA-2
GCCCGCCAGGTTAGCGTCGAGGAACTCTTTCAACTGACC 184 TrxA-1-NotI
GCGGCCGCAAGCGATAAAATTATTCACCTGACTGACG 185 TrxA-2-NotI
GGCGCTGCGGCCGCATCATCATGATCTTTATAATCGCC
[1107] Oligonucleotides SEQ ID NOS.: 171, 172, 173 and 174 were
used to amplify malE residues 1-370 from the E. coli chromosome to
create SEQ ID NO.: 164. Using overlap PCR with the extended NTR
homology, a chimeric translational fusion was made between MalE
(1-370) and NTR residues 43-424 (SEQ ID NO.: 165) to create a SEQ
ID NO.: 166. SEQ ID NO.: 166 was cloned into plasmids pMPX-85,
pMPX-87, pMPX-66 and pMPX-67 (respectively, SEQ ID NOS.: 140, 142,
151 and 153) using SalI and KpnI.
[1108] Three-step PCR with oligonucleotides, SEQ ID NOS.: 175 and
176 as primers was used to amplify NTR residues 43-424 from rat
brain cDNA. SEQ ID NOS.: 177 and 178 were then used with the NTR
(43-424) template to add factor Xa and FLAG sequence. Finally, SEQ
ID NOS.: 177 and 179 were used to add a KpnI site to create SEQ ID
NO.: 165. Using overlap PCR with malE(1-370) containing extended
NTR homology, a chimeric translational fusion was made between NTR
(43-424) and MalE (1-370) (SEQ ID NO.: 164) to create a SEQ ID NO.:
166. SEQ ID NO.: 166 was cloned into SEQ ID NOS.: 140, 142, 151 and
153 using SalI and KpnI.
[1109] Using three-step PCR oligonucleotides SEQ ID NOS.: 175 and
176 were first used to amplify NTR residues 43-424 from rat brain
cDNA. SEQ ID NOS.: 178 and 180 were then used with the NTR (43-424)
template to add factor Xa and FLAG sequence. Finally, SEQ ID NOS.:
179 and 181 were used to add KpnI to create SEQ ID NO.: 167. SEQ ID
NO.: 167 was cloned into SEQ ID NOS.: 140, 142, 151 and 153 using
SalI and KpnI.
[1110] Oligonucleotides SEQ ID NOS.: 182, 183, 184 and 185 were
used to amplify TrxA residues 2-109 from the E. coli chromosome to
create SEQ ID NO.: 168. Using NotI, TrxA residues 2-109 was cloned
into SEQ ID NOS.: 166 and 167 to create SEQ ID NOS.: 169 and 170,
respectively. SEQ ID NO.: 169 and 170 were cloned into SEQ ID NOS.:
140, 142, 151 and 153 using SalI and KpnI.
Example 22
Methods for Functional GPCR Assay
[1111] Functional G-protein-coupled receptor (GPCR) binding assays
in minicells requires expression of a GPCR of interest into the
minicell membrane bilayer and cytoplasmic expression of the
required G-protein. For these purposes, constructs were created to
co-express both a GPCR and a G-protein. To regulate the ratio of
GPCR to G-protein, transcriptional fusions were created. In these
constructs, the GPCR and G-protein are co-transcribed as a
bi-cistronic mRNA. To measure the GPCR-G-protein interaction in the
intact minicell, each protein was created as a chimera with a
transactivation domain. For these studies the N-terminal
DNA-binding, activation domain of the ToxR protein from V. cholerae
was fused to the C-terminus of both the GPCR and G-protein.
Finally, to measure the interaction GPCR-G-protein interaction, the
ToxR-activated ctx promoter region was cloned in front of lacZ.
Dimerization of the ToxR DNA-binding region will bind and activate
the ctx promoter. In this construct, heterodimerization of the GPCR
and G-protein will promote dimerization of ToxR that will be
monitored by LacZ expression. Details of these constructs are shown
in Table 29.
TABLE-US-00031 TABLE 29 FUNCTIONAL HUMAN GPCR CONSTRUCTS SEQ ID
Protein .sup.a, b Construct .sup.a, b NO.: .beta.2AR
SalI-.beta.2AR-PstI, XhoI 186 GS1.alpha. XhoI-GS1.alpha.-XbaI 187
.beta.2AR-GS1.alpha. fusion SalI-.beta.2AR-PstI,
XhoI-GS1.alpha.-XbaI 188 .beta.2AR-stop
SalI-.beta.2AR-PstI-Stop-SD-XhoI 189 .beta.2AR-stop-GS1.alpha.
SalI-.beta.2AR-PstI-Stop-SD-XhoI-GS1.alpha.-XbaI 190 ToxR
ClaI-ToxR-XbaI 191 GS1.alpha. XhoI-GS1.alpha.-ClaI 192 GS2.alpha.
XhoI-GS2.alpha.-C1aI 193 G.alpha.q XhoI-Gq.alpha.-ClaI 194
Gi.alpha. XhoI-Gi.alpha.-ClaI 195 G.alpha.12/13
XhoI-G.alpha.12/13-ClaI 196 GS1.alpha.-ToxR
XhoI-GS1.alpha.-ClaI-ToxR-XbaI 197 GS2.alpha.-ToxR
XhoI-GS2.alpha.-ClaI-ToxR-XbaI 198 G.alpha.q-ToxR XhoI- G.alpha.q
-ClaI-ToxR-XbaI 199 Gi.alpha.-ToxR XhoI-Gi.alpha.-ClaI-ToxR-XbaI
200 G.alpha.12/13-ToxR XhoI- G.alpha.12/13-ClaI-ToxR-XbaI 201 ToxR
PstI-ToxR-XhoI 202 .beta.2AR SalI-.beta.2AR-PstI 203 .beta.2AR-ToxR
SalI-.beta.2AR-PstI-ToxR-Stop-SD-XhoI 204 .beta.2AR-ToxR- stop-
SalI-.beta.2AR-PstI-ToxR-Stop-SD-XhoI- 205 GS1.alpha.-ToxR
GS1.alpha.-ClaI-ToxR-XbaI Pctx XbaI-Pctx-lacZ homology 206 lacZ
Pctx homology-lacZ-XbaI 207 Pctx::lacZ XbaI-Pctx-lacZ-XbaI 208
.sup.a "SD" refers to the Shine-Delgarno ribosome-binding sequence
and "ToxR" refers to the transactivation, DNA-binding domain of the
ToxR protein (residues 5-141). .sup.b All mature constructs were
cloned into SalI and XbaI sites of SEQ ID NOS.: 140, 142, 151 and
153.
TABLE-US-00032 TABLE 30 OLIGONUCLEOTIDE PRIMER SEQUENCES FOR TABLE
29. SEQ ID NO.: Primer name 5' to 3' sequence 209 .beta.2AR-1
GGGGCAACCCGGGAACGGCAGCGCC 210 .beta.2AR-2
GCAGTGAGTCATTTGTACTACAATTCCTCC 211 .beta.2AR-1-SalI
CGCGGTCGACATGGGGCAACCCGGGAACGGCAGCGCC 212 .beta.2AR-2-Link-
GGCTCGAGCTGCAGGTTGGTGACCGTCTGGCCACGCTC XhoI
TAGCAGTGAGTCATTTGTACTACAATTCC 213 GS1.alpha.-1
GGGCTGCCTCGGGAACAGTAAGACCGAGG 214 GS1.alpha.-2
GAGCAGCTCGTACTGACGAAGGTGCATGC 215 GS1.alpha.-1-XhoI
GGAGGCCCTCGAGATGGGCTGCCTCGGGAACAGTAAG ACCGAGG 216 GS1.alpha.-2-XbaI
CCTCTAGATTATTATCGATGAGCAGCTCGTACTGACGA AGGTGCATGC 217
GS1.alpha.-2-ClaI CCATCGATGAGCAGCTCGTACTGACGAAGGTGCATGC 218
G.alpha.12-1 CCGGGGTGGTGCGGACCCTCAGCCGC 219 G.alpha.12-2
CTGCAGCATGATGTCCTTCAGGTTCTCC 220 G.alpha.12-1-XhoI
GCGGGCTCGAGATGTCCGGGGTGGTGCGGACCCTCAGC CGC 221 G.alpha.12-2-ClaI
GCGCCATCGATCTGCAGCATGATGTCCTTCAGGTTCTCC 222 G.alpha.q-1
GACTCTGGAGTCCATCATGGCGTGCTGC 223 G.alpha.q-2
CCAGATTGTACTCCTTCAGGTTCAACTGG 224 G.alpha.q-1-XhoI
ATGACTCTGGAGTCCATCATGGCGTGCTGC 225 G.alpha.q-2-ClaI
GCGCCATCGATGACCAGATTGTACTCCTTCAGGTTCAACTGG 226 Gi.alpha.-1
GGGCTGCACCGTGAGCGCCGAGGACAAGG 227 Gi.alpha.-2
CCTTCAGGTTGTTCTTGATGATGACATCGG 228 Gi.alpha.-1-XhoI
ATGGGCTGCACCGTGAGCGCCGAGGACAAGG 229 Gi.alpha.-2-ClaI
GCGCCATCGATGAAGAGGCCGCAGTCCTTCAGGTTGTTCTTGA TGATGACATCGG 230
GS2.alpha.-1 GGGCTGCCTCGGGAACAGTAAGACCGAGG 231 GS2.alpha.-2
GAGCAGCTCGTACTGACGAAGGTGCATGC 232 GS2.alpha.-1-XhoI
ATGGGCTGCCTCGGGAACAGTAAGACCGAGG 233 GS2.alpha.-2-ClaI
GCGCCATCGATGAGCAGCTCGTACTGACGAAGGTGCATGC 234 .beta.2AR-2-Link-
GGCTCGAGGGCCTCCTTGATTATTACTCGAGGGCCTCC Stop-XhoI
TTGATTATTACTGCAGGTTGGTGACCGTCTGGCCACGC
TCTAGCAGTGAGTCATTTGTACTACAATTCC 235 .beta.2AR-2-Link
CCCTGCAGGTTGGTGACCGTCTGGCCACGCTCTAGCAG TGAGTCATTTGTACTACAATTCC 236
Tox (5-141)- GGACACAACTCAAAAGAGATATCGATGAGTCATATTGG 1B 237 Tox
(5-141)-2 GAGATGTCATGAGCAGCTTCGTTTTCGCG 238 Tox (5-141)-
GCGTGGCCAGACGGTCACCAACCTGCAGGGACACAAC 1-Link TCAAAAGAGATATCG 239
Tox (5-141)- CGGGGATCCTCTAGATTATTAAGAGATGTCATGAGCAG 2-XhoI
CTTCGTTTTCGCG 240 Ctx-1 GGCTGTGGGTAGAAGTGAAACGGGGTTTACCG 241 Ctx-2
CTTTACCATATAATGCTCCCTTTGTTTAACAG 242 Ctx-2-XbaI
CGCGGTCTAGAGGCTGTGGGTAGAAGTGAAACGGGGT TTACCG 243 Ctx-2-LacZ
CGACGGCCAGTGAATCCGTAATCATGGTCTTTACCATA TAATGCTCCCTTTGTTTAACAG 244
LacZ-1 CCATGATTACGGATTCACTGGCCGTCG 245 LacZ-2
CCAGACCAACTGGTAATGGTAGCGACC 246 LacZ-1-Ctx
GGTAAAGACCATGATTACGGATTCACTGGCCGTCG 247 LacZ-2-XbaI
GCGCCTCTAGAAATACGCCCTTTCGGATGAGGGCGTT
ATTATTTTTGACACCAGACCAACTGGTAATGGTAGCG ACC
[1112] Oligonucleotides SEQ ID NOS.: 209, 210, 211 and 212 were
used to amplify human .beta.2AR from human cDNA to create SEQ ID
NO.: 186. Using SalI and XhoI a translational fusion was made
between .beta.2AR and human GS1.alpha. (SEQ ID NO.: 187) to create
a SEQ ID NO.: 188. SEQ ID NO.: 188 was cloned into SEQ ID NOS.:
140, 142, 151 and 153 using SalI and XbaI.
[1113] Oligonucleotides SEQ ID NOS.: 213, 214, 215 and 216 were
used to amplify human GS1.alpha. from human cDNA to create SEQ ID
NO.: 187. Using XhoI and XbaI a translational fusion was made
between GS1.alpha. and human .beta.2AR (SEQ ID NO.: 186) create SEQ
ID NO.: 188. SEQ ID NO.: 188 was cloned into SEQ ID NOS.: 140, 142,
151 and 153 using SalI and XbaI.
[1114] Oligonucleotides SEQ ID NOS.: 213, 214, 215 and 217 were
used to amplify human GS1.alpha. from human cDNA to create SEQ ID
NO.: 192. Using XhoI and XbaI a translational fusion was made with
ToxR residues 5-141 from Vibrio cholerae (SEQ ID NO.: 191) to
create SEQ ID NO.: 197. To be used to create a transcriptional
fusion with .beta.2AR-ToxR chimeras as shown in SEQ ID NO.: 205 and
future GPCR-ToxR chimeras.
[1115] Oligonucleotides SEQ ID NOS.: 218, 219, 220 and 221 were
used to amplify human G.alpha.12/13 from human cDNA to create SEQ
ID NO.: 196. Using XhoI and XbaI a translational fusion was made
with ToxR residues 5-141 from Vibrio cholerae (SEQ ID NO.: 191) to
create SEQ ID NO.: 201. To be used to create future transcriptional
fusions with GPCR-ToxR chimeras as shown in SEQ ID NO.: 205.
[1116] Oligonucleotides SEQ ID NOS.: 222, 223, 224 and 225 were
used to amplify human G.alpha.q from human cDNA to create SEQ ID
NO.: 194. Using XhoI and XbaI a translational fusion was made with
ToxR residues 5-141 from Vibrio cholerae (SEQ ID NO.: 191) to
create SEQ ID NO.: 199. To be used to create future transcriptional
fusions with GPCR-ToxR chimeras as shown in SEQ ID NO.: 205.
[1117] Oligonucleotides SEQ ID NOS.: 226, 227, 228 and 229 were
used to amplify human Gi.alpha. from human cDNA to create SEQ ID
NO.: 195. Using XhoI and XbaI a translational fusion was made with
ToxR residues 5-141 from Vibrio cholerae (SEQ ID NO.: 191) to
create SEQ ID NO.: 200. To be used to create future transcriptional
fusions with GPCR-ToxR chimeras as shown in SEQ ID NO.: 205.
[1118] Oligonucleotides SEQ ID NOS.: 230, 231, 232 and 233 were
used to amplify human GS2.alpha. from human cDNA to create SEQ ID
NO.: 193. Using XhoI and XbaI a translational fusion was made with
ToxR residues 5-141 from Vibrio cholerae (SEQ ID NO.: 191) to
create SEQ ID NO.: 198. To be used to create future transcriptional
fusions with GPCR-ToxR chimeras as shown in SEQ ID NO.: 205.
[1119] Oligonucleotides SEQ ID NOS.: 209, 210, 211 and 234 were
used to amplify human .beta.2AR from human cDNA to create SEQ ID
NO.: 189. Using SalI and XhoI a transcriptional fusion was made
between .beta.2AR and human GS1.alpha. (SEQ ID NO.: 187) to create
a SEQ ID NO.: 190. SEQ ID NO.: 190 was cloned into SEQ ID NOS.:
140, 142, 151 and 153 using SalI and XbaI.
[1120] Oligonucleotides SEQ ID NOS.: 236, 237, 238 and 239 were
used to amplify bases coinciding with ToxR residues 5-141 from
Vibrio Cholerae to create SEQ ID NO.: 202. Using PstI and XhoI a
translational fusion was made between ToxR and human .beta.2AR (SEQ
ID NO.: 203) to create SEQ ID NO.: 204.
[1121] Oligonucleotides SEQ ID NOS.: 209, 210, 211 and 235 were
used to amplify human .beta.2AR from human cDNA to create SEQ ID
NO.: 203. Using SalI and PstI a translational fusion was made
between .beta.2AR and ToxR (SEQ ID NO.: 202) to create SEQ ID NO.:
204.
[1122] Using oligonucleotides SEQ ID NOS.: 197 and 204
transcriptional fusions were created between the .beta.2AR-ToxR
translational fusion (SEQ ID NO.: 204) and the GS1.alpha.-ToxR
translational fusion (SEQ ID NO.: 197) to create SEQ ID NO.:
205.
[1123] Oligonucleotides SEQ ID NOS.: 240, 241, 242 and 243 were
used to amplify the ctx promoter region (Pctx) from Vibrio cholerae
to create SEQ ID NO.: 206. Combining this PCR product in
combination with the SEQ ID NO.: 207 PCR product and amplifying in
the presence of SEQ ID NOS.: 242, 247, SEQ ID NO.: 208 was created.
Using XbaI, the SEQ ID NO.: 208 reporter construct was subsequently
cloned into pACYC184 for co-transformation with the GPCR-G-protein
fusions constructs above.
[1124] Oligonucleotides SEQ ID NOS.: 244, 245, 246 and 247 were
used to amplify the lacZ from E. coli to create SEQ ID NO.: 207.
Combining this PCR product in combination with the SEQ ID NO.: 206
PCR product and amplifying in the presence of SEQ ID NOS.: 242 and
247, SEQ ID NO.: 208 was created. Using XbaI, the 208 reporter
construct was subsequently cloned into pACYC184 for
co-transformation with the GPCR-G-protein fusions constructs
above.
Example 23
Modular Membrane-Targeting and Solubilization Expression
Constructs
[1125] To produce membrane proteins efficiently in minicells it may
be necessary to create chimeric fusions with the membrane protein
of interest. In this Example,various regions of the MalE protein
have been cloned into a modular expression system designed to
create chimeric fusions with direct difficult to target membrane
proteins to produce leader domains that will direct the proteins to
the cytoplasmic membrane (Miller, K., W., et al. 1998. Production
of active chimeric pediocin AcH in Escherichia coli in the absence
of processing and secretion genes from the Pediococcus pap operon.
Appl. Environ. Microbiol. 64:14-20). Similarly, a modified version
of the TrxA protein has also been cloned into this modular
expression system to create chimeric fusions with proteins that are
difficult to maintain in a soluble conformation (LaVallie, E. R.,
et al. 1993. A thioredoxin gene fusion expression system that
circumvents inclusion body formation in the E. coli cytoplasm.
Biotechnology (N.Y.) 11:187-193). Table 31 describes each of these
modular constructs.
TABLE-US-00033 TABLE 31 MODULAR MEMBRANE-TARGETING AND
SOLUBILIZATION EXPRESSION CONSTRUCTS SEQ ID Protein .sup.a
Construct .sup.a NO MalE (1-28) NsiI-MalE(1-28)-Factor Xa-PstI, 248
SalI, XbaI-FLAG, NheI MalE (1-370, del NsiI-MalE(1-370, del
354-364)-Factor 249 354-364) Xa-PstI, SalI, XbaI-FLAG, NheI TrxA
(2-109, del PstI, SalI, XbaI-TrxA(2-109, del 250 103-107)
103-107)-FLAG-NheI MalE (1-28)-TrxA NsiI-MalE(1-28)-Factor Xa-PstI,
SalI, 251 (2-109, del 103-107) XbaI-TrxA (2-109 del 103-107)-FLAG,
NheI MalE (1-370, del NsiI-MalE(1-370, del 354-364)-Factor 252
354-364)-TrxA (2- Xa-PstI, SalI, XbaI-TrxA (2-109 del 109, del
103-107) 103-107)-FLAG, NheI .sup.a The term "del" refers to a
deletion in which amino acid residues following the term "del" are
removed from the sequence.
TABLE-US-00034 TABLE 32 OLIGONUCLEOTIDE PRIMER SEQUENCES FOR TABLE
31. SEQ ID NO.: Primer name 5' to 3' sequence 253 MalE-1-NsiI
CGCGGATGCATATGAAAATAAAAACAGGTGCACGCATCCTCG
CATTATCCGCATTAACGACGATGATGTTTTCCGCCTCGGCTCTC GCC 254 MalE-2-
CGTCGACCGAGGCCTGCAGGCGGGCTTCGATGATTTTGGCGAG middle
AGCCGAGGCGGAAAACATCATCGTCG 255 MalE-3s-NheI
CGAAGCCCGCCTGCAGGCCTCGGTCGACGCCGAATCTAGAGA
TTATAAAGATGACGATGACAAATAATAAGCTAGCGGCGC 256 MalE-4-NheI
GCGCCGCTAGCTTATTATTTGTCATCG 257 MalE-1a GGTGCACGCATCCTCGCATTATCCGC
258 MalE-2a GGCGTTTTCCATGGTGGCGGCAATACGTGG 259 MalE-1-NsiI
CGCGGATGCATATGAAAATAAAAACAGGTGCACGCATCCTC GCATTATCCGC 260
MalE-2-NheI CCGAGGCCTGCAGGCGGGCTTCGATACGCACGGCATACCAG
AAAGCGGACTGGGCGTTTTCCATGGTGGCGGCAATACGTGG 261 MalE-3L-NheI
GCGCCGCTAGCTTATTATTTGTCATCGTCATCTTTATAATCTC
TAGATTCGGCGTCGACCGAGGCCTGCAGGCGGGCTTCGATA CGC 262 TrxA-1a
CCTGACTGACGACAGTTTTGACACGG 263 TrxA-2a
CCTTTAGACAGTGCACCCACTTTGGTTGCCGC 264 TrxA-1a-PstI
CGCGGCTGCAGGCCTCGGTCGACGCCGAATCTAGAAGCGAT
AAAATTATTCACCTGACTGACGACAGTTTTGACACGG 265 TrxA-2-NheI
GCGCCGCTAGCTTATTATTTGTCATCGTCATCTTTATAATCCG
CCAGGTTCTCTTTCAACTGACCTTTAGACAGTGCACCCACTTT GGTTGCCGC
[1126] Oligonucleotides SEQ ID NOS.: 253, 254, 255 and 256 overlap
with each other to form a scaffold template to PCR amplify malE
(1-28) to create a SEQ ID NO.: 248. Following PCR amplification,
SEQ ID NO.: 248 was digested with NsiI and NheI and cloned into SEQ
ID NOS.: 152, 154, 139 and 141 digested with PstI and XbaI. The
resultant products create SEQ ID NOS.: 266, 267, 268 and 269,
respectively, that lose both the 5-prime PstI and 3-prime XbaI
restriction site and retain the PstI, SalI, and XbaI restriction
sites between MalE (1-28) and the FLAG sequence. Insertion of a
protein in alignment with these sites results in a chimeric protein
containing amino-terminal MalE (1-28) and carboxy-terminal
FLAG.
[1127] Oligonucleotides SEQ ID NOS.: 257, 258, 259 and 260 were
used to amplify malE (1-370 with a deletion removing residues
354-364) to create SEQ ID NO.: 249. Following PCR amplification,
SEQ ID NO.: 249 was digested with NsiI and NheI and cloned into SEQ
ID NOS.: 152, 154, 139 and 141 digested with PstI and XbaI. The
resultant products create SEQ ID NOS.: 270, 271, 272 and 273,
respectively, that lose both the 5-prime PstI and 3-prime XbaI
restriction site and retain the PstI, SalI, and XbaI restriction
sites between MalE (1-370, del 354-364) and the FLAG sequence.
Insertion of a protein in alignment with these sites results in a
chimeric protein containing amino-terminal MalE (1-370, del
354-364) and carboxy-terminal FLAG.
[1128] Oligonucleotides SEQ ID NOS.: 262, 263, 264 and 265 were
used to amplify trxA (2-109 with a deletion removing residues
103-107) to create SEQ ID NO.: 250. Following PCR amplification,
SEQ ID NO.: 250 was digested with PstI and NheI and cloned into SEQ
ID NOS.: 152, 154, 139 and 141 digested with PstI and XbaI. to
create SEQ ID NOS.: 274, 275, 276 and 277, respectively. Using
these restriction digestion combinations results in loss of the
XbaI SEQ ID NO.: 249 insertion site.
[1129] The resultant products create SEQ ID NOS.: 274, 275, 276 and
277, respectively, that lose the 3-prime XbaI restriction site and
retain the PstI, SalI, and XbaI restriction sites on the 3-prime
end of the TrxA (1-109, del 103-107) sequence. Insertion of a
protein in alignment with these sites results in a chimeric protein
containing Carboxy-terminal TrxA (1-109, del 103-107)-FLAG.
[1130] SEQ ID NO.: 248 was digested with NsiI and XbaI and cloned
into SEQ ID NOS.: 274, 275, 276 and 277 that were digested with
PstI and XbaI. The resultant products create SEQ ID NOS.: 278, 279,
280 and 281, respectively, that lose the 5 prime PstI restriction
site and retain the PstI, SalI, and XbaI restriction sites between
MalE (1-28) and TrxA (1-109, del 103-107). Insertion of a protein
in alignment with these sites results in a chimeric protein
containing amino-terminal MalE (1-28) and carboxy-terminal TrxA
(1-109, del 103-107)-FLAG.
[1131] SEQ ID NO.: 249 was digested with NsiI and XbaI and cloned
into SEQ ID NOS.: 274, 275, 276 and 277 that were digested with
PstI and XbaI. The resultant products create SEQ ID NOS.: 282, 283,
284 and 285, respectively, that lose the 5 prime PstI restriction
site and retain the PstI, SalI, and XbaI restriction sites between
MalE (1-370, del 354-364) and TrxA (1-109, del 103-107). Insertion
of a protein in alignment with these sites results in a chimeric
protein containing amino-terminal MalE (1-370, del 354-364) and
carboxy-terminal TrxA (1-109, del 103-107)-FLAG.
Example 24
Poroplast.TM. Formation
[1132] Minicells are used to prepare Poroplasts in order to
increase the accessibility of a membrane protein component and/or
domain to the outside environment. Membrane proteins in the inner
membrane are accessible for ligand binding and/or other
interactions in poroplasts, due to the absence of an outer
membrane. The removal of the outer membrane from E. coli whole
cells and minicells to produce poroplasts was carried out using
modifications of previously described protoplast and analysis
protocols (Birdsell et al., Production and Ultrastructure of
Lysozyme and Ethylenediaminetetraacetate-Lysozyme Spheroplasts of
Escherichia coli, J. Bacteriol. 93:427-437, 1967; Weiss et al.,
Protoplast Formation in Escherichia Coli, J. Bacteriol.
128:668-670, 1976; Matsuyama, S-I., et al. SecD is involved in the
release of translocated secretory proteins from the cytoplasmic
membrane of Escherichia coli. 12:265-270, 1993).
[1133] In brief, cells were grown to late-log phase and pelleted at
room temperature. Minicells were also isolated from cultures in
late-log phase. The pellet was washed twice with 50 mM Tris, pH
8.0. Following the second wash, 1.times.10.sup.9 cells were
resuspended in 1 ml 50 mM Tris (pH 8.0) that contained 8% sucrose
and 2 mM EDTA. Cell/EDTA/sucrose mixtures were incubated at
37.degree. C. for 10 min, centrifuged, decanted, and poroplasted
cells were resuspended in 50 mM Tris, pH 8.0 with 8% sucrose.
Incubation with anti-LPS-coated magnetic beads, as described in
Example 14, is used to enrich for poroplasts that lack LPS.
Following incubation with the resuspended protoplasted cells, the
anti-LPS magnetic beads were removed from suspension with a
magnet.
[1134] To examine the range of molecular sizes that can pass
through the cell wall, an IgG molecule was tested for its ability
to pass the intact cell wall. Binding of an antibody to the
ToxR-PhoA chimera expressed on the inner membrane minicell
poroplasts was measured. Briefly, minicell poroplasts with and
without inner membrane-bound ToxR-PhoA were incubated at 37.degree.
C. with anti-PhoA antibody in reaction buffer (50 mM Tris, pH 8.0,
8% sucrose, 1% BSA, and 0.01% Tween-20). Following incubation,
poroplasts were centrifuged, washed 3 times with reaction buffer,
and resuspended in 50 mM Tris, pH 8.0 with 8% sucrose. Following
resuspension, bound proteins from 5.times.10.sup.7 minicells or
minicell poroplasts were separated using denaturing SDS-PAGE,
transferred to nitrocellulose, and developed using with both
anti-PhoA antibody and secondary antibody against both heavy and
light chains of anti-PhoA IgG (Table 33).
TABLE-US-00035 TABLE 33 ANTI-PHOA ACCESSIBILITY TO POROPLAST INNER
MEMBRANE-BOUND TOXR-PHOA EDTA (mM) 0 2 0 2 Lysozyme (mg/ml) 0 0 5 5
Poroplasts (ng Protoplasts (ng antibody bound) antibody bound)
Minicells ToxR- ND .sup.a 0.6 ND .sup.a 12.8 PhoA Minicells only ND
.sup.a ND .sup.a ND .sup.a ND .sup.a .sup.a Non-detectable
[1135] These results demonstrate that the cell wall present on
poroplasts is penetrable by an IgG molecule and that an IgG
molecule is capable of passing the intact cell wall and binding to
an inner membrane protein. From this data it appears that poroplast
operate at .about.10% the efficiency of protoplasts by allowing 0.6
ng of IgG to bind inner membrane-bound ToxR-PhoA compared to 12.8
ng. However, given the large size of IgG (.about.150,000 Daltons)
it is expected that molecules having a smaller molecular weight
will efficiently access inner membrane proteins in poroplasts.
Example 25
Production of Neurotensin Receptor (NTR)
[1136] To demonstrate expression of NTR in isolated minicells,
Ma1E(L)-NTR (SEQ ID NO.: 166 was cloned into pMPX-67 (SEQ ID NO.:
151). Following minicell isolation, 1.5.times.10.sup.9 minicells
were induced with 1 mM Rhamnose for 2 hour at 37.degree. C.
Following induction, the protein produced was visualized via
Western analysis using an anti-MalE antibody following separation
on an SDS-PAGE. The results are shown in FIG. 2.
[1137] These data demonstrates that Ma1E(L)-NTR is induced 87-fold
by addition of 1 mM rhamnose to the minicell induction mixture.
Cross-reactive proteins are host MalE and non-specific binding by
Goat-anti-mouse HRP secondary antibody.
[1138] The contents of the articles, patents, and patent
applications, and all other documents and electronically available
information mentioned or cited herein, are hereby incorporated by
reference in their entirety to the same extent as if each
individual publication was specifically and individually indicated
to be incorporated by reference. Applicants reserve the right to
physically incorporate into this application any and all materials
and information from any such articles, patents, patent
applications, or other documents.
[1139] The inventions illustratively described herein may suitably
be practiced in the absence of any element or elements, limitation
or limitations, not specifically disclosed herein. Thus, for
example, the terms "comprising", "including," containing", etc.
shall be read expansively and without limitation. Additionally, the
terms and expressions employed herein have been used as terms of
description and not of limitation, and there is no intention in the
use of such terms and expressions of excluding any equivalents of
the features shown and described or portions thereof, but it is
recognized that various modifications are possible within the scope
of the invention claimed. Thus, it should be understood that
although the present invention has been specifically disclosed by
preferred embodiments and optional features, modification and
variation of the inventions embodied therein herein disclosed may
be resorted to by those skilled in the art, and that such
modifications and variations are considered to be within the scope
of this invention.
[1140] The invention has been described broadly and generically
herein. Each of the narrower species and subgeneric groupings
falling within the generic disclosure also form part of the
invention. This includes the generic description of the invention
with a proviso or negative limitation removing any subject matter
from the genus, regardless of whether or not the excised material
is specifically recited herein.
[1141] Other embodiments are within the following claims. In
addition, where features or aspects of the invention are described
in terms of Markush groups, those skilled in the art will recognize
that the invention is also thereby described in terms of any
individual member or subgroup of members of the Markush group.
TABLE-US-00036 Lengthy table referenced here
US20120142079A1-20120607-T00001 Please refer to the end of the
specification for access instructions.
TABLE-US-LTS-00001 LENGTHY TABLES The patent application contains a
lengthy table section. A copy of the table is available in
electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20120142079A1).
An electronic copy of the table will also be available from the
USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 370 <210> SEQ ID NO 1 <211> LENGTH: 1260
<212> TYPE: DNA <213> ORGANISM: E. coli <400>
SEQUENCE: 1 ccatacccgt ttttttgggc tagcaggagg aattcaccct gcagatgttt
gaaccaatgg 60 aacttaccaa tgacgcggtg attaaagtca tcggcgtcgg
cggcggcggc ggtaatgctg 120 ttgaacacat ggtgcgcgag cgcattgaag
gtgttgaatt cttcgcggta aataccgatg 180 cacaagcgct gcgtaaaaca
gcggttggac agacgattca aatcggtagc ggtatcacca 240 aaggactggg
cgctggcgct aatccagaag ttggccgcaa tgcggctgat gaggatcgcg 300
atgcattgcg tgcggcgctg gaaggtgcag acatggtctt tattgctgcg ggtatgggtg
360 gtggtaccgg tacaggtgca gcaccagtcg tcgctgaagt ggcaaaagat
ttgggtatcc 420 tgaccgttgc tgtcgtcact aagcctttca actttgaagg
caagaagcgt atggcattcg 480 cggagcaggg gatcactgaa ctgtccaagc
atgtggactc tctgatcact atcccgaacg 540 acaaactgct gaaagttctg
ggccgcggta tctccctgct ggatgcgttt ggcgcagcga 600 acgatgtact
gaaaggcgct gtgcaaggta tcgctgaact gattactcgt ccgggtttga 660
tgaacgtgga ctttgcagac gtacgcaccg taatgtctga gatgggctac gcaatgatgg
720 gttctggcgt ggcgagcggt gaagaccgtg cggaagaagc tgctgaaatg
gctatctctt 780 ctccgctgct ggaagatatc gacctgtctg gcgcgcgcgg
cgtgctggtt aacatcacgg 840 cgggcttcga cctgcgtctg gatgagttcg
aaacggtagg taacaccatc cgtgcatttg 900 cttccgacaa cgcgactgtg
gttatcggta cttctcttga cccggatatg aatgacgagc 960 tgcgcgtaac
cgttgttgcg acaggtatcg gcatggacaa acgtcctgaa atcactctgg 1020
tgaccaataa gcaggttcag cagccagtga tggatcgcta ccagcagcat gggatggctc
1080 cgctgaccca ggagcagaag ccggttgcta aagtcgtgaa tgacaatgcg
ccgcaaactg 1140 cgaaagagcc ggattatctg gatatcccag cattcctgcg
taagcaagct gattaataat 1200 ctagaggatc cccgggtacc gagctcgaat
tcgtaatcat ggtcatagct gtttcctgtg 1260 <210> SEQ ID NO 2
<211> LENGTH: 1260 <212> TYPE: DNA <213>
ORGANISM: E. coli <400> SEQUENCE: 2 gaattcaggc gctttttaga
ctggtcgtaa tgaaattcag caggatcaca ttctgcagat 60 gtttgaacca
atggaactta ccaatgacgc ggtgattaaa gtcatcggcg tcggcggcgg 120
cggcggtaat gctgttgaac acatggtgcg cgagcgcatt gaaggtgttg aattcttcgc
180 ggtaaatacc gatgcacaag cgctgcgtaa aacagcggtt ggacagacga
ttcaaatcgg 240 tagcggtatc accaaaggac tgggcgctgg cgctaatcca
gaagttggcc gcaatgcggc 300 tgatgaggat cgcgatgcat tgcgtgcggc
gctggaaggt gcagacatgg tctttattgc 360 tgcgggtatg ggtggtggta
ccggtacagg tgcagcacca gtcgtcgctg aagtggcaaa 420 agatttgggt
atcctgaccg ttgctgtcgt cactaagcct ttcaactttg aaggcaagaa 480
gcgtatggca ttcgcggagc aggggatcac tgaactgtcc aagcatgtgg actctctgat
540 cactatcccg aacgacaaac tgctgaaagt tctgggccgc ggtatctccc
tgctggatgc 600 gtttggcgca gcgaacgatg tactgaaagg cgctgtgcaa
ggtatcgctg aactgattac 660 tcgtccgggt ttgatgaacg tggactttgc
agacgtacgc accgtaatgt ctgagatggg 720 ctacgcaatg atgggttctg
gcgtggcgag cggtgaagac cgtgcggaag aagctgctga 780 aatggctatc
tcttctccgc tgctggaaga tatcgacctg tctggcgcgc gcggcgtgct 840
ggttaacatc acggcgggct tcgacctgcg tctggatgag ttcgaaacgg taggtaacac
900 catccgtgca tttgcttccg acaacgcgac tgtggttatc ggtacttctc
ttgacccgga 960 tatgaatgac gagctgcgcg taaccgttgt tgcgacaggt
atcggcatgg acaaacgtcc 1020 tgaaatcact ctggtgacca ataagcaggt
tcagcagcca gtgatggatc gctaccagca 1080 gcatgggatg gctccgctga
cccaggagca gaagccggtt gctaaagtcg tgaatgacaa 1140 tgcgccgcaa
actgcgaaag agccggatta tctggatatc ccagcattcc tgcgtaagca 1200
agctgattaa taatctagag gatccccggg taccgagctc gaattcgtaa tcatggtcat
1260 <210> SEQ ID NO 3 <211> LENGTH: 2544 <212>
TYPE: DNA <213> ORGANISM: E. coli <400> SEQUENCE: 3
aagcctgcat tgcggcgctt cagtctccgc tgcatactgt cccgttacca attatgacaa
60 cttgacggct acatcattca ctttttcttc acaaccggca cggaactcgc
tcgggctggc 120 cccggtgcat tttttaaata cccgcgagaa atagagttga
tcgtcaaaac caacattgcg 180 accgacggtg gcgataggca tccgggtggt
gctcaaaagc agcttcgcct ggctgatacg 240 ttggtcctcg cgccagctta
agacgctaat ccctaactgc tggcggaaaa gatgtgacag 300 acgcgacggc
gacaagcaaa catgctgtgc gacgctggcg atatcaaaat tgctgtctgc 360
caggtgatcg ctgatgtact gacaagcctc gcgtacccga ttatccatcg gtggatggag
420 cgactcgtta atcgcttcca tgcgccgcag taacaattgc tcaagcagat
ttatcgccag 480 cagctccgaa tagcgccctt ccccttgccc ggcgttaatg
atttgcccaa acaggtcgct 540 gaaatgcggc tggtgcgctt catccgggcg
aaagaacccc gtattggcaa atattgacgg 600 ccagttaagc cattcatgcc
agtaggcgcg cggacgaaag taaacccact ggtgatacca 660 ttcgcgagcc
tccggatgac gaccgtagtg atgaatctct cctggcggga acagcaaaat 720
atcacccggt cggcaaacaa attctcgtcc ctgatttttc accaccccct gaccgcgaat
780 ggtgagattg agaatataac ctttcattcc cagcggtcgg tcgataaaaa
aatcgagata 840 accgttggcc tcaatcggcg ttaaacccgc caccagatgg
gcattaaacg agtatcccgg 900 cagcagggga tcattttgcg cttcagccat
acttttcata ctcccgccat tcagagaaga 960 aaccaattgt ccatattgca
tcagacattg ccgtcactgc gtcttttact ggctcttctc 1020 gctaaccaaa
ccggtaaccc cgcttattaa aagcattctg taacaaagcg ggaccaaagc 1080
catgacaaaa acgcgtaaca aaagtgtcta taatcacggc agaaaagtcc acattgatta
1140 tttgcacggc gtcacacttt gctatgccat agcattttta tccataagat
tagcggatcc 1200 tacctgacgc tttttatcgc aactctctac tgtttctcca
tacccgtttt tttgggctag 1260 caggaggaat tcaccctgca gatgtttgaa
ccaatggaac ttaccaatga cgcggtgatt 1320 aaagtcatcg gcgtcggcgg
cggcggcggt aatgctgttg aacacatggt gcgcgagcgc 1380 attgaaggtg
ttgaattctt cgcggtaaat accgatgcac aagcgctgcg taaaacagcg 1440
gttggacaga cgattcaaat cggtagcggt atcaccaaag gactgggcgc tggcgctaat
1500 ccagaagttg gccgcaatgc ggctgatgag gatcgcgatg cattgcgtgc
ggcgctggaa 1560 ggtgcagaca tggtctttat tgctgcgggt atgggtggtg
gtaccggtac aggtgcagca 1620 ccagtcgtcg ctgaagtggc aaaagatttg
ggtatcctga ccgttgctgt cgtcactaag 1680 cctttcaact ttgaaggcaa
gaagcgtatg gcattcgcgg agcaggggat cactgaactg 1740 tccaagcatg
tggactctct gatcactatc ccgaacgaca aactgctgaa agttctgggc 1800
cgcggtatct ccctgctgga tgcgtttggc gcagcgaacg atgtactgaa aggcgctgtg
1860 caaggtatcg ctgaactgat tactcgtccg ggtttgatga acgtggactt
tgcagacgta 1920 cgcaccgtaa tgtctgagat gggctacgca atgatgggtt
ctggcgtggc gagcggtgaa 1980 gaccgtgcgg aagaagctgc tgaaatggct
atctcttctc cgctgctgga agatatcgac 2040 ctgtctggcg cgcgcggcgt
gctggttaac atcacggcgg gcttcgacct gcgtctggat 2100 gagttcgaaa
cggtaggtaa caccatccgt gcatttgctt ccgacaacgc gactgtggtt 2160
atcggtactt ctcttgaccc ggatatgaat gacgagctgc gcgtaaccgt tgttgcgaca
2220 ggtatcggca tggacaaacg tcctgaaatc actctggtga ccaataagca
ggttcagcag 2280 ccagtgatgg atcgctacca gcagcatggg atggctccgc
tgacccagga gcagaagccg 2340 gttgctaaag tcgtgaatga caatgcgccg
caaactgcga aagagccgga ttatctggat 2400 atcccagcat tcctgcgtaa
gcaagctgat taataatcta gaggcgttac caattatgac 2460 aacttgacgg
gaagttccta tactttctag agaataggaa cttcccaaag ccagtatcaa 2520
ctcagacaaa ggcaaagcat cttg 2544 <210> SEQ ID NO 4 <211>
LENGTH: 3350 <212> TYPE: DNA <213> ORGANISM: E. coli
<400> SEQUENCE: 4 aagcctgcat tgcggcgctt cagtctccgc tgcatactgt
ccttaatctt tctgcgaatt 60 gagatgacgc cactggctgg gcgtcatccc
ggtttcccgg gtaaacacca ccgaaaaata 120 gttactatct tcaaagccac
attcggtcga aatatcactg attaacaggc ggctatgctg 180 gagaagatat
tgcgcatgac acactctgac ctgtcgcaga tattgattga tggtcattcc 240
agtctgctgg cgaaattgct gacgcaaaac gcgctcactg cacgatgcct catcacaaaa
300 tttatccagc gcaaagggac ttttcaggct agccgccagc cgggtaatca
gcttatccag 360 caacgtttcg ctggatgttg gcggcaacga atcactggtg
taacgatggc gattcagcaa 420 catcaccaac tgcccgaaca gcaactcagc
catttcgtta gcaaacggca catgctgact 480 actttcatgc tcaagctgac
cgataacctg ccgcgcctgc gccatcccca tgctacctaa 540 gcgccagtgt
ggttgccctg cgctggcgtt aaatcccgga atcgccccct gccagtcaag 600
attcagcttc agacgctccg ggcaataaat aatattctgc aaaaccagat cgttaacgga
660 agcgtaggag tgtttatcgt cagcatgaat gtaaaagaga tcgccacggg
taatgcgata 720 agggcgatcg ttgagtacat gcaggccatt accgcgccag
acaatcacca gctcacaaaa 780 atcatgtgta tgttcagcaa agacatcttg
cggataacgg tcagccacag cgactgcctg 840 ctggtcgctg gcaaaaaaat
catctttgag aagttttaac tgatgcgcca ccgtggctac 900 ctcggccaga
gaacgaagtt gattattcgc aatatggcgt acaaatacgt tgagaagatt 960
cgcgttattg cagaaagcca tcccgtccct ggcgaatatc acgcggtgac cagttaaact
1020 ctcggcgaaa aagcgtcgaa aagtggttac tgtcgctgaa tccacagcga
taggcgatgt 1080 cagtaacgct ggcctcgctg tggcgtagca gatgtcgggc
tttcatcagt cgcaggcggt 1140 tcaggtatcg ctgaggcgtc agtcccgttt
gctgcttaag ctgccgatgt agcgtacgca 1200 gtgaaagaga aaattgatcc
gccacggcat cccaattcac ctcatcggca aaatggtcct 1260 ccagccaggc
cagaagcaag ttgagacgtg atgcgctgtt ttccaggttc tcctgcaaac 1320
tgcttttacg cagcaagagc agtaattgca taaacaagat ctcgcgactg gcggtcgagg
1380 gtaaatcatt ttccccttcc tgctgttcca tctgtgcaac cagctgtcgc
acctgctgca 1440 atacgctgtg gttaacgcgc cagtgagacg gatactgccc
atccagctct tgtggcagca 1500 actgattcag cccggcgaga aactgaaatc
gatccggcga gcgatacagc acattggtca 1560 gacacagatt atcggtatgt
tcatacagat gccgatcatg atcgcgtacg aaacagaccg 1620 tgccaccggt
gatggtatag ggctgcccat taaacacatg aatacccgtg ccatgttcga 1680
caatcacaat ttcatgaaaa tcatgatgat gttcaggaaa atccgcctgc gggagccggg
1740 gttctatcgc cacggacgcg ttaccagacg gaaaaaaatc cacactatgt
aatacggtca 1800 tactggcctc ctgatgtcgt caacacggcg aaatagtaat
cacgaggtca ggttcttacc 1860 ttaaattttc gacggaaaac cacgtaaaaa
acgtcgattt ttcaagatac agcgtgaatt 1920 ttcaggaaat gcggtgagca
tcacatcacc acaattcagc aaattgtgaa catcatcacg 1980 ttcatctttc
cctggttgcc aatggcccat tttcctgtca gtaacgagaa ggtcgcgaat 2040
tcaggcgctt tttagactgg tcgtaatgaa attcagcagg atcacatatg tttgaaccaa
2100 tggaacttac caatgacgcg gtgattaaag tcatcggcgt cggcggcggc
ggcggtaatg 2160 ctgttgaaca catggtgcgc gagcgcattg aaggtgttga
attcttcgcg gtaaataccg 2220 atgcacaagc gctgcgtaaa acagcggttg
gacagacgat tcaaatcggt agcggtatca 2280 ccaaaggact gggcgctggc
gctaatccag aagttggccg caatgcggct gatgaggatc 2340 gcgatgcatt
gcgtgcggcg ctggaaggtg cagacatggt ctttattgct gcgggtatgg 2400
gtggtggtac cggtacaggt gcagcaccag tcgtcgctga agtggcaaaa gatttgggta
2460 tcctgaccgt tgctgtcgtc actaagcctt tcaactttga aggcaagaag
cgtatggcat 2520 tcgcggagca ggggatcact gaactgtcca agcatgtgga
ctctctgatc actatcccga 2580 acgacaaact gctgaaagtt ctgggccgcg
gtatctccct gctggatgcg tttggcgcag 2640 cgaacgatgt actgaaaggc
gctgtgcaag gtatcgctga actgattact cgtccgggtt 2700 tgatgaacgt
ggactttgca gacgtacgca ccgtaatgtc tgagatgggc tacgcaatga 2760
tgggttctgg cgtggcgagc ggtgaagacc gtgcggaaga agctgctgaa atggctatct
2820 cttctccgct gctggaagat atcgacctgt ctggcgcgcg cggcgtgctg
gttaacatca 2880 cggcgggctt cgacctgcgt ctggatgagt tcgaaacggt
aggtaacacc atccgtgcat 2940 ttgcttccga caacgcgact gtggttatcg
gtacttctct tgacccggat atgaatgacg 3000 agctgcgcgt aaccgttgtt
gcgacaggta tcggcatgga caaacgtcct gaaatcactc 3060 tggtgaccaa
taagcaggtt cagcagccag tgatggatcg ctaccagcag catgggatgg 3120
ctccgctgac ccaggagcag aagccggttg ctaaagtcgt gaatgacaat gcgccgcaaa
3180 ctgcgaaaga gccggattat ctggatatcc cagcattcct gcgtaagcaa
gctgattaat 3240 aatctagagg cgttaccaat tatgacaact tgacgggaag
ttcctatact ttctagagaa 3300 taggaacttc ccaaagccag tatcaactca
gacaaaggca aagcatcttg 3350 <210> SEQ ID NO 5 <211>
LENGTH: 2280 <212> TYPE: DNA <213> ORGANISM: E. coli
<400> SEQUENCE: 5 aagcctgcat tgcggcgctt cagtctccgc tgcatactgt
ccttaataaa gtgagtcgat 60 attgtctttg ttgaccagta ataccttatg
gaaacggata attcgcttat ccatatctac 120 gtcggcctta cccagattct
gcatttctaa tccaggcttg atctcttcac ccttcagcaa 180 cgtgctggcg
acggctgcga gtgcgtaacc tgcagaggcc ggatcgtaag taatcccttc 240
ggtgatatca ccacttttaa tcagtgatgc cgcctgtgaa gggatcatca tgccatagac
300 tgcgacttta tttttcgccc gtttctcttt caccgcacgt cccgcgccaa
tcggaccgtt 360 tgaaccaaag gagacaaccg ctttcaagtc aggataggtt
ttcatcaggt ccagtgtagt 420 acgacgtgag acatccacac tctcggcaac
cggcatgcgg cgggtaactt catgcatatc 480 cgggtaatgc tctttctggt
atttcaccag caagtcagcc cataagttat gctgcggcac 540 ggtcaaacta
cccacgtaaa tcacatagcc gcccttgcca cccatgcgtt tcgccatatg 600
ctcaacatat tcagcggcaa atttttcgtt atcaatgatt tcgatatccc agttagcact
660 tggctgaccg ggggattcgt tggtcagaac cacaattccg gcatctcgcg
cttttttgaa 720 taccggttcc agcacgttgg catcgtttgg cacgatagta
attgcattaa ccttacgggc 780 gattaaatcc tcaataattt taacttgttg
cggagcatca gtacttgaag gccccacctg 840 tgaggcatta acaccaaagg
ctttacccgc ctcaaccaca ccttcgccca tgcgattaaa 900 ccacggcata
ccatcgactt tagaaatatt caccacgact ttttccgctg cctggagcgg 960
cgcagaaatt agcgcagcgc ctaataacag cgaagacacc atattgataa caaaacgttt
1020 attcatcata tggaacttac caatgacgcg gtgattaaag tcatcggcgt
cggcggcggc 1080 ggcggtaatg ctgttgaaca catggtgcgc gagcgcattg
aaggtgttga attcttcgcg 1140 gtaaataccg atgcacaagc gctgcgtaaa
acagcggttg gacagacgat tcaaatcggt 1200 agcggtatca ccaaaggact
gggcgctggc gctaatccag aagttggccg caatgcggct 1260 gatgaggatc
gcgatgcatt gcgtgcggcg ctggaaggtg cagacatggt ctttattgct 1320
gcgggtatgg gtggtggtac cggtacaggt gcagcaccag tcgtcgctga agtggcaaaa
1380 gatttgggta tcctgaccgt tgctgtcgtc actaagcctt tcaactttga
aggcaagaag 1440 cgtatggcat tcgcggagca ggggatcact gaactgtcca
agcatgtgga ctctctgatc 1500 actatcccga acgacaaact gctgaaagtt
ctgggccgcg gtatctccct gctggatgcg 1560 tttggcgcag cgaacgatgt
actgaaaggc gctgtgcaag gtatcgctga actgattact 1620 cgtccgggtt
tgatgaacgt ggactttgca gacgtacgca ccgtaatgtc tgagatgggc 1680
tacgcaatga tgggttctgg cgtggcgagc ggtgaagacc gtgcggaaga agctgctgaa
1740 atggctatct cttctccgct gctggaagat atcgacctgt ctggcgcgcg
cggcgtgctg 1800 gttaacatca cggcgggctt cgacctgcgt ctggatgagt
tcgaaacggt aggtaacacc 1860 atccgtgcat ttgcttccga caacgcgact
gtggttatcg gtacttctct tgacccggat 1920 atgaatgacg agctgcgcgt
aaccgttgtt gcgacaggta tcggcatgga caaacgtcct 1980 gaaatcactc
tggtgaccaa taagcaggtt cagcagccag tgatggatcg ctaccagcag 2040
catgggatgg ctccgctgac ccaggagcag aagccggttg ctaaagtcgt gaatgacaat
2100 gcgccgcaaa ctgcgaaaga gccggattat ctggatatcc cagcattcct
gcgtaagcaa 2160 gctgattaat aatctagagg cgttaccaat tatgacaact
tgacgggaag ttcctattct 2220 ctagaaagta taggaacttc ccaaagccag
tatcaactca gacaaaggca aagcatcttg 2280 <210> SEQ ID NO 6
<211> LENGTH: 4728 <212> TYPE: DNA <213>
ORGANISM: E. coli <400> SEQUENCE: 6 tcgcgcgttt cggtgatgac
ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct
gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc
ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg
caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt
aaaacgacgg ccagtgccaa gcttaattaa tctttctgcg 420 aattgagatg
acgccactgg ctgggcgtca tcccggtttc ccgggtaaac accaccgaaa 480
aatagttact atcttcaaag ccacattcgg tcgaaatatc actgattaac aggcggctat
540 gctggagaag atattgcgca tgacacactc tgacctgtcg cagatattga
ttgatggtca 600 ttccagtctg ctggcgaaat tgctgacgca aaacgcgctc
actgcacgat gcctcatcac 660 aaaatttatc cagcgcaaag ggacttttca
ggctagccgc cagccgggta atcagcttat 720 ccagcaacgt ttcgctggat
gttggcggca acgaatcact ggtgtaacga tggcgattca 780 gcaacatcac
caactgcccg aacagcaact cagccatttc gttagcaaac ggcacatgct 840
gactactttc atgctcaagc tgaccgataa cctgccgcgc ctgcgccatc cccatgctac
900 ctaagcgcca gtgtggttgc cctgcgctgg cgttaaatcc cggaatcgcc
ccctgccagt 960 caagattcag cttcagacgc tccgggcaat aaataatatt
ctgcaaaacc agatcgttaa 1020 cggaagcgta ggagtgttta tcgtcagcat
gaatgtaaaa gagatcgcca cgggtaatgc 1080 gataagggcg atcgttgagt
acatgcaggc cattaccgcg ccagacaatc accagctcac 1140 aaaaatcatg
tgtatgttca gcaaagacat cttgcggata acggtcagcc acagcgactg 1200
cctgctggtc gctggcaaaa aaatcatctt tgagaagttt taactgatgc gccaccgtgg
1260 ctacctcggc cagagaacga agttgattat tcgcaatatg gcgtacaaat
acgttgagaa 1320 gattcgcgtt attgcagaaa gccatcccgt ccctggcgaa
tatcacgcgg tgaccagtta 1380 aactctcggc gaaaaagcgt cgaaaagtgg
ttactgtcgc tgaatccaca gcgataggcg 1440 atgtcagtaa cgctggcctc
gctgtggcgt agcagatgtc gggctttcat cagtcgcagg 1500 cggttcaggt
atcgctgagg cgtcagtccc gtttgctgct taagctgccg atgtagcgta 1560
cgcagtgaaa gagaaaattg atccgccacg gcatcccaat tcacctcatc ggcaaaatgg
1620 tcctccagcc aggccagaag caagttgaga cgtgatgcgc tgttttccag
gttctcctgc 1680 aaactgcttt tacgcagcaa gagcagtaat tgcataaaca
agatctcgcg actggcggtc 1740 gagggtaaat cattttcccc ttcctgctgt
tccatctgtg caaccagctg tcgcacctgc 1800 tgcaatacgc tgtggttaac
gcgccagtga gacggatact gcccatccag ctcttgtggc 1860 agcaactgat
tcagcccggc gagaaactga aatcgatccg gcgagcgata cagcacattg 1920
gtcagacaca gattatcggt atgttcatac agatgccgat catgatcgcg tacgaaacag
1980 accgtgccac cggtgatggt atagggctgc ccattaaaca catgaatacc
cgtgccatgt 2040 tcgacaatca caatttcatg aaaatcatga tgatgttcag
gaaaatccgc ctgcgggagc 2100 cggggttcta tcgccacgga cgcgttacca
gacggaaaaa aatccacact atgtaatacg 2160 gtcatactgg cctcctgatg
tcgtcaacac ggcgaaatag taatcacgag gtcaggttct 2220 taccttaaat
tttcgacgga aaaccacgta aaaaacgtcg atttttcaag atacagcgtg 2280
aattttcagg aaatgcggtg agcatcacat caccacaatt cagcaaattg tgaacatcat
2340 cacgttcatc tttccctggt tgccaatggc ccattttcct gtcagtaacg
agaaggtcgc 2400 gaattcaggc gctttttaga ctggtcgtaa tgaaattcag
caggatcaca ttctgcaggt 2460 cgactctaga ggatccccgg gtaccgagct
cgaattcgta atcatggtca tagctgtttc 2520 ctgtgtgaaa ttgttatccg
ctcacaattc cacacaacat acgagccgga agcataaagt 2580 gtaaagcctg
gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 2640
ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
2700 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
tcgctgcgct 2760 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
ggcggtaata cggttatcca 2820 cagaatcagg ggataacgca ggaaagaaca
tgtgagcaaa aggccagcaa aaggccagga 2880 accgtaaaaa ggccgcgttg
ctggcgtttt tccataggct ccgcccccct gacgagcatc 2940 acaaaaatcg
acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 3000
cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
3060 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
cgctgtaggt 3120 atctcagttc ggtgtaggtc gttcgctcca agctgggctg
tgtgcacgaa ccccccgttc 3180 agcccgaccg ctgcgcctta tccggtaact
atcgtcttga gtccaacccg gtaagacacg 3240 acttatcgcc actggcagca
gccactggta acaggattag cagagcgagg tatgtaggcg 3300 gtgctacaga
gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg 3360
gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
3420 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
attacgcgca 3480 gaaaaaaagg atctcaagaa gatcctttga tcttttctac
ggggtctgac gctcagtgga 3540 acgaaaactc acgttaaggg attttggtca
tgagattatc aaaaaggatc ttcacctaga 3600 tccttttaaa ttaaaaatga
agttttaaat caatctaaag tatatatgag taaacttggt 3660 ctgacagtta
ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt 3720
catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
3780 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
gatttatcag 3840 caataaacca gccagccgga agggccgagc gcagaagtgg
tcctgcaact ttatccgcct 3900 ccatccagtc tattaattgt tgccgggaag
ctagagtaag tagttcgcca gttaatagtt 3960 tgcgcaacgt tgttgccatt
gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg 4020 cttcattcag
ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca 4080
aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
4140 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
tccgtaagat 4200 gcttttctgt gactggtgag tactcaacca agtcattctg
agaatagtgt atgcggcgac 4260 cgagttgctc ttgcccggcg tcaatacggg
ataataccgc gccacatagc agaactttaa 4320 aagtgctcat cattggaaaa
cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt 4380 tgagatccag
ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt 4440
tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
4500 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
tgaagcattt 4560 atcagggtta ttgtctcatg agcggataca tatttgaatg
tatttagaaa aataaacaaa 4620 taggggttcc gcgcacattt ccccgaaaag
tgccacctga cgtctaagaa accattatta 4680 tcatgacatt aacctataaa
aataggcgta tcacgaggcc ctttcgtc 4728 <210> SEQ ID NO 7
<211> LENGTH: 1440 <212> TYPE: DNA <213>
ORGANISM: E. coli <400> SEQUENCE: 7 gaattcaggc gctttttaga
ctggtcgtaa tgaaattcag caggatcaca ttctgcagat 60 gcctgttctg
gaaaaccggg ctgctcaggg cgatattact gcacccggcg gtgctcgccg 120
tttaacgggt gatcagactg ccgctctgcg tgattctctt agcgataaac ctgcaaaaaa
180 tattattttg ctgattggcg atgggatggg ggactcggaa attactgccg
cacgtaatta 240 tgccgaaggt gcgggcggct tttttaaagg tatagatgcc
ttaccgctta ccgggcaata 300 cactcactat gcgctgaata aaaaaaccgg
caaaccggac tacgtcaccg actcggctgc 360 atcagcaacc gcctggtcaa
ccggtgtcaa aacctataac ggcgcgctgg gcgtcgatat 420 tcacgaaaaa
gatcacccaa cgattctgga aatggcaaaa gccgcaggtc tggcgaccgg 480
taacgtttct accgcagagt tgcaggatgc cacgcccgct gcgctggtgg cacatgtgac
540 ctcgcgcaaa tgctacggtc cgagcgcgac cagtgaaaaa tgtccgggta
acgctctgga 600 aaaaggcgga aaaggatcga ttaccgaaca gctgcttaac
gctcgtgccg acgttacgct 660 tggcggcggc gcaaaaacct ttgctgaaac
ggcaaccgct ggtgaatggc agggaaaaac 720 gctgcgtgaa caggcacagg
cgcgtggtta tcagttggtg agcgatgctg cctcactgaa 780 ttcggtgacg
gaagcgaatc agcaaaaacc cctgcttggc ctgtttgctg acggcaatat 840
gccagtgcgc tggctaggac cgaaagcaac gtaccatggc aatatcgata agcccgcagt
900 cacctgtacg ccaaatccgc aacgtaatga cagtgtacca accctggcgc
agatgaccga 960 caaagccatt gaattgttga gtaaaaatga gaaaggcttt
ttcctgcaag ttgaaggtgc 1020 gtcaatcgat aaacaggatc atgctgcgaa
tccttgtggg caaattggcg agacggtcga 1080 tctcgatgaa gccgtacaac
gggcgctgga attcgctaaa aaggagggta acacgctggt 1140 catagtcacc
gctgatcacg cccacgccag ccagattgtt gcgccggata ccaaagctcc 1200
gggcctcacc caggcgctaa ataccaaaga tggcgcagtg atggtgatga gttacgggaa
1260 ctccgaagag gattcacaag aacataccgg cagtcagttg cgtattgcgg
cgtatggccc 1320 gcatgccgcc aatgttgttg gactgaccga ccagaccgat
ctcttctaca ccatgaaagc 1380 cgctctgggg ctgaaataat aatctagagg
atccccgggt accgagctcg aattcgtaat 1440 <210> SEQ ID NO 8
<211> LENGTH: 1560 <212> TYPE: DNA <213>
ORGANISM: E. coli <400> SEQUENCE: 8 gaattcaggc gctttttaga
ctggtcgtaa tgaaattcag caggatcaca ttctgcagat 60 gtcacggccg
agacttatag tcgctttgtt tttatttttt aatgtatttg tacatggaga 120
aaataaagtg aaacaaagca ctattgcact ggcactctta ccgttactgt ttacccctgt
180 gacaaaagcc cggacaccag aaatgcctgt tctggaaaac cgggctgctc
agggcgatat 240 tactgcaccc ggcggtgctc gccgtttaac gggtgatcag
actgccgctc tgcgtgattc 300 tcttagcgat aaacctgcaa aaaatattat
tttgctgatt ggcgatggga tgggggactc 360 ggaaattact gccgcacgta
attatgccga aggtgcgggc ggctttttta aaggtataga 420 tgccttaccg
cttaccgggc aatacactca ctatgcgctg aataaaaaaa ccggcaaacc 480
ggactacgtc accgactcgg ctgcatcagc aaccgcctgg tcaaccggtg tcaaaaccta
540 taacggcgcg ctgggcgtcg atattcacga aaaagatcac ccaacgattc
tggaaatggc 600 aaaagccgca ggtctggcga ccggtaacgt ttctaccgca
gagttgcagg atgccacgcc 660 cgctgcgctg gtggcacatg tgacctcgcg
caaatgctac ggtccgagcg cgaccagtga 720 aaaatgtccg ggtaacgctc
tggaaaaagg cggaaaagga tcgattaccg aacagctgct 780 taacgctcgt
gccgacgtta cgcttggcgg cggcgcaaaa acctttgctg aaacggcaac 840
cgctggtgaa tggcagggaa aaacgctgcg tgaacaggca caggcgcgtg gttatcagtt
900 ggtgagcgat gctgcctcac tgaattcggt gacggaagcg aatcagcaaa
aacccctgct 960 tggcctgttt gctgacggca atatgccagt gcgctggcta
ggaccgaaag caacgtacca 1020 tggcaatatc gataagcccg cagtcacctg
tacgccaaat ccgcaacgta atgacagtgt 1080 accaaccctg gcgcagatga
ccgacaaagc cattgaattg ttgagtaaaa atgagaaagg 1140 ctttttcctg
caagttgaag gtgcgtcaat cgataaacag gatcatgctg cgaatccttg 1200
tgggcaaatt ggcgagacgg tcgatctcga tgaagccgta caacgggcgc tggaattcgc
1260 taaaaaggag ggtaacacgc tggtcatagt caccgctgat cacgcccacg
ccagccagat 1320 tgttgcgccg gataccaaag ctccgggcct cacccaggcg
ctaaatacca aagatggcgc 1380 agtgatggtg atgagttacg ggaactccga
agaggattca caagaacata ccggcagtca 1440 gttgcgtatt gcggcgtatg
gcccgcatgc cgccaatgtt gttggactga ccgaccagac 1500 cgatctcttc
tacaccatga aagccgctct ggggctgaaa taatctagag gatccccggg 1560
<210> SEQ ID NO 9 <211> LENGTH: 1500 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Nucleic Acid Sequence
<400> SEQUENCE: 9 gaattcaggc gctttttaga ctggtcgtaa tgaaattcag
caggatcaca ttctgcagat 60 gaacttgggg aatcgactgt ttattctgat
agcggtctta cttcccctcg cagtattact 120 gctcatgcct gttctggaaa
accgggctgc tcagggcgat attactgcac ccggcggtgc 180 tcgccgttta
acgggtgatc agactgccgc tctgcgtgat tctcttagcg ataaacctgc 240
aaaaaatatt attttgctga ttggcgatgg gatgggggac tcggaaatta ctgccgcacg
300 taattatgcc gaaggtgcgg gcggcttttt taaaggtata gatgccttac
cgcttaccgg 360 gcaatacact cactatgcgc tgaataaaaa aaccggcaaa
ccggactacg tcaccgactc 420 ggctgcatca gcaaccgcct ggtcaaccgg
tgtcaaaacc tataacggcg cgctgggcgt 480 cgatattcac gaaaaagatc
acccaacgat tctggaaatg gcaaaagccg caggtctggc 540 gaccggtaac
gtttctaccg cagagttgca ggatgccacg cccgctgcgc tggtggcaca 600
tgtgacctcg cgcaaatgct acggtccgag cgcgaccagt gaaaaatgtc cgggtaacgc
660 tctggaaaaa ggcggaaaag gatcgattac cgaacagctg cttaacgctc
gtgccgacgt 720 tacgcttggc ggcggcgcaa aaacctttgc tgaaacggca
accgctggtg aatggcaggg 780 aaaaacgctg cgtgaacagg cacaggcgcg
tggttatcag ttggtgagcg atgctgcctc 840 actgaattcg gtgacggaag
cgaatcagca aaaacccctg cttggcctgt ttgctgacgg 900 caatatgcca
gtgcgctggc taggaccgaa agcaacgtac catggcaata tcgataagcc 960
cgcagtcacc tgtacgccaa atccgcaacg taatgacagt gtaccaaccc tggcgcagat
1020 gaccgacaaa gccattgaat tgttgagtaa aaatgagaaa ggctttttcc
tgcaagttga 1080 aggtgcgtca atcgataaac aggatcatgc tgcgaatcct
tgtgggcaaa ttggcgagac 1140 ggtcgatctc gatgaagccg tacaacgggc
gctggaattc gctaaaaagg agggtaacac 1200 gctggtcata gtcaccgctg
atcacgccca cgccagccag attgttgcgc cggataccaa 1260 agctccgggc
ctcacccagg cgctaaatac caaagatggc gcagtgatgg tgatgagtta 1320
cgggaactcc gaagaggatt cacaagaaca taccggcagt cagttgcgta ttgcggcgta
1380 tggcccgcat gccgccaatg ttgttggact gaccgaccag accgatctct
tctacaccat 1440 gaaagccgct ctggggctga aataataatc tagaggatcc
ccgggtaccg agctcgaatt 1500 <210> SEQ ID NO 10 <211>
LENGTH: 3968 <212> TYPE: DNA <213> ORGANISM: E. coli
<400> SEQUENCE: 10 tcgcgcgttt cggtgatgac ggtgaaaacc
tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat
gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg
tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg
ccagtgccaa gcttcgcagc gctgttcctt 420 tgctcgcctg ctgcgagctg
ggtaagcgga caaattctca ccgtctccgg tggtggggta 480 caggagctca
attaatacac taacggaccg gtaaacaacc gtgcgtgttg tttaccggga 540
taaactcatc aacgtctctg ctaaataact ggcagccaaa tcacggctat tggttaacca
600 atttcagagt gaaaagtata cgaatagagt gtgccttcgc actattcaac
agcaatgata 660 ggcgctcacc tgacaacgcg gtaaactagt tattcacgct
aactataatg gtttaatgat 720 ggataacatg cagactgaag cacaaccgac
acggacccgg atcctcaatg ctgccagaga 780 gattttttca gaaaatggat
ttcacagtgc ctcgatgaaa gccatctgta aatcttgcgc 840 cattagtccc
gggacgctct atcaccattt catctccaaa gaagccttga ttcaggcgat 900
tatcttacag gaccaggaga gggcgctggc ccgtttccgg gaaccgattg aagggattca
960 tttcgttgac tatatggtcg agtccattgt ctctctcacc catgaagcct
ttggacaacg 1020 ggcgctggtg gttgaaatta tggcggaagg gatgcgtaac
ccacaggtcg ccgccatgct 1080 taaaaataag catatgacga tcacggaatt
tgttgcccag cggatgcgtg atgcccagca 1140 aaaaggcgag ataagcccag
acatcaacac ggcaatgact tcacgtttac tgctggatct 1200 gacctacggt
gtactggccg atatcgaagc ggaagacctg gcgcgtgaag cgtcgtttgc 1260
tcagggatta cgcgcgatga ttggcggtat cttaaccgca tcctgattct ctctcttttt
1320 cggcgggctg gtgataactg tgcccgcgtt tcatatcgta atttctctgt
gcaaaaatta 1380 tccttcccgg cttcggagaa ttccccccaa aatattcact
gtagccatat gtcatgagag 1440 tttatcgttc ccaatacgct cgaacgaacg
ttcggttgct tattttatgg cttctgtcaa 1500 cgctgtttta aagattaatg
cgatctatat cacgctgtgg gtattgcagt ttttggtttt 1560 ttgatcgcgg
tgtcagttct ttttatttcc atttctcttc catgggtttc tcacagataa 1620
ctgtgtgcaa cacagaattg gttaactaat cagattaaag gttgaccagt attattatct
1680 taatgaggag tcctgcaggt cgactctaga ggatccccgg gtaccgagct
cgaattcgta 1740 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
ctcacaattc cacacaacat 1800 acgagccgga agcataaagt gtaaagcctg
gggtgcctaa tgagtgagct aactcacatt 1860 aattgcgttg cgctcactgc
ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta 1920 atgaatcggc
caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc 1980
gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
2040 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
tgtgagcaaa 2100 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
ctggcgtttt tccataggct 2160 ccgcccccct gacgagcatc acaaaaatcg
acgctcaagt cagaggtggc gaaacccgac 2220 aggactataa agataccagg
cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 2280 gaccctgccg
cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 2340
tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
2400 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
atcgtcttga 2460 gtccaacccg gtaagacacg acttatcgcc actggcagca
gccactggta acaggattag 2520 cagagcgagg tatgtaggcg gtgctacaga
gttcttgaag tggtggccta actacggcta 2580 cactagaagg acagtatttg
gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 2640 agttggtagc
tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 2700
caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
2760 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
tgagattatc 2820 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
agttttaaat caatctaaag 2880 tatatatgag taaacttggt ctgacagtta
ccaatgctta atcagtgagg cacctatctc 2940 agcgatctgt ctatttcgtt
catccatagt tgcctgactc cccgtcgtgt agataactac 3000 gatacgggag
ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc 3060
accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
3120 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
ctagagtaag 3180 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
gctacaggca tcgtggtgtc 3240 acgctcgtcg tttggtatgg cttcattcag
ctccggttcc caacgatcaa ggcgagttac 3300 atgatccccc atgttgtgca
aaaaagcggt tagctccttc ggtcctccga tcgttgtcag 3360 aagtaagttg
gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac 3420
tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
3480 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
ataataccgc 3540 gccacatagc agaactttaa aagtgctcat cattggaaaa
cgttcttcgg ggcgaaaact 3600 ctcaaggatc ttaccgctgt tgagatccag
ttcgatgtaa cccactcgtg cacccaactg 3660 atcttcagca tcttttactt
tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa 3720 tgccgcaaaa
aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt 3780
tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
3840 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
tgccacctga 3900 cgtctaagaa accattatta tcatgacatt aacctataaa
aataggcgta tcacgaggcc 3960 ctttcgtc 3968 <210> SEQ ID NO 11
<211> LENGTH: 3872 <212> TYPE: DNA <213>
ORGANISM: E. coli <400> SEQUENCE: 11 tcgcgcgttt cggtgatgac
ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct
gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc
ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg
caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt
aaaacgacgg ccagtgccaa gcttttagcc gggaaacgtc 420 tggcggcgct
gttggctaag tttgcggtat tgttgcggcg acatgccgac atatttgccg 480
aacgtgctgt aaaaacgact acttgaacga aagcctgccg tcagggcaat atcgagaata
540 cttttatcgg tatcgctcag taacgcgcga acgtggttga tgcgcatcgc
ggtaatgtac 600 tgtttcatcg tcaattgcat gacccgctgg aatatcccca
ttgcatagtt ggcgttaagt 660 ttgacgtgct cagccacatc gttgatggtc
agcgcctgat catagttttc ggcaataaag 720 cccagcatct ggctaacata
aaattgcgca tggcgcgaga cgctgttttt gtgtgtgcgc 780 gaggttttat
tgaccagaat cggttcccag ccagagaggc taaatcgctt gagcatcagg 840
ccaatttcat caatggcgag ctggcgaatt tgctcgttcg gactgtttaa ttcctgctgc
900 cagcggcgca cttcaaacgg gctaagttgc tgtgtggcca gtgatttgat
caccatgccg 960 tgagtgacgt ggttaatcag gtctttatcc agcggccagg
agagaaacag atgcatcggc 1020 agattaaaaa tcgccatgct ctgacaggtt
ccggtatctg ttagttggtg cggtgtacag 1080 gcccagaaca gcgtgatatg
accctgattg atattcactt tttcattgtt gatcaggtat 1140 tccacatcgc
catcgaaagg cacattcact tcgacctgac catgccagtg gctggtgggc 1200
atgatatgcg gtgcgcgaaa ctcaatctcc atccgctggt attccgaata cagcgacagc
1260 gggctgcggg tctgtttttc gtcgctgctg cacataaacg tatctgtatt
catggatggc 1320 tctctttcct ggaatatcag aattatggca ggagtgaggg
aggatgactg cgagtgggag 1380 cacggttttc accctcttcc cagaggggcg
aggggactct ccgagtatca tgaggccgaa 1440 aactctgctt ttcaggtaat
ttattcccat aaactcagat ttactgctgc ttcacgcagg 1500 atctgagttt
atgggaatgc tcaacctgga agccggaggt tttctgcaga ttcgcctgcc 1560
atgatgaagt tattcaagca agccaggaga tctggtaccc gggtcgactc tagaggatcc
1620 ccgggtaccg agctcgaatt cgtaatcatg gtcatagctg tttcctgtgt
gaaattgtta 1680 tccgctcaca attccacaca acatacgagc cggaagcata
aagtgtaaag cctggggtgc 1740 ctaatgagtg agctaactca cattaattgc
gttgcgctca ctgcccgctt tccagtcggg 1800 aaacctgtcg tgccagctgc
attaatgaat cggccaacgc gcggggagag gcggtttgcg 1860 tattgggcgc
tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 1920
gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
1980 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
aaaaggccgc 2040 gttgctggcg tttttccata ggctccgccc ccctgacgag
catcacaaaa atcgacgctc 2100 aagtcagagg tggcgaaacc cgacaggact
ataaagatac caggcgtttc cccctggaag 2160 ctccctcgtg cgctctcctg
ttccgaccct gccgcttacc ggatacctgt ccgcctttct 2220 cccttcggga
agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 2280
ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
2340 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
cgccactggc 2400 agcagccact ggtaacagga ttagcagagc gaggtatgta
ggcggtgcta cagagttctt 2460 gaagtggtgg cctaactacg gctacactag
aaggacagta tttggtatct gcgctctgct 2520 gaagccagtt accttcggaa
aaagagttgg tagctcttga tccggcaaac aaaccaccgc 2580 tggtagcggt
ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 2640
agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
2700 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
taaattaaaa 2760 atgaagtttt aaatcaatct aaagtatata tgagtaaact
tggtctgaca gttaccaatg 2820 cttaatcagt gaggcaccta tctcagcgat
ctgtctattt cgttcatcca tagttgcctg 2880 actccccgtc gtgtagataa
ctacgatacg ggagggctta ccatctggcc ccagtgctgc 2940 aatgataccg
cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 3000
cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
3060 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
acgttgttgc 3120 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
atggcttcat tcagctccgg 3180 ttcccaacga tcaaggcgag ttacatgatc
ccccatgttg tgcaaaaaag cggttagctc 3240 cttcggtcct ccgatcgttg
tcagaagtaa gttggccgca gtgttatcac tcatggttat 3300 ggcagcactg
cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 3360
tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
3420 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
tcatcattgg 3480 aaaacgttct tcggggcgaa aactctcaag gatcttaccg
ctgttgagat ccagttcgat 3540 gtaacccact cgtgcaccca actgatcttc
agcatctttt actttcacca gcgtttctgg 3600 gtgagcaaaa acaggaaggc
aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 3660 ttgaatactc
atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 3720
catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
3780 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
cattaaccta 3840 taaaaatagg cgtatcacga ggccctttcg tc 3872
<210> SEQ ID NO 12 <211> LENGTH: 3934 <212> TYPE:
DNA <213> ORGANISM: E. coli <400> SEQUENCE: 12
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga
gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat
gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc
aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct
ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gcttcaagcc gtcaattgtc
420 tgattcgtta ccaattatga caacttgacg gctacatcat tcactttttc
ttcacaaccg 480 gcacggaact cgctcgggct ggccccggtg cattttttaa
atacccgcga gaaatagagt 540 tgatcgtcaa aaccaacatt gcgaccgacg
gtggcgatag gcatccgggt ggtgctcaaa 600 agcagcttcg cctggctgat
acgttggtcc tcgcgccagc ttaagacgct aatccctaac 660 tgctggcgga
aaagatgtga cagacgcgac ggcgacaagc aaacatgctg tgcgacgctg 720
gcgatatcaa aattgctgtc tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc
780 cgattatcca tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg
cagtaacaat 840 tgctcaagca gatttatcgc cagcagctcc gaatagcgcc
cttccccttg cccggcgtta 900 atgatttgcc caaacaggtc gctgaaatgc
ggctggtgcg cttcatccgg gcgaaagaac 960 cccgtattgg caaatattga
cggccagtta agccattcat gccagtaggc gcgcggacga 1020 aagtaaaccc
actggtgata ccattcgcga gcctccggat gacgaccgta gtgatgaatc 1080
tctcctggcg ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg tccctgattt
1140 ttcaccaccc cctgaccgcg aatggtgaga ttgagaatat aacctttcat
tcccagcggt 1200 cggtcgataa aaaaatcgag ataaccgttg gcctcaatcg
gcgttaaacc cgccaccaga 1260 tgggcattaa acgagtatcc cggcagcagg
ggatcatttt gcgcttcagc catacttttc 1320 atactcccgc cattcagaga
agaaaccaat tgtccatatt gcatcagaca ttgccgtcac 1380 tgcgtctttt
actggctctt ctcgctaacc aaaccggtaa ccccgcttat taaaagcatt 1440
ctgtaacaaa gcgggaccaa agccatgaca aaaacgcgta acaaaagtgt ctataatcac
1500 ggcagaaaag tccacattga ttatttgcac ggcgtcacac tttgctatgc
catagcattt 1560 ttatccataa gattagcgga tcctacctga cgctttttat
cgcaactctc tactgtttct 1620 ccatacccgt ttttttgggc tagcaggagg
aattcaccct gcaggtcgac tctagaggat 1680 ccccgggtac cgagctcgaa
ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 1740 tatccgctca
caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt 1800
gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
1860 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
aggcggtttg 1920 cgtattgggc gctcttccgc ttcctcgctc actgactcgc
tgcgctcggt cgttcggctg 1980 cggcgagcgg tatcagctca ctcaaaggcg
gtaatacggt tatccacaga atcaggggat 2040 aacgcaggaa agaacatgtg
agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 2100 gcgttgctgg
cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 2160
tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
2220 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
gtccgccttt 2280 ctcccttcgg gaagcgtggc gctttctcat agctcacgct
gtaggtatct cagttcggtg 2340 taggtcgttc gctccaagct gggctgtgtg
cacgaacccc ccgttcagcc cgaccgctgc 2400 gccttatccg gtaactatcg
tcttgagtcc aacccggtaa gacacgactt atcgccactg 2460 gcagcagcca
ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 2520
ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg
2580 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
acaaaccacc 2640 gctggtagcg gtggtttttt tgtttgcaag cagcagatta
cgcgcagaaa aaaaggatct 2700 caagaagatc ctttgatctt ttctacgggg
tctgacgctc agtggaacga aaactcacgt 2760 taagggattt tggtcatgag
attatcaaaa aggatcttca cctagatcct tttaaattaa 2820 aaatgaagtt
ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 2880
tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
2940 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
ccccagtgct 3000 gcaatgatac cgcgagaccc acgctcaccg gctccagatt
tatcagcaat aaaccagcca 3060 gccggaaggg ccgagcgcag aagtggtcct
gcaactttat ccgcctccat ccagtctatt 3120 aattgttgcc gggaagctag
agtaagtagt tcgccagtta atagtttgcg caacgttgtt 3180 gccattgcta
caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 3240
ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
3300 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
actcatggtt 3360 atggcagcac tgcataattc tcttactgtc atgccatccg
taagatgctt ttctgtgact 3420 ggtgagtact caaccaagtc attctgagaa
tagtgtatgc ggcgaccgag ttgctcttgc 3480 ccggcgtcaa tacgggataa
taccgcgcca catagcagaa ctttaaaagt gctcatcatt 3540 ggaaaacgtt
cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 3600
atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
3660 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
gacacggaaa 3720 tgttgaatac tcatactctt cctttttcaa tattattgaa
gcatttatca gggttattgt 3780 ctcatgagcg gatacatatt tgaatgtatt
tagaaaaata aacaaatagg ggttccgcgc 3840 acatttcccc gaaaagtgcc
acctgacgtc taagaaacca ttattatcat gacattaacc 3900 tataaaaata
ggcgtatcac gaggcccttt cgtc 3934 <210> SEQ ID NO 13
<211> LENGTH: 6013 <212> TYPE: DNA <213>
ORGANISM: E. coli <400> SEQUENCE: 13 tagttattaa tagtaatcaa
ttacggggtc attagttcat agcccatata tggagttccg 60 cgttacataa
cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
180 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
atcatatgcc 240 aagtacgccc cctattgacg tcaatgacgg taaatggccc
gcctggcatt atgcccagta 300 catgacctta tgggactttc ctacttggca
gtacatctac gtattagtca tcgctattac 360 catggtgatg cggttttggc
agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420 atttccaagt
ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480
ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
540 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc
gctagcgcta 600 ccggtcgcca ccatggtgag caagggcgag gagctgttca
ccggggtggt gcccatcctg 660 gtcgagctgg acggcgacgt aaacggccac
aagttcagcg tgtccggcga gggcgagggc 720 gatgccacct acggcaagct
gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg 780 ccctggccca
ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc 840
gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag
900 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
gaagttcgag 960 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
acttcaagga ggacggcaac 1020 atcctggggc acaagctgga gtacaactac
aacagccaca acgtctatat catggccgac 1080 aagcagaaga acggcatcaa
ggtgaacttc aagatccgcc acaacatcga ggacggcagc 1140 gtgcagctcg
ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg 1200
cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc
1260 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
catggacgag 1320 ctgtacaagt ccggactcag atctcgagct taataacaag
ccgtcaattg tctgattcgt 1380 taccaattat gacaacttga cggctacatc
attcactttt tcttcacaac cggcacggaa 1440 ctcgctcggg ctggccccgg
tgcatttttt aaatacccgc gagaaataga gttgatcgtc 1500 aaaaccaaca
ttgcgaccga cggtggcgat aggcatccgg gtggtgctca aaagcagctt 1560
cgcctggctg atacgttggt cctcgcgcca gcttaagacg ctaatcccta actgctggcg
1620 gaaaagatgt gacagacgcg acggcgacaa gcaaacatgc tgtgcgacgc
tggcgatatc 1680 aaaattgctg tctgccaggt gatcgctgat gtactgacaa
gcctcgcgta cccgattatc 1740 catcggtgga tggagcgact cgttaatcgc
ttccatgcgc cgcagtaaca attgctcaag 1800 cagatttatc gccagcagct
ccgaatagcg cccttcccct tgcccggcgt taatgatttg 1860 cccaaacagg
tcgctgaaat gcggctggtg cgcttcatcc gggcgaaaga accccgtatt 1920
ggcaaatatt gacggccagt taagccattc atgccagtag gcgcgcggac gaaagtaaac
1980 ccactggtga taccattcgc gagcctccgg atgacgaccg tagtgatgaa
tctctcctgg 2040 cgggaacagc aaaatatcac ccggtcggca aacaaattct
cgtccctgat ttttcaccac 2100 cccctgaccg cgaatggtga gattgagaat
ataacctttc attcccagcg gtcggtcgat 2160 aaaaaaatcg agataaccgt
tggcctcaat cggcgttaaa cccgccacca gatgggcatt 2220 aaacgagtat
cccggcagca ggggatcatt ttgcgcttca gccatacttt tcatactccc 2280
gccattcaga gaagaaacca attgtccata ttgcatcaga cattgccgtc actgcgtctt
2340 ttactggctc ttctcgctaa ccaaaccggt aaccccgctt attaaaagca
ttctgtaaca 2400 aagcgggacc aaagccatga caaaaacgcg taacaaaagt
gtctataatc acggcagaaa 2460 agtccacatt gattatttgc acggcgtcac
actttgctat gccatagcat ttttatccat 2520 aagattagcg gatcctacct
gacgcttttt atcgcaactc tctactgttt ctccataccc 2580 gtttttttgg
gctagcagga ggaattcacc atggtacccg gggatcctct agagtcgacc 2640
tgcaggcatg caagcttggc ccgcgggccc gggatccacc ggatctagat aactgatcat
2700 aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc
cacacctccc 2760 cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt
aacttgttta ttgcagctta 2820 taatggttac aaataaagca atagcatcac
aaatttcaca aataaagcat ttttttcact 2880 gcattctagt tgtggtttgt
ccaaactcat caatgtatct taacgcgtaa attgtaagcg 2940 ttaatatttt
gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat 3000
aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg
3060 ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
gtcaaagggc 3120 gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
atcaccctaa tcaagttttt 3180 tggggtcgag gtgccgtaaa gcactaaatc
ggaaccctaa agggagcccc cgatttagag 3240 cttgacgggg aaagccggcg
aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg 3300 gcgctagggc
gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc 3360
ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc
3420 ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
caataaccct 3480 gataaatgct tcaataatat tgaaaaagga agagtcctga
ggcggaaaga accagctgtg 3540 gaatgtgtgt cagttagggt gtggaaagtc
cccaggctcc ccagcaggca gaagtatgca 3600 aagcatgcat ctcaattagt
cagcaaccag gtgtggaaag tccccaggct ccccagcagg 3660 cagaagtatg
caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc 3720
gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
3780 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc
agaagtagtg 3840 aggaggcttt tttggaggcc taggcttttg caaagatcga
tcaagagaca ggatgaggat 3900 cgtttcgcat gattgaacaa gatggattgc
acgcaggttc tccggccgct tgggtggaga 3960 ggctattcgg ctatgactgg
gcacaacaga caatcggctg ctctgatgcc gccgtgttcc 4020 ggctgtcagc
gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga 4080
atgaactgca agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
4140 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg
ggcgaagtgc 4200 cggggcagga tctcctgtca tctcaccttg ctcctgccga
gaaagtatcc atcatggctg 4260 atgcaatgcg gcggctgcat acgcttgatc
cggctacctg cccattcgac caccaagcga 4320 aacatcgcat cgagcgagca
cgtactcgga tggaagccgg tcttgtcgat caggatgatc 4380 tggacgaaga
gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca 4440
tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
4500 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
gcggaccgct 4560 atcaggacat agcgttggct acccgtgata ttgctgaaga
gcttggcggc gaatgggctg 4620 accgcttcct cgtgctttac ggtatcgccg
ctcccgattc gcagcgcatc gccttctatc 4680 gccttcttga cgagttcttc
tgagcgggac tctggggttc gaaatgaccg accaagcgac 4740 gcccaacctg
ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt 4800
cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
4860 gttcttcgcc caccctaggg ggaggctaac tgaaacacgg aaggagacaa
taccggaagg 4920 aacccgcgct atgacggcaa taaaaagaca gaataaaacg
cacggtgttg ggtcgtttgt 4980 tcataaacgc ggggttcggt cccagggctg
gcactctgtc gataccccac cgagacccca 5040 ttggggccaa tacgcccgcg
tttcttcctt ttccccaccc caccccccaa gttcgggtga 5100 aggcccaggg
ctcgcagcca acgtcggggc ggcaggccct gccatagcct caggttactc 5160
atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat
5220 cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
actgagcgtc 5280 agaccccgta gaaaagatca aaggatcttc ttgagatcct
ttttttctgc gcgtaatctg 5340 ctgcttgcaa acaaaaaaac caccgctacc
agcggtggtt tgtttgccgg atcaagagct 5400 accaactctt tttccgaagg
taactggctt cagcagagcg cagataccaa atactgtcct 5460 tctagtgtag
ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct 5520
cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
5580 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
cggggggttc 5640 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
ctgagatacc tacagcgtga 5700 gctatgagaa agcgccacgc ttcccgaagg
gagaaaggcg gacaggtatc cggtaagcgg 5760 cagggtcgga acaggagagc
gcacgaggga gcttccaggg ggaaacgcct ggtatcttta 5820 tagtcctgtc
gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg 5880
ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
5940 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
ataaccgtat 6000 taccgccatg cat 6013 <210> SEQ ID NO 14
<211> LENGTH: 1380 <212> TYPE: DNA <213>
ORGANISM: Rattus norvegicus <400> SEQUENCE: 14 gaattcaggc
gctttttaga ctggtcgtaa tgaaattcag caggatcaca ttctgcaggt 60
cgacatggca accacgcacg cgcagggcca cccgccagtc ttggggaatg atactctccg
120 ggaacattat gattacgtgg ggaagctggc aggcaggctg cgggatcccc
ctgagggtag 180 caccctcatc accaccatcc tcttcttggt cacctgtagc
ttcatcgtct tggagaacct 240 gatggttttg attgccatct ggaaaaacaa
taaatttcat aaccgcatgt actttttcat 300 cggcaacttg gctctctgcg
acctgctggc cggcatagcc tacaaggtca acattctgat 360 gtccggtagg
aagacgttca gcctgtctcc aacagtgtgg ttcctcaggg agggcagtat 420
gttcgtagcc ctgggcgcat ccacatgcag cttattggcc attgccattg agcggcacct
480 gaccatgatc aagatgaggc cgtacgacgc caacaagaag caccgcgtgt
tccttctgat 540 tgggatgtgc tggctaattg ccttctcgct gggtgccctg
cccatcctgg gctggaactg 600 cctggaaaac tttcccgact gctctaccat
cttgcccctc tactccaaga aatacattgc 660 ctttctcatc agcatcttca
tagccattct ggtgaccatc gtcatcttgt acgcgcgcat 720 ctacttcctg
gtcaagtcca gcagccgcag ggtggccaac cacaactccg agagatccat 780
ggcccttctg cggaccgtag tgatcgtggt gagcgtgttc atcgcctgtt ggtcccccct
840 tttcatcctc ttcctcatcg atgtggcctg cagggcgaag gagtgctcca
tcctcttcaa 900 gagtcagtgg ttcatcatgc tggctgtcct caactcggcc
atgaaccctg tcatctacac 960 gctggccagc aaagagatgc ggcgtgcttt
cttccggttg gtgtgcggct gtctggtcaa 1020 gggcaagggg acccaggcct
ccccgatgca gcctgctctt gacccgagca gaagtaaatc 1080 aagctccagt
aacaacagca gcagccactc tccaaaggtc aaggaagacc tgccccatgt 1140
ggctacctct tcctgcgtta ctgacaaaac gaggtcgctt cagaatgggg tcctctgcaa
1200 gaagggcaat tctgcagata tccagcacag tggcggccgc tcgagtctag
agggcccgcg 1260 gttcgaaggt aagcctatcc ctaaccctct cctcggtctc
gattctacgc gtaccggtca 1320 tcatcaccat caccattgat aaggtaccga
gctcgaattc gtaatcatgg tcatagctgt 1380 <210> SEQ ID NO 15
<211> LENGTH: 1320 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 15 gaattcaggc
gctttttaga ctggtcgtaa tgaaattcag caggatcaca ttctgcaggt 60
cgacatgggg caacccggga acggcagcgc cttcttgctg gcacccaatg gaagccatgc
120 gccggaccac gacgtcacgc agcaaaggga cgaggtgtgg gtggtgggca
tgggcatcgt 180 catgtctctc atcgtcctgg ccatcgtgtt tggcaatgtg
ctggtcatca cagccattgc 240 caagttcgag cgtctgcaga cggtcaccaa
ctacttcatc acttcactgg cctgtgctga 300 tctggtcatg ggcctagcag
tggtgccctt tggggccgcc catattctta tgaaaatgtg 360 gacttttggc
aacttctggt gcgagttttg gacttccatt gatgtgctgt gcgtcacggc 420
cagcattgag accctgtgcg tgatcgcagt ggatcgctac tttgccatta cttcaccttt
480 caagtaccag agcctgctga ccaagaataa ggcccgggtg atcattctga
tggtgtggat 540 tgtgtcaggc cttayctcct tcttgcccat tcagatgcac
tggtacaggg ccacccacca 600 ggaagccatc aactgctatg ccaatgagac
ctgctgtgac ttcttcacga accaagccta 660 tgccattgcc tcttccatcg
tgtccttcta cgttcccctg gtgatcatgg tcttcgtcta 720 ctccagggtc
tttcaggagg ccaaaaggca gctccagaag attgacaaat ctgagggccg 780
cttccatgtc cagaacctta gccaggtgga gcaggatggg cggacggggc atggactccg
840 cagatcttcc aagttctgct tgaaggagca caaagccctc aagacgttag
gcatcatcat 900 gggcactttc accctctgct ggctgccctt cttcatcgtt
aacattgtgc atgtgatcca 960 ggataacctc atccgtaagg aagtttacat
cctcctaaat tggataggct atgtcaattc 1020 tggtttcaat ccccttatct
actgccggag cccagatttc aggattgcct tccaggagct 1080 tctgtgcctg
cgcaggtctt ctttgaaggc ctatggcaat ggctactcca gcaacggcaa 1140
cacaggggag cagagtggat atcacgtgga acaggagaaa gaaaataaac tgctgtgtga
1200 agacctccca ggcacggaag actttgtggg ccatcaaggt actgtgccta
gcgataacat 1260 tgattcacaa gggaggaatt gtagtacaaa tgactcactg
ctataataag gatccccggg 1320 <210> SEQ ID NO 16 <211>
LENGTH: 52 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide sequence <400> SEQUENCE: 16
aattggtacc tcaatgatga tgatgatgat gcttgcagag gaccccattc tg 52
<210> SEQ ID NO 17 <211> LENGTH: 1320 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 17
tcgcaactct ctactgtttc tccatacccg tttttttggg ctagcaggag gaattcacca
60 tggatagtgt gtgtccccaa ggaaaatata tccaccctca aaataattcg
atttgctgta 120 ccaagtgcca caaaggaacc tacttgtaca atgactgtcc
aggcccgggg caggatacgg 180 actgcaggga gtgtgagagc ggctccttca
ccgcttcaga aaaccacctc agacactgcc 240 tcagctgctc caaatgccga
aaggaaatgg gtcaggtgga gatctcttct tgcacagtgg 300 accgggacac
cgtgtgtggc tgcaggaaga accagtaccg gcattattgg agtgaaaacc 360
ttttccagtg cttcaattgc agcctctgcc tcaatgggac cgtgcacctc tcctgccagg
420 agaaacagaa caccgtgtgc acctgccatg caggtttctt tctaagagaa
aacgagtgtg 480 tctcctgtag taactgtaag aaaagcctgg agtgcacgaa
gttgtgccta ccccagattg 540 agaatgttaa gggcactgag gactcaggca
ccacagtgct gttgcccctg gtcattttct 600 ttggtctttg ccttttatcc
ctcctcttca ttggtttaat gtatcgctac caacggtgga 660 agtccaagct
ctactccatt gtttgtggga aatcgacacc tgaaaaagag ggggagcttg 720
aaggaactac tactaagccc ctggccccaa acccaagctt cagtcccact ccaggcttca
780 cccccaccct gggcttcagt cccgtgccca gttccacctt cacctccagc
tccacctata 840 cccccggtga ctgtcccaac tttgcggctc cccgcagaga
ggtggcacca ccctatcagg 900 gggctgaccc catccttgcg acagccctcg
cctccgaccc catccccaac ccccttcaga 960 agtgggagga cagcgcccac
aagccacaga gcctagacac tgatgacccc gcgacgctgt 1020 acgccgtggt
ggagaacgtg cccccgttgc gctggaagga attcgtgcgg cgcctagggc 1080
tgagcgacca cgagatcgat cggctggagc tgcagaacgg gcgctgcctg cgcgaggcgc
1140 aatacagcat gctggcgacc tggaggcggc gcacgccgcg gcgcgaggcc
acgctggagc 1200 tgctgggacg cgtgctccgc gacatggacc tgctgggctg
cctggaggac atcgaggagg 1260 cgctttgcgg ccccgccgcc ctcccgcccg
cgcccagtct tctcagatga tctagagtcg 1320 <210> SEQ ID NO 18
<211> LENGTH: 1380 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 18 ccatacccgt
ttttttgggc tagcaggagg aattcaccct gcaggtcgac atgggactgg 60
tccctcacct aggggacagg gagaagagag atagtgtgtg tccccaagga aaatatatcc
120 accctcaaaa taattcgatt tgctgtacca agtgccacaa aggaacctac
ttgtacaatg 180 actgtccagg cccggggcag gatacggact gcagggagtg
tgagagcggc tccttcaccg 240 cttcagaaaa ccacctcaga cactgcctca
gctgctccaa atgccgaaag gaaatgggtc 300 aggtggagat ctcttcttgc
acagtggacc gggacaccgt gtgtggctgc aggaagaacc 360 agtaccggca
ttattggagt gaaaaccttt tccagtgctt caattgcagc ctctgcctca 420
atgggaccgt gcacctctcc tgccaggaga aacagaacac cgtgtgcacc tgccatgcag
480 gtttctttct aagagaaaac gagtgtgtct cctgtagtaa ctgtaagaaa
agcctggagt 540 gcacgaagtt gtgcctaccc cagattgaga atgttaaggg
cactgaggac tcaggcacca 600 cagtgctgtt gcccctggtc attttctttg
gtctttgcct tttatccctc ctcttcattg 660 gtttaatgta tcgctaccaa
cggtggaagt ccaagctcta ctccattgtt tgtgggaaat 720 cgacacctga
aaaagagggg gagcttgaag gaactactac taagcccctg gccccaaacc 780
caagcttcag tcccactcca ggcttcaccc ccaccctggg cttcagtccc gtgcccagtt
840 ccaccttcac ctccagctcc acctataccc ccggtgactg tcccaacttt
gcggctcccc 900 gcagagaggt ggcaccaccc tatcaggggg ctgaccccat
ccttgcgaca gccctcgcct 960 ccgaccccat ccccaacccc cttcagaagt
gggaggacag cgcccacaag ccacagagcc 1020 tagacactga tgaccccgcg
acgctgtacg ccgtggtgga gaacgtgccc ccgttgcgct 1080 ggaaggaatt
cgtgcggcgc ctagggctga gcgaccacga gatcgatcgg ctggagctgc 1140
agaacgggcg ctgcctgcgc gaggcgcaat acagcatgct ggcgacctgg aggcggcgca
1200 cgccgcggcg cgaggccacg ctggagctgc tgggacgcgt gctccgcgac
atggacctgc 1260 tgggctgcct ggaggacatc gaggaggcgc tttgcggccc
cgccgccctc ccgcccgcgc 1320 ccagtcttct cagataataa ggtaccgagc
tcgaattcgt aatcatggtc atagctgttt 1380 <210> SEQ ID NO 19
<211> LENGTH: 780 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 19 gtttttttgg gctagcagga
ggaattcatg agcactgaaa gcatgatccg ggacgtggag 60 ctggccgagg
aggcgctccc caagaagaca ggggggcccc agggctccag gcggtgcttg 120
ttcctcagcc tcttctcctt cctgatcgtg gcaggcgcca ccacgctctt ctgcctgctg
180 cactttggag tgatcggccc ccagagggaa gagttcccca gggacctctc
tctaatcagc 240 cctctggccc aggcagtcag atcatcttct cgaaccccga
gtgacaagcc tgtagcccat 300 gttgtagcaa accctcaagc tgaggggcag
ctccagtggc tgaaccgccg ggccaatgcc 360 ctcctggcca atggcgtgga
gctgagagat aaccagctgg tggtgccatc agagggcctg 420 tacctcatct
actcccaggt cctcttcaag ggccaaggct gcccctccac ccatgtgctc 480
ctcacccaca ccatcagccg catcgccgtc tcctaccaga ccaaggtcaa cctcctctct
540 gccatcaaga gcccctgcca gagggagacc ccagaggggg ctgaggccaa
gccctggtat 600 gagcccatct atctgggagg ggtcttccag ctggagaagg
gtgaccgact cagcgctgag 660 atcaatcggc ccgactatct cgactttgcc
gagtctgggc aggtctactt tgggatcatt 720 gccctgtgat aagcttggcc
cgcgggcccg ggatccaccg gatctagata actgatcata 780 <210> SEQ ID
NO 20 <211> LENGTH: 300 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic nucleic acid sequence <400>
SEQUENCE: 20 gtttttttgg gctagcagga ggaattcacc atggtaccat gaacttgggg
aatcgactgt 60 ttattctgat agcggtctta cttcccctcg cagtattact
gctcaatagt gactctgaat 120 gtcccctgtc ccacgatggg tactgcctcc
atgatggtgt gtgcatgtat attgaagcat 180 tggacaagta tgcatgcaac
tgtgttgttg gctacatcgg ggagcgatgt cagtaccgag 240 acctgaagtg
gtgggaactg cgctaataag cttggcccgc gggcccggga tccaccggat 300
<210> SEQ ID NO 21 <211> LENGTH: 1620 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleic acid sequence
<400> SEQUENCE: 21 gtttttttgg gctagcagga ggaattcacc
atgaacttgg ggaatcgact gtttattctg 60 atagcggtct tacttcccct
cgcagtatta ctgctctcat tcacattgag cgtcaccgtt 120 cagcagcctc
agttgacatt aacggcggcc gtcattggtg atggcgcacc ggctaatggg 180
aaaactgcaa tcaccgttga gttcaccgtt gctgattttg aggggaaacc cttagccggg
240 caggaggtgg tgataaccac caataatggt gcgctaccga ataaaatcac
ggaaaagaca 300 gatgcaaatg gcgtcgcgcg cattgcatta accaatacga
cagatggcgt gacggtagtc 360 acagcagaag tggaggggca acggcaaagt
gttgataccc actttgttaa gggtactatc 420 gcggcggata aatccactct
ggctgcggta ccgacatcta tcatcgctga tggtctaatg 480 gcttcaacca
tcacgttgga gttgaaggat acctatgggg acccgcaggc tggcgcgaat 540
gtggcttttg acacaacctt aggcaatatg ggcgttatca cggatcacaa tgacggcact
600 tatagcgcac cattgaccag taccacgttg ggggtagcaa cagtaacggt
gaaagtggat 660 ggggctgcgt tcagtgtgcc gagtgtgacg gttaatttca
cggcagatcc tattccagat 720 gctggccgct ccagtttcac cgtctccaca
ccggatatct tggctgatgg cacgatgagt 780 tccacattat cctttgtccc
tgtcgataag aatggccatt ttatcagtgg gatgcagggc 840 ttgagtttta
ctcaaaacgg tgtgccggtg agtattagcc ccattaccga gcagccagat 900
agctataccg cgacggtggt tgggaatagt gtcggtgatg tcacaatcac gccgcaggtt
960 gataccctga tactgagtac attgcagaaa aaaatatccc tattcccggt
acctacgctg 1020 accggtattc tggttaacgg gcaaaatttc gctacggata
aagggttccc gaaaacgatc 1080 tttaaaaacg ccacattcca gttacagatg
gataacgatg ttgctaataa tactcagtat 1140 gagtggtcgt cgtcattcac
acccaatgta tcggttaacg atcagggtca ggtgacgatt 1200 acctaccaaa
cctatagcga agtggctgtg acggcgaaaa gtaaaaaatt cccaagttat 1260
tcggtgagtt atcggttcta cccaaatcgg tggatatacg atggcggcag atcgctggta
1320 tccagtctcg aggccagcag acaatgccaa ggttcagata tgtctgcggt
tcttgaatcc 1380 tcacgtgcaa ccaacggaac gcgtgcgcct gacgggacat
tgtggggcga gtgggggagc 1440 ttgaccgcgt atagttctga ttggcaatct
ggtgaatatt gggtcaaaaa gaccagcacg 1500 gattttgaaa ccatgaatat
ggacacaggc gcactgcaac cagggcctgc atacttggcg 1560 ttcccgctct
gtgcgctgtc aatataactg caggcatgca agcttggccc gcgggcccgg 1620
<210> SEQ ID NO 22 <211> LENGTH: 208 <212> TYPE:
DNA <213> ORGANISM: E. coli <400> SEQUENCE: 22
gaattcaggc gctttttaga ctggtcgtaa tgaaattcag caggatcaca ttctgcagat
60 gtcacggccg agacttatag tcgctttgtt tttatttttt aatgtatttg
tacatggaga 120 aaataaagtg aaacaaagca ctattgcact ggcactctta
ccgttactgt ttacccctgt 180 gacaaaagcc cggacaccag aatctaga 208
<210> SEQ ID NO 23 <211> LENGTH: 1546 <212> TYPE:
DNA <213> ORGANISM: E. coli <400> SEQUENCE: 23
gaattcaggc gctttttaga ctggtcgtaa tgaaattcag caggatcaca ttctgcagat
60 gtcacggccg agacttatag tcgctttgtt tttatttttt aatgtatttg
tacatggaga 120 aaataaagtg aaacaaagca ctattgcact ggcactctta
ccgttactgt ttacccctgt 180 gacaaaagcc cggacaccag aaatgcctgt
tctggaaaac cgggctgctc agggcgatat 240 tactgcaccc ggcggtgctc
gccgtttaac gggtgatcag actgccgctc tgcgtgattc 300 tcttagcgat
aaacctgcaa aaaatattat tttgctgatt ggcgatggga tgggggactc 360
ggaaattact gccgcacgta attatgccga aggtgcgggc ggctttttta aaggtataga
420 tgccttaccg cttaccgggc aatacactca ctatgcgctg aataaaaaaa
ccggcaaacc 480 ggactacgtc accgactcgg ctgcatcagc aaccgcctgg
tcaaccggtg tcaaaaccta 540 taacggcgcg ctgggcgtcg atattcacga
aaaagatcac ccaacgattc tggaaatggc 600 aaaagccgca ggtctggcga
ccggtaacgt ttctaccgca gagttgcagg atgccacgcc 660 cgctgcgctg
gtggcacatg tgacctcgcg caaatgctac ggtccgagcg cgaccagtga 720
aaaatgtccg ggtaacgctc tggaaaaagg cggaaaagga tcgattaccg aacagctgct
780 taacgctcgt gccgacgtta cgcttggcgg cggcgcaaaa acctttgctg
aaacggcaac 840 cgctggtgaa tggcagggaa aaacgctgcg tgaacaggca
caggcgcgtg gttatcagtt 900 ggtgagcgat gctgcctcac tgaattcggt
gacggaagcg aatcagcaaa aacccctgct 960 tggcctgttt gctgacggca
atatgccagt gcgctggcta ggaccgaaag caacgtacca 1020 tggcaatatc
gataagcccg cagtcacctg tacgccaaat ccgcaacgta atgacagtgt 1080
accaaccctg gcgcagatga ccgacaaagc cattgaattg ttgagtaaaa atgagaaagg
1140 ctttttcctg caagttgaag gtgcgtcaat cgataaacag gatcatgctg
cgaatccttg 1200 tgggcaaatt ggcgagacgg tcgatctcga tgaagccgta
caacgggcgc tggaattcgc 1260 taaaaaggag ggtaacacgc tggtcatagt
caccgctgat cacgcccacg ccagccagat 1320 tgttgcgccg gataccaaag
ctccgggcct cacccaggcg ctaaatacca aagatggcgc 1380 agtgatggtg
atgagttacg ggaactccga agaggattca caagaacata ccggcagtca 1440
gttgcgtatt gcggcgtatg gcccgcatgc cgccaatgtt gttggactga ccgaccagac
1500 cgatctcttc tacaccatga aagccgctct ggggctgaaa tctaga 1546
<210> SEQ ID NO 24 <211> LENGTH: 148 <212> TYPE:
DNA <213> ORGANISM: E. coli <400> SEQUENCE: 24
gaattcaggc gctttttaga ctggtcgtaa tgaaattcag caggatcaca ttctgcagat
60 gaaaataaaa acaggtgcac gcatcctcgc attatccgca ttaacgacga
tgatgttttc 120 cgcctcggct ctcgccaaaa tctctaga 148 <210> SEQ
ID NO 25 <211> LENGTH: 1174 <212> TYPE: DNA <213>
ORGANISM: E. coli <400> SEQUENCE: 25 gaattcaggc gctttttaga
ctggtcgtaa tgaaattcag caggatcaca ttctgcagat 60 gaaaataaaa
acaggtgcac gcatcctcgc attatccgca ttaacgacga tgatgttttc 120
cgcctcggct ctcgccaaaa tcgaagaagg taaactggta atctggatta acggcgataa
180 aggctataac ggtctcgctg aagtcggtaa gaaattcgag aaagataccg
gaattaaagt 240 caccgttgag catccggata aactggaaga gaaattccca
caggttgcgg caactggcga 300 tggccctgac attatcttct gggcacacga
ccgctttggt ggctacgctc aatctggcct 360 gttggctgaa atcaccccgg
acaaagcgtt ccaggacaag ctgtatccgt ttacctggga 420 tgccgtacgt
tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct 480
gatttataac aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct
540 ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc
aagaaccgta 600 cttcacctgg ccgctgattg ctgctgacgg gggttatgcg
ttcaagtatg aaaacggcaa 660 gtacgacatt aaagacgtgg gcgtggataa
cgctggcgcg aaagcgggtc tgaccttcct 720 ggttgacctg attaaaaaca
aacacatgaa tgcagacacc gattactcca tcgcagaagc 780 tgcctttaat
aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat 840
cgacaccagc aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc
900 caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc gccagtccga
acaaagagct 960 ggcgaaagag ttcctcgaaa actatctgct gactgatgaa
ggtctggaag cggttaataa 1020 agacaaaccg ctgggtgccg tagcgctgaa
gtcttacgag gaagagttgg cgaaagatcc 1080 acgtattgcc gccaccatgg
aaaacgccca gaaaggtgaa atcatgccga acatcccgca 1140 gatgtccgct
ttctggtatg ccgtgcgttc taga 1174 <210> SEQ ID NO 26
<211> LENGTH: 3840 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic nucleic acid sequence <400>
SEQUENCE: 26 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
atcaggcgcc 60 atacgcgaca gcgcgcaata accgttctcg actcataaaa
gtgatgccgc tataatgccg 120 cgtcctattt gaatgctttc gggatgattc
tggtaacagg gaatgtgatt gattataaga 180 acatcccggt tccgcgaagc
caacaacctg tgcttgcggg gtaagagttg accgagcact 240 gtgatttttt
gaggtaacaa gatgcaagtt tcagttgaaa ccactcaagg ccttggccgc 300
cgtgtaacga ttactatcgc tgctgacagc atcgagaccg ctgttaaaag cgagctggtc
360 aacgttgcga aaaaagtacg tattgacggc ttccgcaaag gcaaagtgcc
aatgaatatc 420 gttgctcagc gttatggcgc gtctgtacgc caggacgttc
tgggtgacct gatgagccgt 480 aacttcattg acgccatcat taaagaaaaa
atcaatccgg ctggcgcacc gacttatgtt 540 ccgggcgaat acaagctggg
tgaagacttc acttactctg tagagtttga agtttatccg 600 gaagttgaac
tgcagggtct ggaagcgatc gaagttgaaa aaccgatcgt tgaagtgacc 660
gacgctgacg ttgacggcat gctggatact ctgcgtaaac agcaggcgac ctggaaagaa
720 aaagacggcg ctgttgaagc agaagaccgc gtaaccatcg acttcaccgg
ttctgtagac 780 ggcgaagagt tcgaaggcgg taaagcgtct gatttcgtac
tggcgatggg ccagggtcgt 840 atgatcccgg gctttgaaga cggtatcaaa
ggccacaaag ctggcgaaga gttcaccatc 900 gacgtgacct tcccggaaga
ataccacgca gaaaacctga aaggtaaagc agcgaaattc 960 gctatcaacc
tgaagaaagt tgaagagcgt gaactgccgg aactgactgc agaattcatc 1020
aaacgtttcg gcgttgaaga tggttccgta gaaggtctgc gcgctgaagt gcgtaaaaac
1080 atggagcgcg agctgaagag cgccatccgt aaccgcgtta agtctcaggc
gatcgaaggt 1140 ctggtaaaag ctaacgacat cgacgtaccg gctgcgctga
tcgacagcga aatcgacgtt 1200 ctgcgtcgcc aggctgcaca gcgtttcggt
ggcaacgaaa aacaagctct ggaactgccg 1260 cgcgaactgt tcgaagaaca
ggctaaacgc cgcgtagttg ttggcctgct gctgggcgaa 1320 gttatccgca
ccaacgagct gaaagctgac gaagagcgcg tgaaaggcct gatcgaagag 1380
atggcttctg cgtacgaaga tccgaaagaa gttatcgagt tctacagcaa aaacaaagaa
1440 ctgatggaca acatgcgcaa tgttgctctg gaagaacagg ctgttgaagc
tgtactggcg 1500 aaagcgaaag tgactgaaaa agaaaccact ttcaacgagc
tgatgaacca gcaggcgtaa 1560 taataatcta gaggtagcac aatcagattc
gcttatgacg gcgatgaaga aattgcgatg 1620 aaatgtgagg tgaatcaggg
ttttcacccg attttgtgct gatcagaatt ttttttcttt 1680 ttcccccttg
aaggggcgaa gcctcatccc catttctctg gtcaccagcc gggaaaccac 1740
gtaagctccg gcgtcaccca taacagatac ggactttctc aaaggagagt tatcaatgaa
1800 tattcgtcca ttgcatgatc gcgtgatcgt caagcgtaaa gaagttgaaa
ctaaatctgc 1860 tggcggcatc gttctgaccg gctctgcagc ggctaaatcc
acccgcggcg aagtgctggc 1920 tgtcggcaat ggccgtatcc ttgaaaatgg
cgaagtgaag ccgctggatg tgaaagttgg 1980 cgacatcgtt attttcaacg
atggctacgg tgtgaaatct gagaagatcg acaatgaaga 2040 agtgttgatc
atgtccgaaa gcgacattct ggcaattgtt gaagcgtaat ccgcgcacga 2100
cactgaacat acgaatttaa ggaataaaga taatggcagc taaagacgta aaattcggta
2160 acgacgctcg tgtgaaaatg ctgcgcggcg taaacgtact ggcagatgca
gtgaaagtta 2220 ccctcggtcc aaaaggccgt aacgtagttc tggataaatc
tttcggtgca ccgaccatca 2280 ccaaagatgg tgtttccgtt gctcgtgaaa
tcgaactgga agacaagttc gaaaatatgg 2340 gtgcgcagat ggtgaaagaa
gttgcctcta aagcaaacga cgctgcaggc gacggtacca 2400 ccactgcaac
cgtactggct caggctatca tcactgaagg tctgaaagct gttgctgcgg 2460
gcatgaaccc gatggacctg aaacgtggta tcgacaaagc ggttaccgct gcagttgaag
2520 aactgaaagc gctgtccgta ccatgctctg actctaaagc gattgctcag
gttggtacca 2580 tctccgctaa ctccgacgaa accgtaggta aactgatcgc
tgaagcgatg gacaaagtcg 2640 gtaaagaagg cgttatcacc gttgaagacg
gtaccggtct gcaggacgaa ctggacgtgg 2700 ttgaaggtat gcagttcgac
cgtggctacc tgtctcctta cttcatcaac aagccggaaa 2760 ctggcgcagt
agaactggaa agcccgttca tcctgctggc tgacaagaaa atctccaaca 2820
tccgcgaaat gctgccggtt ctggaagctg ttgccaaagc aggcaaaccg ctgctgatca
2880 tcgctgaaga tgtagaaggc gaagcgctgg caactctggt tgttaacacc
atgcgtggca 2940 tcgtgaaagt cgctgcggtt aaagcaccgg gcttcggcga
tcgtcgtaaa gctatgctgc 3000 aggatatcgc aaccctgact ggcggtaccg
tgatctctga agagatcggt atggagctgg 3060 aaaaagcaac cctggaagac
ctgggtcagg ctaaacgtgt tgtgatcaac aaagacacca 3120 ccactatcat
cgatggcgtg ggtgaagaag ctgcaatcca gggccgtgtt gctcagatcc 3180
gtcagcagat tgaagaagca acttctgact acgaccgtga aaaactgcag gaacgcgtag
3240 cgaaactggc aggcggcgtt gcagttatca aagtgggtgc tgctaccgaa
gttgaaatga 3300 aagagaaaaa agcacgcgtt gaagatgccc tgcacgcgac
ccgtgctgcg gtagaagaag 3360 gcgtggttgc tggtggtggt gttgcgctga
tccgcgtagc gtctaaactg gctgacctgc 3420 gtggtcagaa cgaagaccag
aacgtgggta tcaaagttgc actgcgtgca atggaagctc 3480 cgctgcgtca
gatcgtattg aactgcggcg aagaaccgtc tgttgttgct aacaccgtta 3540
aaggcggcga cggcaactac ggttacaacg cagcaaccga agaatacggc aacatgatcg
3600 acatgggtat cctggatcca accaaagtaa ctcgttctgc tctgcagtac
gcagcttctg 3660 tggctggcct gatgatcacc accgaatgca tggttaccga
cctgccgaaa aacgatgcag 3720 ctgacttagg cgctgctggc ggtatgggcg
gcatgggtgg catgggcggc atgatgtaat 3780 aataagcttg catgcctgca
ggtcgactct agaggatccc cgggtaccga gctcgaattc 3840 <210> SEQ ID
NO 27 <211> LENGTH: 480 <212> TYPE: DNA <213>
ORGANISM: E. coli <400> SEQUENCE: 27 gaattcaggc gctttttaga
ctggtcgtaa tgaaattcag caggatcaca ttctgcagat 60 gatcgaagcc
cgctctagac tcgagagcga taaaattatt cacctgactg acgacagttt 120
tgacacggat gtactcaaag cggacggggc gatcctcgtc gatttctggg cagagtggtg
180 cggtccgtgc aaaatgatcg ccccgattct ggatgaaatc gctgacgaat
atcagggcaa 240 actgaccgtt gcaaaactga acatcgatca aaaccctggc
actgcgccga aatatggcat 300 ccgtggtatc ccgactctgc tgctgttcaa
aaacggtgaa gtggcggcaa ccaaagtggg 360 tgcactgtct aaaggtcagt
tgaaagagtt cctcgacgct aacctggcgc tcgaggatta 420 taaagatcat
gatggcgatt ataaagatca tgatgattaa taaggatccc cgggtaccga 480
<210> SEQ ID NO 28 <211> LENGTH: 1140 <212> TYPE:
DNA <213> ORGANISM: Rattus norvegicus <400> SEQUENCE:
28 atggcaacca cgcacgcgca ggggcacccg ccagtcttgg ggaatgatac
tctccgggaa 60 cattatgatt acgtggggaa gctggcaggc aggctgcggg
atccccctga gggtagcacc 120 ctcatcacca ccatcctctt cttggtcacc
tgtagcttca tcgtcttgga gaacctgatg 180 gttttgattg ccatctggaa
aaacaataaa tttcataacc gcatgtactt tttcatcggc 240 aacttggctc
tctgcgacct gctggccggc atagcctaca aggtcaacat tctgatgtcc 300
ggtaggaaga cgttcagcct gtctccaaca gtgtggttcc tcagggaggg cagtatgttc
360 gtagccctgg gcgcatccac atgcagctta ttggccattg ccattgagcg
gcacctgacc 420 atgatcaaga tgaggccgta cgacgccaac aagaagcacc
gcgtgttcct tctgattggg 480 atgtgctggc taattgcctt ctcgctgggt
gccctgccca tcctgggctg gaactgcctg 540 gagaactttc ccgactgctc
taccatcttg cccctctact ccaagaaata cattgccttt 600 ctcatcagca
tcttcacagc cattctggtg accatcgtca tcttgtacgc gcgcatctac 660
ttcctggtca agtccagcag ccgcagggtg gccaaccaca actccgagag atccatggcc
720 cttctgcgga ccgtagtgat cgtggtgagc gtgttcatcg cctgttggtc
cccccttttc 780 atcctcttcc tcatcgatgt ggcctgcagg gcgaaggagt
gctccatcct cttcaagagt 840 cagtggttca tcatgctggc tgtcctcaac
tcggccatga accctgtcat ctacacgctg 900 gccagcaaag agatgcggcg
tgctttcttc cggttggtgt gcggctgtct ggtcaagggc 960 aaggggaccc
aggcctcccc gatgcagcct gctcttgacc cgagcagaag taaatcaagc 1020
tccagtaaca acagcagcag ccactctcca aaggtcaagg aagacctgcc ccatgtggct
1080 acctcttcct gcgtcactga caaaacgagg tcgcttcaga atggggtcct
ctgcaagtga 1140 <210> SEQ ID NO 29 <211> LENGTH: 379
<212> TYPE: PRT <213> ORGANISM: Rattus norvegicus
<400> SEQUENCE: 29 Met Ala Thr Thr His Ala Gln Gly His Pro
Pro Val Leu Gly Asn Asp 1 5 10 15 Thr Leu Arg Glu His Tyr Asp Tyr
Val Gly Lys Leu Ala Gly Arg Leu 20 25 30 Arg Asp Pro Pro Glu Gly
Ser Thr Leu Ile Thr Thr Ile Leu Phe Leu 35 40 45 Val Thr Cys Ser
Phe Ile Val Leu Glu Asn Leu Met Val Leu Ile Ala 50 55 60 Ile Trp
Lys Asn Asn Lys Phe His Asn Arg Met Tyr Phe Phe Ile Gly 65 70 75 80
Asn Leu Ala Leu Cys Asp Leu Leu Ala Gly Ile Ala Tyr Lys Val Asn 85
90 95 Ile Leu Met Ser Gly Arg Lys Thr Phe Ser Leu Ser Pro Thr Val
Trp 100 105 110 Phe Leu Arg Glu Gly Ser Met Phe Val Ala Leu Gly Ala
Ser Thr Cys 115 120 125 Ser Leu Leu Ala Ile Ala Ile Glu Arg His Leu
Thr Met Ile Lys Met 130 135 140 Arg Pro Tyr Asp Ala Asn Lys Lys His
Arg Val Phe Leu Leu Ile Gly 145 150 155 160 Met Cys Trp Leu Ile Ala
Phe Ser Leu Gly Ala Leu Pro Ile Leu Gly 165 170 175 Trp Asn Cys Leu
Glu Asn Phe Pro Asp Cys Ser Thr Ile Leu Pro Leu 180 185 190 Tyr Ser
Lys Lys Tyr Ile Ala Phe Leu Ile Ser Ile Phe Thr Ala Ile 195 200 205
Leu Val Thr Ile Val Ile Leu Tyr Ala Arg Ile Tyr Phe Leu Val Lys 210
215 220 Ser Ser Ser Arg Arg Val Ala Asn His Asn Ser Glu Arg Ser Met
Ala 225 230 235 240 Leu Leu Arg Thr Val Val Ile Val Val Ser Val Phe
Ile Ala Cys Trp 245 250 255 Ser Pro Leu Phe Ile Leu Phe Leu Ile Asp
Val Ala Cys Arg Ala Lys 260 265 270 Glu Cys Ser Ile Leu Phe Lys Ser
Gln Trp Phe Ile Met Leu Ala Val 275 280 285 Leu Asn Ser Ala Met Asn
Pro Val Ile Tyr Thr Leu Ala Ser Lys Glu 290 295 300 Met Arg Arg Ala
Phe Phe Arg Leu Val Cys Gly Cys Leu Val Lys Gly 305 310 315 320 Lys
Gly Thr Gln Ala Ser Pro Met Gln Pro Ala Leu Asp Pro Ser Arg 325 330
335 Ser Lys Ser Ser Ser Ser Asn Asn Ser Ser Ser His Ser Pro Lys Val
340 345 350 Lys Glu Asp Leu Pro His Val Ala Thr Ser Ser Cys Val Thr
Asp Lys 355 360 365 Thr Arg Ser Leu Gln Asn Gly Val Leu Cys Lys 370
375 <210> SEQ ID NO 30 <400> SEQUENCE: 30 000
<210> SEQ ID NO 31 <211> LENGTH: 32 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleic acid primer
sequence <400> SEQUENCE: 31 aattgctagc tccaccagca tcccagtggt
ta 32 <210> SEQ ID NO 32 <211> LENGTH: 32 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic nucleic acid
primer sequence <400> SEQUENCE: 32 aattggatcc ttaagaagaa
gaattgacgt tt 32 <210> SEQ ID NO 33 <211> LENGTH: 32
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleic acid primer sequence <400> SEQUENCE: 33 aattggatcc
agaagaagaa ttgacgtttc ca 32 <210> SEQ ID NO 34 <211>
LENGTH: 52 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleic acid primer sequence <400> SEQUENCE: 34
aattggatcc ttaatgatga tgatgatgat gagaagaaga attgacgttt cc 52
<210> SEQ ID NO 35 <211> LENGTH: 24 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleic acid primer
sequence <400> SEQUENCE: 35 ttatggcaac cacgcacgcg cagg 24
<210> SEQ ID NO 36 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleic acid primer
sequence <400> SEQUENCE: 36 agaccgtcac ttgcagagga c 21
<210> SEQ ID NO 37 <211> LENGTH: 32 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleic acid primer
sequence <400> SEQUENCE: 37 aattgctagc acgcacgcgc aggggcaccc
gc 32 <210> SEQ ID NO 38 <211> LENGTH: 34 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic nucleic acid
primer sequence <400> SEQUENCE: 38 aattggtacc tcacttgcag
aggaccccat tctg 34 <210> SEQ ID NO 39 <211> LENGTH: 32
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleic acid primer sequence <400> SEQUENCE: 39 aattgctagc
acgcacgcgc aggggcaccc gc 32 <210> SEQ ID NO 40 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleic acid primer sequence <400> SEQUENCE: 40
ggtcgccacc atggtgagca a 21 <210> SEQ ID NO 41 <211>
LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleic acid primer sequence <400> SEQUENCE: 41
ttaaggatcc ttacttgtac agctcgtcca t 31 <210> SEQ ID NO 42
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic nucleic acid primer sequence <400>
SEQUENCE: 42 ggtcgccacc atggtgagca a 21 <210> SEQ ID NO 43
<211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic nucleic acid primer sequence <400>
SEQUENCE: 43 ttaaggatcc cttgtacagc tcgtccatgc c 31 <210> SEQ
ID NO 44 <211> LENGTH: 25 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 44 ccaatggaac ttaccaatga cgcgg 25 <210>
SEQ ID NO 45 <211> LENGTH: 24 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 45 gcttgcttac gcaggaatgc tggg 24
<210> SEQ ID NO 46 <211> LENGTH: 45 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 46 cgcggctgca gatgtttgaa ccaatggaac
ttaccaatga cgcgg 45 <210> SEQ ID NO 47 <211> LENGTH: 45
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 47 gcgcctctag
attattaatc agcttgctta cgcaggaatg ctggg 45 <210> SEQ ID NO 48
<211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 48 gctagactgg gcggttttat ggacagcaag c 31 <210> SEQ
ID NO 49 <211> LENGTH: 35 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 49 gcgttaataa ttcagaagaa ctcgtcaaga aggcg 35
<210> SEQ ID NO 50 <211> LENGTH: 99 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 50 gcgcctactg acgtagttcg accgtcggac
tagcgaagtt cctatacttt ctagagaata 60 ggaacttcgc tagactgggc
ggttttatgg acagcaagc 99 <210> SEQ ID NO 51 <211>
LENGTH: 109 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 51
caagatgctt tgcctttgtc tgagttgata ctggctttgg gaagttccta ttctctagaa
60 agtataggaa cttcgcgtta ataattcaga agaactcgtc aagaaggcg 109
<210> SEQ ID NO 52 <211> LENGTH: 26 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 52 cgttaccaat tatgacaact tgacgg 26
<210> SEQ ID NO 53 <211> LENGTH: 29 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 53 ttaatctttc tgcgaattga gatgacgcc
29 <210> SEQ ID NO 54 <211> LENGTH: 28 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 54 gtgagtcgat attgtctttg
ttgaccag 28 <210> SEQ ID NO 55 <211> LENGTH: 66
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 55 gcctgcattg
cggcgcttca gtctccgctg catactgtcc cgttaccaat tatgacaact 60 tgacgg 66
<210> SEQ ID NO 56 <211> LENGTH: 69 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 56 gcctgcattg cggcgcttca gtctccgctg
catactgtcc ttaatctttc tgcgaattga 60 gatgacgcc 69 <210> SEQ ID
NO 57 <211> LENGTH: 76 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 57 gcctgcattg cggcgcttca gtctccgctg
catactgtcc ttaataaagt gagtcgatat 60 tgtctttgtt gaccag 76
<210> SEQ ID NO 58 <211> LENGTH: 66 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 58 gcctgcattg cggcgcttca gtctccgctg
catactgtcc cgttaccaat tatgacaact 60 tgacgg 66 <210> SEQ ID NO
59 <211> LENGTH: 30 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 59 gcctgttctg gaaaaccggg ctgctcaggg 30
<210> SEQ ID NO 60 <211> LENGTH: 28 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 60 gcggctttca tggtgtagaa gagatcgg 28
<210> SEQ ID NO 61 <211> LENGTH: 43 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 61 ccgcgctgca gatgcctgtt ctggaaaacc
gggctgctca ggg 43 <210> SEQ ID NO 62 <211> LENGTH: 58
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 62 gcgcctctag
attattattt cagccccaga gcggctttca tggtgtagaa gagatcgg 58 <210>
SEQ ID NO 63 <211> LENGTH: 24 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 63 gtcacggccg agacttatag tcgc 24
<210> SEQ ID NO 64 <211> LENGTH: 28 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 64 gcggctttca tggtgtagaa gagatcgg 28
<210> SEQ ID NO 65 <211> LENGTH: 37 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 65 ccgcgctgca gatgtcacgg ccgagactta
tagtcgc 37 <210> SEQ ID NO 66 <211> LENGTH: 58
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 66 gcgcctctag
attattattt cagccccaga gcggctttca tggtgtagaa gagatcgg 58 <210>
SEQ ID NO 67 <211> LENGTH: 109 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 67 ccgcgctgca gatgaacttg gggaatcgac
tgtttattct gatagcggtc ttacttcccc 60 tcgcagtatt actgctcatg
cctgttctgg aaaaccgggc tgctcaggg 109 <210> SEQ ID NO 68
<211> LENGTH: 58 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 68 gcgcctctag attattattt cagccccaga gcggctttca tggtgtagaa
gagatcgg 58 <210> SEQ ID NO 69 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 69 gcgaattgag
atgacgccac tggc 24 <210> SEQ ID NO 70 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 70 cctgctgaat
ttcattaacg accag 25 <210> SEQ ID NO 71 <211> LENGTH: 48
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 71 cggcgaagct
taattaatct ttctgcgaat tgagatgacg ccactggc 48 <210> SEQ ID NO
72 <211> LENGTH: 53 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 72 cgccgtaatc gccgctgcag aatgtgatcc
tgctgaattt cattaacgac cag 53 <210> SEQ ID NO 73 <211>
LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 73
cgcagcgctg ttcctttgct cg 22 <210> SEQ ID NO 74 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 74
cctcattaag ataataatac tgg 23 <210> SEQ ID NO 75 <211>
LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 75
gccgcaagct tcgcagcgct gttcctttgc tcg 33 <210> SEQ ID NO 76
<211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 76 ccaatgcatt ggttctgcag gactcctcat taagataata atactgg 47
<210> SEQ ID NO 77 <211> LENGTH: 19 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 77 cgtctttagc cgggaaacg 19
<210> SEQ ID NO 78 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 78 gcagatctcc tggcttgc 18
<210> SEQ ID NO 79 <211> LENGTH: 30 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 79 gccgcaagct tcgtctttag ccgggaaacg
30 <210> SEQ ID NO 80 <211> LENGTH: 26 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 80 cggtcgacgc agatctcctg
gcttgc 26 <210> SEQ ID NO 81 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 81 caagccgtca
attgtctgat tcg 23 <210> SEQ ID NO 82 <211> LENGTH: 22
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 82 ggtgaattcc
tcctgctagc cc 22 <210> SEQ ID NO 83 <211> LENGTH: 34
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 83 gcgccaagct
tcaagccgtc aattgtctga ttcg 34 <210> SEQ ID NO 84 <211>
LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 84
ctgcagggtg aattcctcct gctagccc 28 <210> SEQ ID NO 85
<211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 85 gcttaactcg agcttaataa caagccgtca attgtctgat tc 42
<210> SEQ ID NO 86 <211> LENGTH: 34 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 86 gcttaaccgc gggccaagct tgcatgcctg
ctcc 34 <210> SEQ ID NO 87 <211> LENGTH: 26 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 87 ggcaaccacg cacgcgcagg
gccacc 26 <210> SEQ ID NO 88 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 88 caatggtgat
ggtgatgatg accgg 25 <210> SEQ ID NO 89 <211> LENGTH: 38
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 89 cgcggtcgac
atggcaacca cgcacgcgca gggccacc 38 <210> SEQ ID NO 90
<211> LENGTH: 40 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 90 gcgccggtac cttatcaatg gtgatggtga tgatgaccgg 40
<210> SEQ ID NO 91 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 91 ggggcaaccc gggaacggca gcgcc 25
<210> SEQ ID NO 92 <211> LENGTH: 30 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 92 gcagtgagtc atttgtacta caattcctcc
30 <210> SEQ ID NO 93 <211> LENGTH: 37 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 93 cgcggtcgac atggggcaac
ccgggaacgg cagcgcc 37 <210> SEQ ID NO 94 <211> LENGTH:
49 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 94 gcgccggatc
cttattatag cagtgagtca tttgtactac aattcctcc 49 <210> SEQ ID NO
95 <211> LENGTH: 28 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 95 ggactggtcc ctcacctagg ggacaggg 28
<210> SEQ ID NO 96 <211> LENGTH: 27 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 96 ctgagaagac tgggcgcggg cgggagg 27
<210> SEQ ID NO 97 <211> LENGTH: 42 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 97 cgcgggtcga catgggactg gtccctcacc
taggggacag gg 42 <210> SEQ ID NO 98 <211> LENGTH: 44
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 98 gcgccggtac
cttattactg agaagactgg gcgcgggcgg gagg 44 <210> SEQ ID NO 99
<211> LENGTH: 16 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 99 gatagtgtgt gtcccc 16 <210> SEQ ID NO 100
<211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 100 ctgagaagac tgggcgc 17 <210> SEQ ID NO 101
<211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 101 gggagaccat ggatagtgtg tgtcccc 27 <210> SEQ ID
NO 102 <211> LENGTH: 32 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 102 gcctcatcta gattactgag aagactgggc gc 32
<210> SEQ ID NO 103 <211> LENGTH: 26 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 103 gagcactgaa agcatgatcc gggacg 26
<210> SEQ ID NO 104 <211> LENGTH: 28 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 104 cagggcaatg atcccaaagt agacctgc
28 <210> SEQ ID NO 105 <211> LENGTH: 39 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 105 ccgcggaatt catgagcact
gaaagcatga tccgggacg 39 <210> SEQ ID NO 106 <211>
LENGTH: 43 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 106
ggcgcaagct tatcacaggg caatgatccc aaagtagacc tgc 43 <210> SEQ
ID NO 107 <211> LENGTH: 110 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 107 tctgatagcg gtcttacttc ccctcgcagt
attactgctc aatagtgact ctgaatgtcc 60 cctgtcccac gatgggtact
gcctccatga tggtgtgtgc atgtatattg 110 <210> SEQ ID NO 108
<211> LENGTH: 101 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 108 aggtctcggt actgacatcg ctccccgatg tagccaacaa
cacagttgca tgcatacttg 60 tccaatgctt caatatacat gcacacacca
tcatggaggc a 101 <210> SEQ ID NO 109 <211> LENGTH: 63
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 109
ccgcgggtac catgaacttg gggaatcgac tgtttattct gatagcggtc ttacttcccc
60 tcg 63 <210> SEQ ID NO 110 <211> LENGTH: 61
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 110
gcgccaagct tattagcgca gttcccacca cttcaggtct cggtactgac atcgctcccc
60 g 61 <210> SEQ ID NO 111 <211> LENGTH: 22
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 111
tcattcacat tgagcgtcac cg 22 <210> SEQ ID NO 112 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 112
ttatattgac agcgcacaga gcgg 24 <210> SEQ ID NO 113 <211>
LENGTH: 101 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 113
gcaagaattc accatgaact tggggaatcg actgtttatt ctgatagcgg tcttacttcc
60 cctcgcagta ttactgctct cattcacatt gagcgtcacc g 101 <210>
SEQ ID NO 114 <211> LENGTH: 47 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 114 cgcggttacg taagcaactg cagttatatt
gacagcgcac agagcgg 47 <210> SEQ ID NO 115 <211> LENGTH:
24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 115
gtcacggccg agacttatag tcgc 24 <210> SEQ ID NO 116 <400>
SEQUENCE: 116 000 <210> SEQ ID NO 117 <211> LENGTH: 37
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 117
cgcggctgca gatgtcacgg ccgagactta tagtcgc 37 <210> SEQ ID NO
118 <211> LENGTH: 38 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 118 cgcggtctag attctggtgt ccgggctttt gtcacagg
38 <210> SEQ ID NO 119 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 119 cagccccaga gcggctttca tgg
23 <210> SEQ ID NO 120 <211> LENGTH: 37 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 120 cgcggtctag atttcagccc
cagagcggct ttcatgg 37 <210> SEQ ID NO 121 <211> LENGTH:
106 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 121
cgcggctgca gatgaaaata aaaacaggtg cacgcatcct cgcattatcc gcattaacga
60 cgatgatgtt ttccgcctcg gctctcgcca aaatctctag acgcgg 106
<210> SEQ ID NO 122 <211> LENGTH: 106 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 122 ccgcgtctag agattttggc gagagccgag
gcggaaaaca tcatcgtcgt taatgcggat 60 aatgcgagga tgcgtgcacc
tgtttttatt ttcatctgca gccgcg 106 <210> SEQ ID NO 123
<211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 123 ggtgcacgca tcctcgcatt atccgc 26 <210> SEQ ID NO
124 <211> LENGTH: 26 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 124 cggcatacca gaaagcggac atctgc 26
<210> SEQ ID NO 125 <211> LENGTH: 52 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 125 cgcggctgca gatgaaaata aaaacaggtg
cacgcatcct cgcattatcc gc 52 <210> SEQ ID NO 126 <211>
LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 126
cgcggtctag aacgcacggc ataccagaaa gcggacatct gc 42 <210> SEQ
ID NO 127 <211> LENGTH: 27 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 127 cgcgacagcg cgcaataacc gttctcg 27
<210> SEQ ID NO 128 <211> LENGTH: 27 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 128 gctggttcat cagctcgttg aaagtgg 27
<210> SEQ ID NO 129 <211> LENGTH: 41 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 129 gcgccggcgc catacgcgac agcgcgcaat
aaccgttctc g 41 <210> SEQ ID NO 130 <211> LENGTH: 52
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 130
ggcgctctag attattatta cgcctgctgg ttcatcagct cgttgaaagt gg 52
<210> SEQ ID NO 131 <211> LENGTH: 29 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 131 ggtagcacaa tcagattcgc ttatgacgg
29 <210> SEQ ID NO 132 <211> LENGTH: 25 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 132 gccgcccatg ccacccatgc
cgccc 25 <210> SEQ ID NO 133 <211> LENGTH: 38
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 133
gcgtctagag gtagcacaat cagattcgct tatgacgg 38 <210> SEQ ID NO
134 <211> LENGTH: 49 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 134 ggcgcaagct tattattaca tcatgccgcc
catgccaccc atgccgccc 49 <210> SEQ ID NO 135 <211>
LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 135
gcgataaaat tattcacctg actgacg 27 <210> SEQ ID NO 136
<211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 136 gcgtcgagga actctttcaa ctgacc 26 <210> SEQ ID NO
137 <211> LENGTH: 66 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 137 cgcggctgca gatgatcgaa gcccgctcta
gactcgagag cgataaaatt attcacctga 60 ctgacg 66 <210> SEQ ID NO
138 <211> LENGTH: 101 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 138 ccgcgggatc cttattaatc atcatgatct
ttataatcgc catcatgatc tttataatcc 60 tcgagcgcca ggttagcgtc
gaggaactct ttcaactgac c 101 <210> SEQ ID NO 139 <400>
SEQUENCE: 139 000 <210> SEQ ID NO 140 <400> SEQUENCE:
140 000 <210> SEQ ID NO 141 <400> SEQUENCE: 141 000
<210> SEQ ID NO 142 <400> SEQUENCE: 142 000 <210>
SEQ ID NO 143 <211> LENGTH: 65 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 143 tatgtaagga ggttgtcgac cggctcagtc
tagaggtacc cgccctcatc cgaaagggcg 60 tattg 65 <210> SEQ ID NO
144 <211> LENGTH: 67 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 144 gatccaatac gccctttcgg atgagggcgg
gtacctctag actgagccgg tcgacaacct 60 ccttaca 67 <210> SEQ ID
NO 145 <211> LENGTH: 65 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 145 tatgtaagga ggttctgcag cggctcagtc
tagaggtacc cgccctcatc cgaaagggcg 60 tattg 65 <210> SEQ ID NO
146 <211> LENGTH: 67 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 146 gatccaatac gccctttcgg atgagggcgg
gtacctctag actgagccgc tgcagaacct 60 ccttaca 67 <210> SEQ ID
NO 147 <211> LENGTH: 66 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 147 gatcctaagg aggttgtcga ccggctcagt
ctagaggtac ccgccctcat ccgaaagggc 60 gtattc 66 <210> SEQ ID NO
148 <211> LENGTH: 66 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 148 tcgagaatac gccctttcgg atgagggcgg
gtacctctag actgagccgg tcgacaacct 60 ccttag 66 <210> SEQ ID NO
149 <211> LENGTH: 66 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 149 gatcctaagg aggttctgca gcggctcagt
ctagaggtac ccgccctcat ccgaaagggc 60 gtattc 66 <210> SEQ ID NO
150 <211> LENGTH: 66 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 150 tcgagaatac gccctttcgg atgagggcgg
gtacctctag actgagccgc tgcagaacct 60 ccttag 66 <210> SEQ ID NO
151 <211> LENGTH: 4740 <212> TYPE: DNA <213>
ORGANISM: E. coli <400> SEQUENCE: 151 tcgcgcgttt cggtgatgac
ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct
gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc
ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg
caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt
aaaacgacgg ccagtgccaa gcttaattaa tctttctgcg 420 aattgagatg
acgccactgg ctgggcgtca tcccggtttc ccgggtaaac accaccgaaa 480
aatagttact atcttcaaag ccacattcgg tcgaaatatc actgattaac aggcggctat
540 gctggagaag atattgcgca tgacacactc tgacctgtcg cagatattga
ttgatggtca 600 ttccagtctg ctggcgaaat tgctgacgca aaacgcgctc
actgcacgat gcctcatcac 660 aaaatttatc cagcgcaaag ggacttttca
ggctagccgc cagccgggta atcagcttat 720 ccagcaacgt ttcgctggat
gttggcggca acgaatcact ggtgtaacga tggcgattca 780 gcaacatcac
caactgcccg aacagcaact cagccatttc gttagcaaac ggcacatgct 840
gactactttc atgctcaagc tgaccgataa cctgccgcgc ctgcgccatc cccatgctac
900 ctaagcgcca gtgtggttgc cctgcgctgg cgttaaatcc cggaatcgcc
ccctgccagt 960 caagattcag cttcagacgc tccgggcaat aaataatatt
ctgcaaaacc agatcgttaa 1020 cggaagcgta ggagtgttta tcgtcagcat
gaatgtaaaa gagatcgcca cgggtaatgc 1080 gataagggcg atcgttgagt
acatgcaggc cattaccgcg ccagacaatc accagctcac 1140 aaaaatcatg
tgtatgttca gcaaagacat cttgcggata acggtcagcc acagcgactg 1200
cctgctggtc gctggcaaaa aaatcatctt tgagaagttt taactgatgc gccaccgtgg
1260 ctacctcggc cagagaacga agttgattat tcgcaatatg gcgtacaaat
acgttgagaa 1320 gattcgcgtt attgcagaaa gccatcccgt ccctggcgaa
tatcacgcgg tgaccagtta 1380 aactctcggc gaaaaagcgt cgaaaagtgg
ttactgtcgc tgaatccaca gcgataggcg 1440 atgtcagtaa cgctggcctc
gctgtggcgt agcagatgtc gggctttcat cagtcgcagg 1500 cggttcaggt
atcgctgagg cgtcagtccc gtttgctgct taagctgccg atgtagcgta 1560
cgcagtgaaa gagaaaattg atccgccacg gcatcccaat tcacctcatc ggcaaaatgg
1620 tcctccagcc aggccagaag caagttgaga cgtgatgcgc tgttttccag
gttctcctgc 1680 aaactgcttt tacgcagcaa gagcagtaat tgcataaaca
agatctcgcg actggcggtc 1740 gagggtaaat cattttcccc ttcctgctgt
tccatctgtg caaccagctg tcgcacctgc 1800 tgcaatacgc tgtggttaac
gcgccagtga gacggatact gcccatccag ctcttgtggc 1860 agcaactgat
tcagcccggc gagaaactga aatcgatccg gcgagcgata cagcacattg 1920
gtcagacaca gattatcggt atgttcatac agatgccgat catgatcgcg tacgaaacag
1980 accgtgccac cggtgatggt atagggctgc ccattaaaca catgaatacc
cgtgccatgt 2040 tcgacaatca caatttcatg aaaatcatga tgatgttcag
gaaaatccgc ctgcgggagc 2100 cggggttcta tcgccacgga cgcgttacca
gacggaaaaa aatccacact atgtaatacg 2160 gtcatactgg cctcctgatg
tcgtcaacac ggcgaaatag taatcacgag gtcaggttct 2220 taccttaaat
tttcgacgga aaaccacgta aaaaacgtcg atttttcaag atacagcgtg 2280
aattttcagg aaatgcggtg agcatcacat caccacaatt cagcaaattg tgaacatcat
2340 cacgttcatc tttccctggt tgccaatggc ccattttcct gtcagtaacg
agaaggtcgc 2400 gaattcaggc gctttttaga ctggtcgtaa tgaaattcag
gaggttgtcg actctagagg 2460 atccccgcgc cctcatccga aagggcgtat
tggtaccgag ctcgaattcg taatcatggt 2520 catagctgtt tcctgtgtga
aattgttatc cgctcacaat tccacacaac atacgagccg 2580 gaagcataaa
gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt 2640
tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
2700 gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
tcgctcactg 2760 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
agctcactca aaggcggtaa 2820 tacggttatc cacagaatca ggggataacg
caggaaagaa catgtgagca aaaggccagc 2880 aaaaggccag gaaccgtaaa
aaggccgcgt tgctggcgtt tttccatagg ctccgccccc 2940 ctgacgagca
tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat 3000
aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
3060 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
tctcatagct 3120 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
caagctgggc tgtgtgcacg 3180 aaccccccgt tcagcccgac cgctgcgcct
tatccggtaa ctatcgtctt gagtccaacc 3240 cggtaagaca cgacttatcg
ccactggcag cagccactgg taacaggatt agcagagcga 3300 ggtatgtagg
cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa 3360
ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
3420 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
tgcaagcagc 3480 agattacgcg cagaaaaaaa ggatctcaag aagatccttt
gatcttttct acggggtctg 3540 acgctcagtg gaacgaaaac tcacgttaag
ggattttggt catgagatta tcaaaaagga 3600 tcttcaccta gatcctttta
aattaaaaat gaagttttaa atcaatctaa agtatatatg 3660 agtaaacttg
gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct 3720
gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
3780 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
tcaccggctc 3840 cagatttatc agcaataaac cagccagccg gaagggccga
gcgcagaagt ggtcctgcaa 3900 ctttatccgc ctccatccag tctattaatt
gttgccggga agctagagta agtagttcgc 3960 cagttaatag tttgcgcaac
gttgttgcca ttgctacagg catcgtggtg tcacgctcgt 4020 cgtttggtat
ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc 4080
ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
4140 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
actgtcatgc 4200 catccgtaag atgcttttct gtgactggtg agtactcaac
caagtcattc tgagaatagt 4260 gtatgcggcg accgagttgc tcttgcccgg
cgtcaatacg ggataatacc gcgccacata 4320 gcagaacttt aaaagtgctc
atcattggaa aacgttcttc ggggcgaaaa ctctcaagga 4380 tcttaccgct
gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag 4440
catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
4500 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
tttcaatatt 4560 attgaagcat ttatcagggt tattgtctca tgagcggata
catatttgaa tgtatttaga 4620 aaaataaaca aataggggtt ccgcgcacat
ttccccgaaa agtgccacct gacgtctaag 4680 aaaccattat tatcatgaca
ttaacctata aaaataggcg tatcacgagg ccctttcgtc 4740 <210> SEQ ID
NO 152 <211> LENGTH: 4746 <212> TYPE: DNA <213>
ORGANISM: E. coli <400> SEQUENCE: 152 tcgcgcgttt cggtgatgac
ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct
gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc
ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg
caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt
aaaacgacgg ccagtgccaa gcttaattaa tctttctgcg 420 aattgagatg
acgccactgg ctgggcgtca tcccggtttc ccgggtaaac accaccgaaa 480
aatagttact atcttcaaag ccacattcgg tcgaaatatc actgattaac aggcggctat
540 gctggagaag atattgcgca tgacacactc tgacctgtcg cagatattga
ttgatggtca 600 ttccagtctg ctggcgaaat tgctgacgca aaacgcgctc
actgcacgat gcctcatcac 660 aaaatttatc cagcgcaaag ggacttttca
ggctagccgc cagccgggta atcagcttat 720 ccagcaacgt ttcgctggat
gttggcggca acgaatcact ggtgtaacga tggcgattca 780 gcaacatcac
caactgcccg aacagcaact cagccatttc gttagcaaac ggcacatgct 840
gactactttc atgctcaagc tgaccgataa cctgccgcgc ctgcgccatc cccatgctac
900 ctaagcgcca gtgtggttgc cctgcgctgg cgttaaatcc cggaatcgcc
ccctgccagt 960 caagattcag cttcagacgc tccgggcaat aaataatatt
ctgcaaaacc agatcgttaa 1020 cggaagcgta ggagtgttta tcgtcagcat
gaatgtaaaa gagatcgcca cgggtaatgc 1080 gataagggcg atcgttgagt
acatgcaggc cattaccgcg ccagacaatc accagctcac 1140 aaaaatcatg
tgtatgttca gcaaagacat cttgcggata acggtcagcc acagcgactg 1200
cctgctggtc gctggcaaaa aaatcatctt tgagaagttt taactgatgc gccaccgtgg
1260 ctacctcggc cagagaacga agttgattat tcgcaatatg gcgtacaaat
acgttgagaa 1320 gattcgcgtt attgcagaaa gccatcccgt ccctggcgaa
tatcacgcgg tgaccagtta 1380 aactctcggc gaaaaagcgt cgaaaagtgg
ttactgtcgc tgaatccaca gcgataggcg 1440 atgtcagtaa cgctggcctc
gctgtggcgt agcagatgtc gggctttcat cagtcgcagg 1500 cggttcaggt
atcgctgagg cgtcagtccc gtttgctgct taagctgccg atgtagcgta 1560
cgcagtgaaa gagaaaattg atccgccacg gcatcccaat tcacctcatc ggcaaaatgg
1620 tcctccagcc aggccagaag caagttgaga cgtgatgcgc tgttttccag
gttctcctgc 1680 aaactgcttt tacgcagcaa gagcagtaat tgcataaaca
agatctcgcg actggcggtc 1740 gagggtaaat cattttcccc ttcctgctgt
tccatctgtg caaccagctg tcgcacctgc 1800 tgcaatacgc tgtggttaac
gcgccagtga gacggatact gcccatccag ctcttgtggc 1860 agcaactgat
tcagcccggc gagaaactga aatcgatccg gcgagcgata cagcacattg 1920
gtcagacaca gattatcggt atgttcatac agatgccgat catgatcgcg tacgaaacag
1980 accgtgccac cggtgatggt atagggctgc ccattaaaca catgaatacc
cgtgccatgt 2040 tcgacaatca caatttcatg aaaatcatga tgatgttcag
gaaaatccgc ctgcgggagc 2100 cggggttcta tcgccacgga cgcgttacca
gacggaaaaa aatccacact atgtaatacg 2160 gtcatactgg cctcctgatg
tcgtcaacac ggcgaaatag taatcacgag gtcaggttct 2220 taccttaaat
tttcgacgga aaaccacgta aaaaacgtcg atttttcaag atacagcgtg 2280
aattttcagg aaatgcggtg agcatcacat caccacaatt cagcaaattg tgaacatcat
2340 cacgttcatc tttccctggt tgccaatggc ccattttcct gtcagtaacg
agaaggtcgc 2400 gaattcaggc gctttttaga ctggtcgtaa tgaaattcag
gaggttctgc aggtcgactc 2460 tagaggatcc ccgcgccctc atccgaaagg
gcgtattggt accgagctcg aattcgtaat 2520 catggtcata gctgtttcct
gtgtgaaatt gttatccgct cacaattcca cacaacatac 2580 gagccggaag
cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa 2640
ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
2700 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
gcttcctcgc 2760 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
ggtatcagct cactcaaagg 2820 cggtaatacg gttatccaca gaatcagggg
ataacgcagg aaagaacatg tgagcaaaag 2880 gccagcaaaa ggccaggaac
cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc 2940 gcccccctga
cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag 3000
gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
3060 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
gcgctttctc 3120 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
tcgctccaag ctgggctgtg 3180 tgcacgaacc ccccgttcag cccgaccgct
gcgccttatc cggtaactat cgtcttgagt 3240 ccaacccggt aagacacgac
ttatcgccac tggcagcagc cactggtaac aggattagca 3300 gagcgaggta
tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca 3360
ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
3420 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
tttgtttgca 3480 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
tcctttgatc ttttctacgg 3540 ggtctgacgc tcagtggaac gaaaactcac
gttaagggat tttggtcatg agattatcaa 3600 aaaggatctt cacctagatc
cttttaaatt aaaaatgaag ttttaaatca atctaaagta 3660 tatatgagta
aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag 3720
cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
3780 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
ccacgctcac 3840 cggctccaga tttatcagca ataaaccagc cagccggaag
ggccgagcgc agaagtggtc 3900 ctgcaacttt atccgcctcc atccagtcta
ttaattgttg ccgggaagct agagtaagta 3960 gttcgccagt taatagtttg
cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac 4020 gctcgtcgtt
tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat 4080
gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
4140 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
tctcttactg 4200 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
ctcaaccaag tcattctgag 4260 aatagtgtat gcggcgaccg agttgctctt
gcccggcgtc aatacgggat aataccgcgc 4320 cacatagcag aactttaaaa
gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct 4380 caaggatctt
accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat 4440
cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
4500 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
ttcctttttc 4560 aatattattg aagcatttat cagggttatt gtctcatgag
cggatacata tttgaatgta 4620 tttagaaaaa taaacaaata ggggttccgc
gcacatttcc ccgaaaagtg ccacctgacg 4680 tctaagaaac cattattatc
atgacattaa cctataaaaa taggcgtatc acgaggccct 4740 ttcgtc 4746
<210> SEQ ID NO 153 <211> LENGTH: 3946 <212>
TYPE: DNA <213> ORGANISM: E. coli <400> SEQUENCE: 153
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga
gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat
gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc
aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct
ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gcttcaagcc gtcaattgtc
420 tgattcgtta ccaattatga caacttgacg gctacatcat tcactttttc
ttcacaaccg 480 gcacggaact cgctcgggct ggccccggtg cattttttaa
atacccgcga gaaatagagt 540 tgatcgtcaa aaccaacatt gcgaccgacg
gtggcgatag gcatccgggt ggtgctcaaa 600 agcagcttcg cctggctgat
acgttggtcc tcgcgccagc ttaagacgct aatccctaac 660 tgctggcgga
aaagatgtga cagacgcgac ggcgacaagc aaacatgctg tgcgacgctg 720
gcgatatcaa aattgctgtc tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc
780 cgattatcca tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg
cagtaacaat 840 tgctcaagca gatttatcgc cagcagctcc gaatagcgcc
cttccccttg cccggcgtta 900 atgatttgcc caaacaggtc gctgaaatgc
ggctggtgcg cttcatccgg gcgaaagaac 960 cccgtattgg caaatattga
cggccagtta agccattcat gccagtaggc gcgcggacga 1020 aagtaaaccc
actggtgata ccattcgcga gcctccggat gacgaccgta gtgatgaatc 1080
tctcctggcg ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg tccctgattt
1140 ttcaccaccc cctgaccgcg aatggtgaga ttgagaatat aacctttcat
tcccagcggt 1200 cggtcgataa aaaaatcgag ataaccgttg gcctcaatcg
gcgttaaacc cgccaccaga 1260 tgggcattaa acgagtatcc cggcagcagg
ggatcatttt gcgcttcagc catacttttc 1320 atactcccgc cattcagaga
agaaaccaat tgtccatatt gcatcagaca ttgccgtcac 1380 tgcgtctttt
actggctctt ctcgctaacc aaaccggtaa ccccgcttat taaaagcatt 1440
ctgtaacaaa gcgggaccaa agccatgaca aaaacgcgta acaaaagtgt ctataatcac
1500 ggcagaaaag tccacattga ttatttgcac ggcgtcacac tttgctatgc
catagcattt 1560 ttatccataa gattagcgga tcctacctga cgctttttat
cgcaactctc tactgtttct 1620 ccatacccgt ttttttgggc tagcaggagg
ccgtcgactc tagaggatcc ccgcgccctc 1680 atccgaaagg gcgtattggt
accgagctcg aattcgtaat catggtcata gctgtttcct 1740 gtgtgaaatt
gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 1800
aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
1860 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
acgcgcgggg 1920 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
tcactgactc gctgcgctcg 1980 gtcgttcggc tgcggcgagc ggtatcagct
cactcaaagg cggtaatacg gttatccaca 2040 gaatcagggg ataacgcagg
aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 2100 cgtaaaaagg
ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 2160
aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
2220 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
taccggatac 2280 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
atagctcacg ctgtaggtat 2340 ctcagttcgg tgtaggtcgt tcgctccaag
ctgggctgtg tgcacgaacc ccccgttcag 2400 cccgaccgct gcgccttatc
cggtaactat cgtcttgagt ccaacccggt aagacacgac 2460 ttatcgccac
tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 2520
gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
2580 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
ttgatccggc 2640 aaacaaacca ccgctggtag cggtggtttt tttgtttgca
agcagcagat tacgcgcaga 2700 aaaaaaggat ctcaagaaga tcctttgatc
ttttctacgg ggtctgacgc tcagtggaac 2760 gaaaactcac gttaagggat
tttggtcatg agattatcaa aaaggatctt cacctagatc 2820 cttttaaatt
aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 2880
gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
2940 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
cttaccatct 3000 ggccccagtg ctgcaatgat accgcgagac ccacgctcac
cggctccaga tttatcagca 3060 ataaaccagc cagccggaag ggccgagcgc
agaagtggtc ctgcaacttt atccgcctcc 3120 atccagtcta ttaattgttg
ccgggaagct agagtaagta gttcgccagt taatagtttg 3180 cgcaacgttg
ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 3240
tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
3300 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
cgcagtgtta 3360 tcactcatgg ttatggcagc actgcataat tctcttactg
tcatgccatc cgtaagatgc 3420 ttttctgtga ctggtgagta ctcaaccaag
tcattctgag aatagtgtat gcggcgaccg 3480 agttgctctt gcccggcgtc
aatacgggat aataccgcgc cacatagcag aactttaaaa 3540 gtgctcatca
ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 3600
agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
3660 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
gggaataagg 3720 gcgacacgga aatgttgaat actcatactc ttcctttttc
aatattattg aagcatttat 3780 cagggttatt gtctcatgag cggatacata
tttgaatgta tttagaaaaa taaacaaata 3840 ggggttccgc gcacatttcc
ccgaaaagtg ccacctgacg tctaagaaac cattattatc 3900 atgacattaa
cctataaaaa taggcgtatc acgaggccct ttcgtc 3946 <210> SEQ ID NO
154 <211> LENGTH: 3952 <212> TYPE: DNA <213>
ORGANISM: E. coli <400> SEQUENCE: 154 tcgcgcgttt cggtgatgac
ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct
gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc
ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg
caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt
aaaacgacgg ccagtgccaa gcttcaagcc gtcaattgtc 420 tgattcgtta
ccaattatga caacttgacg gctacatcat tcactttttc ttcacaaccg 480
gcacggaact cgctcgggct ggccccggtg cattttttaa atacccgcga gaaatagagt
540 tgatcgtcaa aaccaacatt gcgaccgacg gtggcgatag gcatccgggt
ggtgctcaaa 600 agcagcttcg cctggctgat acgttggtcc tcgcgccagc
ttaagacgct aatccctaac 660 tgctggcgga aaagatgtga cagacgcgac
ggcgacaagc aaacatgctg tgcgacgctg 720 gcgatatcaa aattgctgtc
tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc 780 cgattatcca
tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg cagtaacaat 840
tgctcaagca gatttatcgc cagcagctcc gaatagcgcc cttccccttg cccggcgtta
900 atgatttgcc caaacaggtc gctgaaatgc ggctggtgcg cttcatccgg
gcgaaagaac 960 cccgtattgg caaatattga cggccagtta agccattcat
gccagtaggc gcgcggacga 1020 aagtaaaccc actggtgata ccattcgcga
gcctccggat gacgaccgta gtgatgaatc 1080 tctcctggcg ggaacagcaa
aatatcaccc ggtcggcaaa caaattctcg tccctgattt 1140 ttcaccaccc
cctgaccgcg aatggtgaga ttgagaatat aacctttcat tcccagcggt 1200
cggtcgataa aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacc cgccaccaga
1260 tgggcattaa acgagtatcc cggcagcagg ggatcatttt gcgcttcagc
catacttttc 1320 atactcccgc cattcagaga agaaaccaat tgtccatatt
gcatcagaca ttgccgtcac 1380 tgcgtctttt actggctctt ctcgctaacc
aaaccggtaa ccccgcttat taaaagcatt 1440 ctgtaacaaa gcgggaccaa
agccatgaca aaaacgcgta acaaaagtgt ctataatcac 1500 ggcagaaaag
tccacattga ttatttgcac ggcgtcacac tttgctatgc catagcattt 1560
ttatccataa gattagcgga tcctacctga cgctttttat cgcaactctc tactgtttct
1620 ccatacccgt ttttttgggc tagcaggagg ccctgcaggt cgactctaga
ggatccccgc 1680 gccctcatcc gaaagggcgt attggtaccg agctcgaatt
cgtaatcatg gtcatagctg 1740 tttcctgtgt gaaattgtta tccgctcaca
attccacaca acatacgagc cggaagcata 1800 aagtgtaaag cctggggtgc
ctaatgagtg agctaactca cattaattgc gttgcgctca 1860 ctgcccgctt
tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 1920
gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
1980 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
aatacggtta 2040 tccacagaat caggggataa cgcaggaaag aacatgtgag
caaaaggcca gcaaaaggcc 2100 aggaaccgta aaaaggccgc gttgctggcg
tttttccata ggctccgccc ccctgacgag 2160 catcacaaaa atcgacgctc
aagtcagagg tggcgaaacc cgacaggact ataaagatac 2220 caggcgtttc
cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 2280
ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
2340 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
cgaacccccc 2400 gttcagcccg accgctgcgc cttatccggt aactatcgtc
ttgagtccaa cccggtaaga 2460 cacgacttat cgccactggc agcagccact
ggtaacagga ttagcagagc gaggtatgta 2520 ggcggtgcta cagagttctt
gaagtggtgg cctaactacg gctacactag aaggacagta 2580 tttggtatct
gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 2640
tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
2700 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
tgacgctcag 2760 tggaacgaaa actcacgtta agggattttg gtcatgagat
tatcaaaaag gatcttcacc 2820 tagatccttt taaattaaaa atgaagtttt
aaatcaatct aaagtatata tgagtaaact 2880 tggtctgaca gttaccaatg
cttaatcagt gaggcaccta tctcagcgat ctgtctattt 2940 cgttcatcca
tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 3000
ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
3060 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
aactttatcc 3120 gcctccatcc agtctattaa ttgttgccgg gaagctagag
taagtagttc gccagttaat 3180 agtttgcgca acgttgttgc cattgctaca
ggcatcgtgg tgtcacgctc gtcgtttggt 3240 atggcttcat tcagctccgg
ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 3300 tgcaaaaaag
cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 3360
gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
3420 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
gtgtatgcgg 3480 cgaccgagtt gctcttgccc ggcgtcaata cgggataata
ccgcgccaca tagcagaact 3540 ttaaaagtgc tcatcattgg aaaacgttct
tcggggcgaa aactctcaag gatcttaccg 3600 ctgttgagat ccagttcgat
gtaacccact cgtgcaccca actgatcttc agcatctttt 3660 actttcacca
gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 3720
ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
3780 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
gaaaaataaa 3840 caaatagggg ttccgcgcac atttccccga aaagtgccac
ctgacgtcta agaaaccatt 3900 attatcatga cattaaccta taaaaatagg
cgtatcacga ggccctttcg tc 3952 <210> SEQ ID NO 155 <211>
LENGTH: 3886 <212> TYPE: DNA <213> ORGANISM: E. coli
<400> SEQUENCE: 155 tcgcgcgttt cggtgatgac ggtgaaaacc
tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat
gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg
tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg
ccagtgccaa gcttttagcc gggaaacgtc 420 tggcggcgct gttggctaag
tttgcggtat tgttgcggcg acatgccgac atatttgccg 480 aacgtgctgt
aaaaacgact acttgaacga aagcctgccg tcagggcaat atcgagaata 540
cttttatcgg tatcgctcag taacgcgcga acgtggttga tgcgcatcgc ggtaatgtac
600 tgtttcatcg tcaattgcat gacccgctgg aatatcccca ttgcatagtt
ggcgttaagt 660 ttgacgtgct cagccacatc gttgatggtc agcgcctgat
catagttttc ggcaataaag 720 cccagcatct ggctaacata aaattgcgca
tggcgcgaga cgctgttttt gtgtgtgcgc 780 gaggttttat tgaccagaat
cggttcccag ccagagaggc taaatcgctt gagcatcagg 840 ccaatttcat
caatggcgag ctggcgaatt tgctcgttcg gactgtttaa ttcctgctgc 900
cagcggcgca cttcaaacgg gctaagttgc tgtgtggcca gtgatttgat caccatgccg
960 tgagtgacgt ggttaatcag gtctttatcc agcggccagg agagaaacag
atgcatcggc 1020 agattaaaaa tcgccatgct ctgacaggtt ccggtatctg
ttagttggtg cggtgtacag 1080 gcccagaaca gcgtgatatg accctgattg
atattcactt tttcattgtt gatcaggtat 1140 tccacatcgc catcgaaagg
cacattcact tcgacctgac catgccagtg gctggtgggc 1200 atgatatgcg
gtgcgcgaaa ctcaatctcc atccgctggt attccgaata cagcgacagc 1260
gggctgcggg tctgtttttc gtcgctgctg cacataaacg tatctgtatt catggatggc
1320 tctctttcct ggaatatcag aattatggca ggagtgaggg aggatgactg
cgagtgggag 1380 cacggttttc accctcttcc cagaggggcg aggggactct
ccgagtatca tgaggccgaa 1440 aactctgctt ttcaggtaat ttattcccat
aaactcagat ttactgctgc ttcacgcagg 1500 atctgagttt atgggaatgc
tcaacctgga agccggaggt tttctgcaga ttcgcctgcc 1560 atgatgaagt
tattcaagca agccaggagg tcgtcgactc tagaggatcc ccgcgccctc 1620
atccgaaagg gcgtattggt accgagctcg aattcgtaat catggtcata gctgtttcct
1680 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
cataaagtgt 1740 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
ttgcgttgcg ctcactgccc 1800 gctttccagt cgggaaacct gtcgtgccag
ctgcattaat gaatcggcca acgcgcgggg 1860 agaggcggtt tgcgtattgg
gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 1920 gtcgttcggc
tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 1980
gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
2040 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
cgagcatcac 2100 aaaaatcgac gctcaagtca gaggtggcga aacccgacag
gactataaag ataccaggcg 2160 tttccccctg gaagctccct cgtgcgctct
cctgttccga ccctgccgct taccggatac 2220 ctgtccgcct ttctcccttc
gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 2280 ctcagttcgg
tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 2340
cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
2400 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
tgtaggcggt 2460 gctacagagt tcttgaagtg gtggcctaac tacggctaca
ctagaaggac agtatttggt 2520 atctgcgctc tgctgaagcc agttaccttc
ggaaaaagag ttggtagctc ttgatccggc 2580 aaacaaacca ccgctggtag
cggtggtttt tttgtttgca agcagcagat tacgcgcaga 2640 aaaaaaggat
ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 2700
gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
2760 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
aacttggtct 2820 gacagttacc aatgcttaat cagtgaggca cctatctcag
cgatctgtct atttcgttca 2880 tccatagttg cctgactccc cgtcgtgtag
ataactacga tacgggaggg cttaccatct 2940 ggccccagtg ctgcaatgat
accgcgagac ccacgctcac cggctccaga tttatcagca 3000 ataaaccagc
cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 3060
atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
3120 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
tggtatggct 3180 tcattcagct ccggttccca acgatcaagg cgagttacat
gatcccccat gttgtgcaaa 3240 aaagcggtta gctccttcgg tcctccgatc
gttgtcagaa gtaagttggc cgcagtgtta 3300 tcactcatgg ttatggcagc
actgcataat tctcttactg tcatgccatc cgtaagatgc 3360 ttttctgtga
ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 3420
agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
3480 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
accgctgttg 3540 agatccagtt cgatgtaacc cactcgtgca cccaactgat
cttcagcatc ttttactttc 3600 accagcgttt ctgggtgagc aaaaacagga
aggcaaaatg ccgcaaaaaa gggaataagg 3660 gcgacacgga aatgttgaat
actcatactc ttcctttttc aatattattg aagcatttat 3720 cagggttatt
gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata 3780
ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
3840 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc 3886
<210> SEQ ID NO 156 <211> LENGTH: 28 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 156 gcagaacctc ctgaatttca ttacgacc
28 <210> SEQ ID NO 157 <211> LENGTH: 86 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 157 ccgcgggtac caatacgccc
tttcggatga gggcgcgggg atcctctaga gtcgacgtcg 60 acaacctcct
gaatttcatt acgacc 86 <210> SEQ ID NO 158 <211> LENGTH:
86 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 158
ccgcgggtac caatacgccc tttcggatga gggcgcgggg atcctctaga gtcgacctgc
60 agaacctcct gaatttcatt acgacc 86 <210> SEQ ID NO 159
<211> LENGTH: 38 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 159 ctgcagggcc tcctgctagc ccaaaaaaac gggtatgg 38
<210> SEQ ID NO 160 <211> LENGTH: 94 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 160 ccgcgggtac caatacgccc tttcggatga
gggcgcgggg atcctctaga gtcgacgtcg 60 acggcctcct gctagcccaa
aaaaacgggt atgg 94 <210> SEQ ID NO 161 <211> LENGTH: 94
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 161
ccgcgggtac caatacgccc tttcggatga gggcgcgggg atcctctaga gtcgacctgc
60 agggcctcct gctagcccaa aaaaacgggt atgg 94 <210> SEQ ID NO
162 <211> LENGTH: 32 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 162 cctcctggct tgcttgaata acttcatcat gg 32
<210> SEQ ID NO 163 <211> LENGTH: 91 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 163 ccgcgggtac caatacgccc tttcggatga
gggcgcgggg atcctctaga gtcgaccccc 60 tcctggcttg cttgaataac
ttcatcatgg c 91 <210> SEQ ID NO 164 <400> SEQUENCE: 164
000 <210> SEQ ID NO 165 <400> SEQUENCE: 165 000
<210> SEQ ID NO 166 <211> LENGTH: 2319 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic nucleic acid
sequence <400> SEQUENCE: 166 gtcgacatga aaataaaaac aggtgcacgc
atcctcgcat tatccgcatt aacgacgatg 60 atgttttccg cctcggctct
cgccaaaatc gaagaaggta aactggtaat ctggattaac 120 ggcgataaag
gctataacgg tctcgctgaa gtcggtaaga aattcgagaa agataccgga 180
attaaagtca ccgttgagca tccggataaa ctggaagaga aattcccaca ggttgcggca
240 actggcgatg gccctgacat tatcttctgg gcacacgacc gctttggtgg
ctacgctcaa 300 tctggcctgt tggctgaaat caccccggac aaagcgttcc
aggacaagct gtatccgttt 360 acctgggatg ccgtacgtta caacggcaag
ctgattgctt acccgatcgc tgttgaagcg 420 ttatcgctga tttataacaa
agatctgctg ccgaacccgc caaaaacctg ggaagagatc 480 ccggcgctgg
ataaagaact gaaagcgaaa ggtaagagcg cgctgatgtt caacctgcaa 540
gaaccgtact tcacctggcc gctgattgct gctgacgggg gttatgcgtt caagtatgaa
600 aacggcaagt acgacattaa agacgtgggc gtggataacg ctggcgcgaa
agcgggtctg 660 accttcctgg ttgacctgat taaaaacaaa cacatgaatg
cagacaccga ttactccatc 720 gcagaagctg cctttaataa aggcgaaaca
gcgatgacca tcaacggccc gtgggcatgg 780 tccaacatcg acaccagcaa
agtgaattat ggtgtaacgg tactgccgac cttcaagggt 840 caaccatcca
aaccgttcgt tggcgtgctg agcgcaggta ttaacgccgc cagtccgaac 900
aaagagctgg cgaaagagtt cctcgaaaac tatctgctga ctgatgaagg tctggaagcg
960 gttaataaag acaaaccgct gggtgccgta gcgctgaagt cttacgagga
agagttggcg 1020 aaagatccac gtattgccgc caccatggaa aacgcccaga
aaggtgaaat catgccgaac 1080 atcccgcaga tgtccgcttt ctggtatgcc
gtgctgatcg aagcccgcac ctcggaatcc 1140 gacacggcag ggcccaacag
cgacctggac gtgaacactg acatttattc caaggtgctg 1200 gtgactgcta
tatacctggc actcttcgtg gtgggcactg tgggcaactc cgtgacagcc 1260
ttcactctag cgcggaagaa gtcactgcag agcctgcaga gcactgtgca ttaccacctg
1320 ggcagcctgg cactgtcgga cctgcttatc cttctgctgg ccatgcccgt
ggagctatac 1380 aacttcatct gggtacacca tccctgggcc tttggggacg
ctggctgccg tggctactat 1440 ttcctgcgtg atgcctgcac ctatgccaca
gccctcaatg tagccagcct gagtgtggag 1500 cgctacttgg ccatctgcca
tcccttcaag gccaagaccc tcatgtcccg cagccgcacc 1560 aagaaattca
tcagtgccat atggctagct tcggcgctgc tggctatacc catgcttttc 1620
accatgggcc tgcagaaccg cagtggtgac ggcacgcacc ctggcggcct ggtgtgcaca
1680 cccattgtgg acacagccac tgtcaaggtc gtcatccagg ttaacacctt
catgtccttc 1740 ctgtttccca tgttggtcat ctccatccta aacaccgtga
ttgccaacaa actgacagtc 1800 atggtgcacc aggccgccga gcagggccga
gtgtgcaccg tgggcacaca caacggttta 1860 gagcacagca cgttcaacat
gaccatcgag ccgggtcgtg tccaggccct gcgccacgga 1920 gtcctcgtct
tacgtgctgt ggtcattgcc tttgtggtct gctggctgcc ctaccacgtg 1980
cgacgcctga tgttctgcta tatctcggat gaacagtgga ctacgttcct cttcgatttc
2040 taccactatt tctacatgct aaccaacgct ctcttctacg tcagctccgc
catcaatccc 2100 atcctctaca acctggtctc cgccaacttc cgccaggtct
ttctgtccac gctggcctgc 2160 ctttgtcctg ggtggcgcca ccgccgaaag
aagaggccaa cgttctccag gaagcccaac 2220 agcatgtcca gcaaccatgc
cttttccacc agcgccaccc gggagaccct gtacgcggcc 2280 gcagattata
aagatgacga tgacaaataa taaggtacc 2319 <210> SEQ ID NO 167
<211> LENGTH: 1293 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic nucleic acid sequence <400>
SEQUENCE: 167 gtcgacatga aaataaaaac aggtgcacgc atcctcgcat
tatccgcatt aacgacgatg 60 atgttttccg cctcggctct cgccaaaatc
atcgaagccc gcacctcgga atccgacacg 120 gcagggccca acagcgacct
ggacgtgaac actgacattt attccaaggt gctggtgact 180 gctatatacc
tggcactctt cgtggtgggc actgtgggca actccgtgac agccttcact 240
ctagcgcgga agaagtcact gcagagcctg cagagcactg tgcattacca cctgggcagc
300 ctggcactgt cggacctgct tatccttctg ctggccatgc ccgtggagct
atacaacttc 360 atctgggtac accatccctg ggcctttggg gacgctggct
gccgtggcta ctatttcctg 420 cgtgatgcct gcacctatgc cacagccctc
aatgtagcca gcctgagtgt ggagcgctac 480 ttggccatct gccatccctt
caaggccaag accctcatgt cccgcagccg caccaagaaa 540 ttcatcagtg
ccatatggct agcttcggcg ctgctggcta tacccatgct tttcaccatg 600
ggcctgcaga accgcagtgg tgacggcacg caccctggcg gcctggtgtg cacacccatt
660 gtggacacag ccactgtcaa ggtcgtcatc caggttaaca ccttcatgtc
cttcctgttt 720 cccatgttgg tcatctccat cctaaacacc gtgattgcca
acaaactgac agtcatggtg 780 caccaggccg ccgagcaggg ccgagtgtgc
accgtgggca cacacaacgg tttagagcac 840 agcacgttca acatgaccat
cgagccgggt cgtgtccagg ccctgcgcca cggagtcctc 900 gtcttacgtg
ctgtggtcat tgcctttgtg gtctgctggc tgccctacca cgtgcgacgc 960
ctgatgttct gctatatctc ggatgaacag tggactacgt tcctcttcga tttctaccac
1020 tatttctaca tgctaaccaa cgctctcttc tacgtcagct ccgccatcaa
tcccatcctc 1080 tacaacctgg tctccgccaa cttccgccag gtctttctgt
ccacgctggc ctgcctttgt 1140 cctgggtggc gccaccgccg aaagaagagg
ccaacgttct ccaggaagcc caacagcatg 1200 tccagcaacc atgccttttc
caccagcgcc acccgggaga ccctgtacgc ggccgcagat 1260 tataaagatg
acgatgacaa ataataaggt acc 1293 <210> SEQ ID NO 168
<400> SEQUENCE: 168 000 <210> SEQ ID NO 169 <211>
LENGTH: 2652 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleic acid sequence <400> SEQUENCE: 169
gtcgacatga aaataaaaac aggtgcacgc atcctcgcat tatccgcatt aacgacgatg
60 atgttttccg cctcggctct cgccaaaatc gaagaaggta aactggtaat
ctggattaac 120 ggcgataaag gctataacgg tctcgctgaa gtcggtaaga
aattcgagaa agataccgga 180 attaaagtca ccgttgagca tccggataaa
ctggaagaga aattcccaca ggttgcggca 240 actggcgatg gccctgacat
tatcttctgg gcacacgacc gctttggtgg ctacgctcaa 300 tctggcctgt
tggctgaaat caccccggac aaagcgttcc aggacaagct gtatccgttt 360
acctgggatg ccgtacgtta caacggcaag ctgattgctt acccgatcgc tgttgaagcg
420 ttatcgctga tttataacaa agatctgctg ccgaacccgc caaaaacctg
ggaagagatc 480 ccggcgctgg ataaagaact gaaagcgaaa ggtaagagcg
cgctgatgtt caacctgcaa 540 gaaccgtact tcacctggcc gctgattgct
gctgacgggg gttatgcgtt caagtatgaa 600 aacggcaagt acgacattaa
agacgtgggc gtggataacg ctggcgcgaa agcgggtctg 660 accttcctgg
ttgacctgat taaaaacaaa cacatgaatg cagacaccga ttactccatc 720
gcagaagctg cctttaataa aggcgaaaca gcgatgacca tcaacggccc gtgggcatgg
780 tccaacatcg acaccagcaa agtgaattat ggtgtaacgg tactgccgac
cttcaagggt 840 caaccatcca aaccgttcgt tggcgtgctg agcgcaggta
ttaacgccgc cagtccgaac 900 aaagagctgg cgaaagagtt cctcgaaaac
tatctgctga ctgatgaagg tctggaagcg 960 gttaataaag acaaaccgct
gggtgccgta gcgctgaagt cttacgagga agagttggcg 1020 aaagatccac
gtattgccgc caccatggaa aacgcccaga aaggtgaaat catgccgaac 1080
atcccgcaga tgtccgcttt ctggtatgcc gtgctgatcg aagcccgcac ctcggaatcc
1140 gacacggcag ggcccaacag cgacctggac gtgaacactg acatttattc
caaggtgctg 1200 gtgactgcta tatacctggc actcttcgtg gtgggcactg
tgggcaactc cgtgacagcc 1260 ttcactctag cgcggaagaa gtcactgcag
agcctgcaga gcactgtgca ttaccacctg 1320 ggcagcctgg cactgtcgga
cctgcttatc cttctgctgg ccatgcccgt ggagctatac 1380 aacttcatct
gggtacacca tccctgggcc tttggggacg ctggctgccg tggctactat 1440
ttcctgcgtg atgcctgcac ctatgccaca gccctcaatg tagccagcct gagtgtggag
1500 cgctacttgg ccatctgcca tcccttcaag gccaagaccc tcatgtcccg
cagccgcacc 1560 aagaaattca tcagtgccat atggctagct tcggcgctgc
tggctatacc catgcttttc 1620 accatgggcc tgcagaaccg cagtggtgac
ggcacgcacc ctggcggcct ggtgtgcaca 1680 cccattgtgg acacagccac
tgtcaaggtc gtcatccagg ttaacacctt catgtccttc 1740 ctgtttccca
tgttggtcat ctccatccta aacaccgtga ttgccaacaa actgacagtc 1800
atggtgcacc aggccgccga gcagggccga gtgtgcaccg tgggcacaca caacggttta
1860 gagcacagca cgttcaacat gaccatcgag ccgggtcgtg tccaggccct
gcgccacgga 1920 gtcctcgtct tacgtgctgt ggtcattgcc tttgtggtct
gctggctgcc ctaccacgtg 1980 cgacgcctga tgttctgcta tatctcggat
gaacagtgga ctacgttcct cttcgatttc 2040 taccactatt tctacatgct
aaccaacgct ctcttctacg tcagctccgc catcaatccc 2100 atcctctaca
acctggtctc cgccaacttc cgccaggtct ttctgtccac gctggcctgc 2160
ctttgtcctg ggtggcgcca ccgccgaaag aagaggccaa cgttctccag gaagcccaac
2220 agcatgtcca gcaaccatgc cttttccacc agcgccaccc gggagaccct
gtacgcggcc 2280 gcaagcgata aaattattca cctgactgac gacagttttg
acacggatgt actcaaagcg 2340 gacggggcga tcctcgtcga tttctgggca
gagtggtgcg gtccgtgcaa aatgatcgcc 2400 ccgattctgg atgaaatcgc
tgacgaatat cagggcaaac tgaccgttgc aaaactgaac 2460 atcgatcaaa
accctggcac tgcgccgaaa tatggcatcc gtggtatccc gactctgctg 2520
ctgttcaaaa acggtgaagt ggcggcaacc aaagtgggtg cactgtctaa aggtcagttg
2580 aaagagttcc tcgacgctaa cctggcggcg gccgcagatt ataaagatga
cgatgacaaa 2640 taataaggta cc 2652 <210> SEQ ID NO 170
<211> LENGTH: 1626 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic nucleic acid sequence <400>
SEQUENCE: 170 gtcgacatga aaataaaaac aggtgcacgc atcctcgcat
tatccgcatt aacgacgatg 60 atgttttccg cctcggctct cgccaaaatc
atcgaagccc gcacctcgga atccgacacg 120 gcagggccca acagcgacct
ggacgtgaac actgacattt attccaaggt gctggtgact 180 gctatatacc
tggcactctt cgtggtgggc actgtgggca actccgtgac agccttcact 240
ctagcgcgga agaagtcact gcagagcctg cagagcactg tgcattacca cctgggcagc
300 ctggcactgt cggacctgct tatccttctg ctggccatgc ccgtggagct
atacaacttc 360 atctgggtac accatccctg ggcctttggg gacgctggct
gccgtggcta ctatttcctg 420 cgtgatgcct gcacctatgc cacagccctc
aatgtagcca gcctgagtgt ggagcgctac 480 ttggccatct gccatccctt
caaggccaag accctcatgt cccgcagccg caccaagaaa 540 ttcatcagtg
ccatatggct agcttcggcg ctgctggcta tacccatgct tttcaccatg 600
ggcctgcaga accgcagtgg tgacggcacg caccctggcg gcctggtgtg cacacccatt
660 gtggacacag ccactgtcaa ggtcgtcatc caggttaaca ccttcatgtc
cttcctgttt 720 cccatgttgg tcatctccat cctaaacacc gtgattgcca
acaaactgac agtcatggtg 780 caccaggccg ccgagcaggg ccgagtgtgc
accgtgggca cacacaacgg tttagagcac 840 agcacgttca acatgaccat
cgagccgggt cgtgtccagg ccctgcgcca cggagtcctc 900 gtcttacgtg
ctgtggtcat tgcctttgtg gtctgctggc tgccctacca cgtgcgacgc 960
ctgatgttct gctatatctc ggatgaacag tggactacgt tcctcttcga tttctaccac
1020 tatttctaca tgctaaccaa cgctctcttc tacgtcagct ccgccatcaa
tcccatcctc 1080 tacaacctgg tctccgccaa cttccgccag gtctttctgt
ccacgctggc ctgcctttgt 1140 cctgggtggc gccaccgccg aaagaagagg
ccaacgttct ccaggaagcc caacagcatg 1200 tccagcaacc atgccttttc
caccagcgcc acccgggaga ccctgtacgc ggccgcaagc 1260 gataaaatta
ttcacctgac tgacgacagt tttgacacgg atgtactcaa agcggacggg 1320
gcgatcctcg tcgatttctg ggcagagtgg tgcggtccgt gcaaaatgat cgccccgatt
1380 ctggatgaaa tcgctgacga atatcagggc aaactgaccg ttgcaaaact
gaacatcgat 1440 caaaaccctg gcactgcgcc gaaatatggc atccgtggta
tcccgactct gctgctgttc 1500 aaaaacggtg aagtggcggc aaccaaagtg
ggtgcactgt ctaaaggtca gttgaaagag 1560 ttcctcgacg ctaacctggc
agcggccgca gattataaag atgacgatga caaataataa 1620 ggtacc 1626
<210> SEQ ID NO 171 <211> LENGTH: 26 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 171 ggtgcacgca tcctcgcatt atccgc 26
<210> SEQ ID NO 172 <211> LENGTH: 31 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 172 cgcacggcat accagaaagc ggacatctgc
g 31 <210> SEQ ID NO 173 <211> LENGTH: 43 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 173 ccgcggtcga catgaaaata
aaaacaggtg cacgcatcct cgc 43 <210> SEQ ID NO 174 <211>
LENGTH: 66 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 174
gccgtgtcgg attccgaggt gcggccttcg atacgcacgg cataccaaga aagcgggatg
60 ttcggc 66 <210> SEQ ID NO 175 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 175
cctcggaatc cgacacggca gggc 24 <210> SEQ ID NO 176 <211>
LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 176
gtacagggtc tcccgggtgg cgctgg 26 <210> SEQ ID NO 177
<211> LENGTH: 43 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 177 ccgcgatcga aggccgcacc tcggaatccg acacggcagg gcc 43
<210> SEQ ID NO 178 <211> LENGTH: 73 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 178 ggcgcggtac ctttgtcatc gtcatcttta
taatctgcgg ccgcgtacag ggtctcccgg 60 gtggcgctgg tgg 73 <210>
SEQ ID NO 179 <211> LENGTH: 51 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 179 gcggcggtac cttattattt gtcatcgtca
tctttataat ctgcggccgc g 51 <210> SEQ ID NO 180 <211>
LENGTH: 79 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 180
ccgcattaac gacgatgatg ttttccgcct cggctctcgc caaaatcatc gaaggccgca
60 cctcggaatc cgacacggc 79 <210> SEQ ID NO 181 <211>
LENGTH: 82 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 181
ccgcggtcga catgaaaata aaaacaggtg cacgcatcct cgcattatcc gcattaacga
60 cgatgatgtt ttccgcctcg gc 82 <210> SEQ ID NO 182
<211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 182 ccgcgagcga taaaattatt cacctgactg acg 33 <210>
SEQ ID NO 183 <211> LENGTH: 39 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 183 gcccgccagg ttagcgtcga ggaactcttt
caactgacc 39 <210> SEQ ID NO 184 <211> LENGTH: 37
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 184
gcggccgcaa gcgataaaat tattcacctg actgacg 37 <210> SEQ ID NO
185 <211> LENGTH: 38 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 185 ggcgctgcgg ccgcatcatc atgatcttta taatcgcc
38 <210> SEQ ID NO 186 <400> SEQUENCE: 186 000
<210> SEQ ID NO 187 <400> SEQUENCE: 187 000 <210>
SEQ ID NO 188 <211> LENGTH: 2465 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleic acid sequence
<400> SEQUENCE: 188 gtcgacatgg ggcaacccgg gaacggcagc
gccttcttgc tggcacccaa tggaagccat 60 gcgccggacc acgacgtcac
gcagcaaagg gacgaggtgt gggtggtggg catgggcatc 120 gtcatgtctc
tcatcgtcct ggccatcgtg tttggcaatg tgctggtcat cacagccatt 180
gccaagttcg agcgtctgca gacggtcacc aactacttca tcacttcact ggcctgtgct
240 gatctggtca tgggcctagc agtggtgccc tttggggccg cccatattct
tatgaaaatg 300 tggacttttg gcaacttctg gtgcgagttt tggacttcca
ttgatgtgct gtgcgtcacg 360 gccagcattg agaccctgtg cgtgatcgca
gtggatcgct actttgccat tacttcacct 420 ttcaagtacc agagcctgct
gaccaagaat aaggcccggg tgatcattct gatggtgtgg 480 attgtgtcag
gccttayctc cttcttgccc attcagatgc actggtacag ggccacccac 540
caggaagcca tcaactgcta tgccaatgag acctgctgtg acttcttcac gaaccaagcc
600 tatgccattg cctcttccat cgtgtccttc tacgttcccc tggtgatcat
ggtcttcgtc 660 tactccaggg tctttcagga ggccaaaagg cagctccaga
agattgacaa atctgagggc 720 cgcttccatg tccagaacct tagccaggtg
gagcaggatg ggcggacggg gcatggactc 780 cgcagatctt ccaagttctg
cttgaaggag cacaaagccc tcaagacgtt aggcatcatc 840 atgggcactt
tcaccctctg ctggctgccc ttcttcatcg ttaacattgt gcatgtgatc 900
caggataacc tcatccgtaa ggaagtttac atcctcctaa attggatagg ctatgtcaat
960 tctggtttca atccccttat ctactgccgg agcccagatt tcaggattgc
cttccaggag 1020 cttctgtgcc tgcgcaggtc ttctttgaag gcctatggca
atggctactc cagcaacggc 1080 aacacagggg agcagagtgg atatcacgtg
gaacaggaga aagaaaataa actgctgtgt 1140 gaagacctcc caggcacgga
agactttgtg ggccatcaag gtactgtgcc tagcgataac 1200 attgattcac
aagggaggaa ttgtagtaca aatgactcac tgctagagcg tggccagacg 1260
gtcaccaacc tgcagctcga gggctgcctc gggaacagta agaccgagga ccagcgcaac
1320 gaggagaagg cgcagcgtga ggccaacaaa aagatcgaga agcagctgca
gaaggacaag 1380 caggtctacc gggccacgca ccgcctgctg ctgctgggtg
ctggagaatc tggtaaaagc 1440 accattgtga agcagatgag gatcctgcat
gttaatgggt ttaatggaga cagtgagaag 1500 gcaaccaaag tgcaggacat
caaaaacaac ctgaaagagg cgattgaaac cattgtggcc 1560 gccatgagca
acctggtgcc ccccgtggag ctggccaacc ccgagaacca gttcagagtg 1620
gactacatcc tgagtgtgat gaacgtgcct gactttgact tccctcccga attctatgag
1680 catgccaagg ctctgtggga ggatgaagga gtgcgtgcct gctacgaacg
ctccaacgag 1740 taccagctga ttgactgtgc ccagtacttc ctggacaaga
tcgacgtgat caagcaggct 1800 gactatgtgc cgagcgatca ggacctgctt
cgctgccgtg tcctgacttc tggaatcttt 1860 gagaccaagt tccaggtgga
caaagtcaac ttccacatgt ttgacgtggg tggccagcgc 1920 gatgaacgcc
gcaagtggat ccagtgcttc aacgatgtga ctgccatcat cttcgtggtg 1980
gccagcagca gctacaacat ggtcatccgg gaggacaacc agaccaaccg cctgcaggag
2040 gctctgaacc tcttcaagag catctggaac aacagatggc tgcgcaccat
ctctgtgatc 2100 ctgttcctca acaagcaaga tctgctcgct gagaaagtcc
ttgctgggaa atcgaagatt 2160 gaggactact ttccagaatt tgctcgctac
actactcctg aggatgctac tcccgagccc 2220 ggagaggacc cacgcgtgac
ccgggccaag tacttcattc gagatgagtt tctgaggatc 2280 agcactgcca
gtggagatgg gcgtcactac tgctaccctc atttcacctg cgctgtggac 2340
actgagaaca tccgccgtgt gttcaacgac tgccgtgaca tcattcagcg catgcacctt
2400 cgtcagtacg agctgctcat cgattaataa tctagaggat ccccgcgccc
tcatccgaaa 2460 gggcg 2465 <210> SEQ ID NO 189 <400>
SEQUENCE: 189 000 <210> SEQ ID NO 190 <211> LENGTH:
2485 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleic acid sequence <400> SEQUENCE: 190
gtcgacatgg ggcaacccgg gaacggcagc gccttcttgc tggcacccaa tggaagccat
60 gcgccggacc acgacgtcac gcagcaaagg gacgaggtgt gggtggtggg
catgggcatc 120 gtcatgtctc tcatcgtcct ggccatcgtg tttggcaatg
tgctggtcat cacagccatt 180 gccaagttcg agcgtctgca gacggtcacc
aactacttca tcacttcact ggcctgtgct 240 gatctggtca tgggcctagc
agtggtgccc tttggggccg cccatattct tatgaaaatg 300 tggacttttg
gcaacttctg gtgcgagttt tggacttcca ttgatgtgct gtgcgtcacg 360
gccagcattg agaccctgtg cgtgatcgca gtggatcgct actttgccat tacttcacct
420 ttcaagtacc agagcctgct gaccaagaat aaggcccggg tgatcattct
gatggtgtgg 480 attgtgtcag gccttayctc cttcttgccc attcagatgc
actggtacag ggccacccac 540 caggaagcca tcaactgcta tgccaatgag
acctgctgtg acttcttcac gaaccaagcc 600 tatgccattg cctcttccat
cgtgtccttc tacgttcccc tggtgatcat ggtcttcgtc 660 tactccaggg
tctttcagga ggccaaaagg cagctccaga agattgacaa atctgagggc 720
cgcttccatg tccagaacct tagccaggtg gagcaggatg ggcggacggg gcatggactc
780 cgcagatctt ccaagttctg cttgaaggag cacaaagccc tcaagacgtt
aggcatcatc 840 atgggcactt tcaccctctg ctggctgccc ttcttcatcg
ttaacattgt gcatgtgatc 900 caggataacc tcatccgtaa ggaagtttac
atcctcctaa attggatagg ctatgtcaat 960 tctggtttca atccccttat
ctactgccgg agcccagatt tcaggattgc cttccaggag 1020 cttctgtgcc
tgcgcaggtc ttctttgaag gcctatggca atggctactc cagcaacggc 1080
aacacagggg agcagagtgg atatcacgtg gaacaggaga aagaaaataa actgctgtgt
1140 gaagacctcc caggcacgga agactttgtg ggccatcaag gtactgtgcc
tagcgataac 1200 attgattcac aagggaggaa ttgtagtaca aatgactcac
tgctagagcg tggccagacg 1260 gtcaccaacc tgcagtaata atcaaggagg
ccctcgagat gggctgcctc gggaacagta 1320 agaccgagga ccagcgcaac
gaggagaagg cgcagcgtga ggccaacaaa aagatcgaga 1380 agcagctgca
gaaggacaag caggtctacc gggccacgca ccgcctgctg ctgctgggtg 1440
ctggagaatc tggtaaaagc accattgtga agcagatgag gatcctgcat gttaatgggt
1500 ttaatggaga cagtgagaag gcaaccaaag tgcaggacat caaaaacaac
ctgaaagagg 1560 cgattgaaac cattgtggcc gccatgagca acctggtgcc
ccccgtggag ctggccaacc 1620 ccgagaacca gttcagagtg gactacatcc
tgagtgtgat gaacgtgcct gactttgact 1680 tccctcccga attctatgag
catgccaagg ctctgtggga ggatgaagga gtgcgtgcct 1740 gctacgaacg
ctccaacgag taccagctga ttgactgtgc ccagtacttc ctggacaaga 1800
tcgacgtgat caagcaggct gactatgtgc cgagcgatca ggacctgctt cgctgccgtg
1860 tcctgacttc tggaatcttt gagaccaagt tccaggtgga caaagtcaac
ttccacatgt 1920 ttgacgtggg tggccagcgc gatgaacgcc gcaagtggat
ccagtgcttc aacgatgtga 1980 ctgccatcat cttcgtggtg gccagcagca
gctacaacat ggtcatccgg gaggacaacc 2040 agaccaaccg cctgcaggag
gctctgaacc tcttcaagag catctggaac aacagatggc 2100 tgcgcaccat
ctctgtgatc ctgttcctca acaagcaaga tctgctcgct gagaaagtcc 2160
ttgctgggaa atcgaagatt gaggactact ttccagaatt tgctcgctac actactcctg
2220 aggatgctac tcccgagccc ggagaggacc cacgcgtgac ccgggccaag
tacttcattc 2280 gagatgagtt tctgaggatc agcactgcca gtggagatgg
gcgtcactac tgctaccctc 2340 atttcacctg cgctgtggac actgagaaca
tccgccgtgt gttcaacgac tgccgtgaca 2400 tcattcagcg catgcacctt
cgtcagtacg agctgctcat cgattaataa tctagaggat 2460 ccccgcgccc
tcatccgaaa gggcg 2485 <210> SEQ ID NO 191 <400>
SEQUENCE: 191 000 <210> SEQ ID NO 192 <211> LENGTH:
1152 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 192 ctcgagatgg gctgcctcgg gaacagtaag
accgaggacc agcgcaacga ggagaaggcg 60 cagcgtgagg ccaacaaaaa
gatcgagaag cagctgcaga aggacaagca ggtctaccgg 120 gccacgcacc
gcctgctgct gctgggtgct ggagaatctg gtaaaagcac cattgtgaag 180
cagatgagga tcctgcatgt taatgggttt aatggagaca gtgagaaggc aaccaaagtg
240 caggacatca aaaacaacct gaaagaggcg attgaaacca ttgtggccgc
catgagcaac 300 ctggtgcccc ccgtggagct ggccaacccc gagaaccagt
tcagagtgga ctacatcctg 360 agtgtgatga acgtgcctga ctttgacttc
cctcccgaat tctatgagca tgccaaggct 420 ctgtgggagg atgaaggagt
gcgtgcctgc tacgaacgct ccaacgagta ccagctgatt 480 gactgtgccc
agtacttcct ggacaagatc gacgtgatca agcaggctga ctatgtgccg 540
agcgatcagg acctgcttcg ctgccgtgtc ctgacttctg gaatctttga gaccaagttc
600 caggtggaca aagtcaactt ccacatgttt gacgtgggtg gccagcgcga
tgaacgccgc 660 aagtggatcc agtgcttcaa cgatgtgact gccatcatct
tcgtggtggc cagcagcagc 720 tacaacatgg tcatccggga ggacaaccag
accaaccgcc tgcaggaggc tctgaacctc 780 ttcaagagca tctggaacaa
cagatggctg cgcaccatct ctgtgatcct gttcctcaac 840 aagcaagatc
tgctcgctga gaaagtcctt gctgggaaat cgaagattga ggactacttt 900
ccagaatttg ctcgctacac tactcctgag gatgctactc ccgagcccgg agaggaccca
960 cgcgtgaccc gggccaagta cttcattcga gatgagtttc tgaggatcag
cactgccagt 1020 ggagatgggc gtcactactg ctaccctcat ttcacctgcg
ctgtggacac tgagaacatc 1080 cgccgtgtgt tcaacgactg ccgtgacatc
attcagcgca tgcaccttcg tcagtacgag 1140 ctgctcatcg at 1152
<210> SEQ ID NO 193 <211> LENGTH: 1194 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:
193 ctcgagatgg gctgcctcgg gaacagtaag accgaggacc agcgcaacga
ggagaaggcg 60 cagcgtgagg ccaacaaaaa gatcgagaag cagctgcaga
aggacaagca ggtctaccgg 120 gccacgcacc gcctgctgct gctgggtgct
ggagaatctg gtaaaagcac cattgtgaag 180 cagatgagga tcctgcatgt
taatgggttt aatggagagg gcggcgaaga ggacccgcag 240 gctgcaagga
gcaacagcga tggtgagaag gcaaccaaag tgcaggacat caaaaacaac 300
ctgaaagagg cgattgaaac cattgtggcc gccatgagca acctggtgcc ccccgtggag
360 ctggccaacc ccgagaacca gttcagagtg gactacatcc tgagtgtgat
gaacgtgcct 420 gactttgact tccctcccga attctatgag catgccaagg
ctctgtggga ggatgaagga 480 gtgcgtgcct gctacgaacg ctccaacgag
taccagctga ttgactgtgc ccagtacttc 540 ctggacaaga tcgacgtgat
caagcaggct gactatgtgc cgagcgatca ggacctgctt 600 cgctgccgtg
tcctgacttc tggaatcttt gagaccaagt tccaggtgga caaagtcaac 660
ttccacatgt ttgacgtggg tggccagcgc gatgaacgcc gcaagtggat ccagtgcttc
720 aacgatgtga ctgccatcat cttcgtggtg gccagcagca gctacaacat
ggtcatccgg 780 gaggacaacc agaccaaccg cctgcaggag gctctgaacc
tcttcaagag catctggaac 840 aacagatggc tgcgcaccat ctctgtgatc
ctgttcctca acaagcaaga tctgctcgct 900 gagaaagtcc ttgctgggaa
atcgaagatt gaggactact ttccagaatt tgctcgctac 960 actactcctg
aggatgctac tcccgagccc ggagaggacc cacgcgtgac ccgggccaag 1020
tacttcattc gagatgagtt tctgaggatc agcactgcca gtggagatgg gcgtcactac
1080 tgctaccctc atttcacctg cgctgtggac actgagaaca tccgccgtgt
gttcaacgac 1140 tgccgtgaca tcattcagcg catgcacctt cgtcagtacg
agctgctcat cgat 1194 <210> SEQ ID NO 194 <211> LENGTH:
1089 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 194 ctcgagatga ctctggagtc catcatggcg
tgctgcctga gcgaggaggc caaggaagcc 60 cggcggatca acgacgagat
cgagcggcag ctccgcaggg acaagcggga cgcccgccgg 120 gagctcaagc
tgctgctgct cgggacagga gagagtggca agagtacgtt tatcaagcag 180
atgagaatca tccatgggtc aggatactct gatgaagata aaaggggctt caccaagctg
240 gtgtatcaga acatcttcac ggccatgcag gccatgatca gagccatgga
cacactcaag 300 atcccataca agtatgagca caataaggct catgcacaat
tagttcgaga agttgatgtg 360 gagaaggtgt ctgcttttga gaatccatat
gtagatgcaa taaagagttt atggaatgat 420 cctggaatcc aggaatgcta
tgatagacga cgagaatatc aattatctga ctctaccaaa 480 tactatctta
atgacttgga ccgcgtagct gaccctgcct acctgcctac gcaacaagat 540
gtgcttagag ttcgagtccc caccacaggg atcatcgaat acccctttga cttacaaagt
600 gtcattttca gaatggtcga tgtagggggc caaaggtcag agagaagaaa
atggatacac 660 tgctttgaaa atgtcacctc tatcatgttt ctagtagcgc
ttagtgaata tgatcaagtt 720 ctcgtggagt cagacaatga gaaccgaatg
gaggaaagca aggctctctt tagaacaatt 780 atcacatacc cctggttcca
gaactcctcg gttattctgt tcttaaacaa gaaagatctt 840 ctagaggaga
aaatcatgta ttcccatcta gtcgactact tcccagaata tgatggaccc 900
cagagagatg cccaggcagc ccgagaattc attctgaaga tgttcgtgga cctgaaccca
960 gacagtgaca aaattatcta ctcccacttc acgtgcgcca cagacaccga
gaatatccgc 1020 tttgtctttg ctgccgtcaa ggacaccatc ctccagttga
acctgaagga gtacaatctg 1080 gtcatcgat 1089 <210> SEQ ID NO 195
<211> LENGTH: 1077 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 195 ctcgagatgg
gctgcaccgt gagcgccgag gacaaggcgg cggccgagcg ctctaagatg 60
atcgacaaga acctgcggga ggacggagag aaggcggcgc gggaggtgaa gttgctgctg
120 ttgggtgctg gggagtcagg gaagagcacc atcgtcaagc agatgaagat
catccacgag 180 gatggctact ccgaggagga atgccggcag taccgggcgg
ttgtctacag caacaccatc 240 cagtccatca tggccattgt caaagccatg
ggaaacctgc agatcgactt tgccgacccc 300 tccagagcgg acgacgccag
gcagctattt gcactgtcct gcaccgccga ggagcaaggc 360 gtgctccctg
atgacctgtc cggcgtcatc cggaggctct gggctgacca tggtgtgcag 420
gcctgctttg gccgctcaag ggaataccag ctcaacgact cagctgccta ctacctgaac
480 gacctggagc gtattgcaca gagtgactac atccccacac agcaagatgt
gctacggacc 540 cgcgtaaaga ccacggggat cgtggagaca cacttcacct
tcaaggacct acacttcaag 600 atgtttgatg tgggtggtca gcggtctgag
cggaagaagt ggatccactg ctttgagggc 660 gtcacagcca tcatcttctg
cgtagccttg agcgcctatg acttggtgct agctgaggac 720 gaggagatga
accgcatgca tgagagcatg aagctattcg atagcatctg caacaacaag 780
tggttcacag acacgtccat catcctcttc ctcaacaaga aggacctgtt tgaggagaag
840 atcacacaca gtcccctgac catctgcttc cctgagtaca caggggccaa
caaatatgat 900 gaggcagcca gctacatcca gagtaagttt gaggacctga
ataagcgcaa agacaccaag 960 gagatctaca cgcacttcac gtgcgccacc
gacaccaaga acgtgcagtt cgtgtttgac 1020 gccgtcaccg atgtcatcat
caagaacaac ctgaaggact gcggcctctt catgcat 1077 <210> SEQ ID NO
196 <211> LENGTH: 1155 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 196 ctcgagatgt
ccggggtggt gcggaccctc agccgctgcc tgctgccggc cgaggccggc 60
ggggcccgcg agcgcagggc gggcagcggc gcgcgcgacg cggagcgcga ggcccggagg
120 cgtagccgcg acatcgacgc gctgctggcc cgcgagcggc gcgcggtccg
gcgcctggtg 180 aagatcctgc tgctgggcgc gggcgagagc ggcaagtcca
cgttcctcaa gcagatgcgc 240 atcatccacg gccgcgagtt cgaccagaag
gcgctgctgg agttccgcga caccatcttc 300 gacaacatcc tcaagggctc
aagggttctt gttgatgcac gagataagct tggcattcct 360 tggcagtatt
ctgaaaatga gaagcatggg atgttcctga tggccttcga gaacaaggcg 420
gggctgcctg tggagccggc caccttccag ctgtacgtcc cggccctgag cgcactctgg
480 agggattctg gcatcaggga ggctttcagc cggagaagcg agtttcagct
gggggagtcg 540 gtgaagtact tcctggacaa cttggaccgg atcggccagc
tgaattactt tcctagtaag 600 caagatatcc tgctggctag gaaagccacc
aagggaattg tggagcatga cttcgttatt 660 aagaagatcc cctttaagat
ggtggatgtg ggcggccagc ggtcccagcg ccagaagtgg 720 ttccagtgct
tcgacgggat cacgtccatc ctgttcatgg tctcctccag cgagtacgac 780
caggtcctca tggaggacag gcgcaccaac cggctggtgg agtccatgaa catcttcgag
840 accatcgtca acaacaagct cttcttcaac gtctccatca ttctcttcct
caacaagatg 900 gacctcctgg tggagaaggt gaagaccgtg agcatcaaga
agcacttccc ggacttcagg 960 ggcgacccgc accagctgga ggacgtccag
cgctacctgg tccagtgctt cgacaggaag 1020 agacggaacc gcagcaagcc
actcttccac cacttcacca ccgccatcga caccgagaac 1080 gtccgcttcg
tgttccatgc tgtgaaagac accatcctgc aggagaacct gaaggacatc 1140
atgctgcaga tcgat 1155 <210> SEQ ID NO 197 <400>
SEQUENCE: 197 000 <210> SEQ ID NO 198 <400> SEQUENCE:
198 000 <210> SEQ ID NO 199 <400> SEQUENCE: 199 000
<210> SEQ ID NO 200 <400> SEQUENCE: 200 000 <210>
SEQ ID NO 201 <400> SEQUENCE: 201 000 <210> SEQ ID NO
202 <400> SEQUENCE: 202 000 <210> SEQ ID NO 203
<400> SEQUENCE: 203 000 <210> SEQ ID NO 204 <400>
SEQUENCE: 204 000 <210> SEQ ID NO 205 <211> LENGTH:
3307 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleic acid sequence <400> SEQUENCE: 205
gtcgacatgg ggcaacccgg gaacggcagc gccttcttgc tggcacccaa tggaagccat
60 gcgccggacc acgacgtcac gcagcaaagg gacgaggtgt gggtggtggg
catgggcatc 120 gtcatgtctc tcatcgtcct ggccatcgtg tttggcaatg
tgctggtcat cacagccatt 180 gccaagttcg agcgtctgca gacggtcacc
aactacttca tcacttcact ggcctgtgct 240 gatctggtca tgggcctagc
agtggtgccc tttggggccg cccatattct tatgaaaatg 300 tggacttttg
gcaacttctg gtgcgagttt tggacttcca ttgatgtgct gtgcgtcacg 360
gccagcattg agaccctgtg cgtgatcgca gtggatcgct actttgccat tacttcacct
420 ttcaagtacc agagcctgct gaccaagaat aaggcccggg tgatcattct
gatggtgtgg 480 attgtgtcag gccttayctc cttcttgccc attcagatgc
actggtacag ggccacccac 540 caggaagcca tcaactgcta tgccaatgag
acctgctgtg acttcttcac gaaccaagcc 600 tatgccattg cctcttccat
cgtgtccttc tacgttcccc tggtgatcat ggtcttcgtc 660 tactccaggg
tctttcagga ggccaaaagg cagctccaga agattgacaa atctgagggc 720
cgcttccatg tccagaacct tagccaggtg gagcaggatg ggcggacggg gcatggactc
780 cgcagatctt ccaagttctg cttgaaggag cacaaagccc tcaagacgtt
aggcatcatc 840 atgggcactt tcaccctctg ctggctgccc ttcttcatcg
ttaacattgt gcatgtgatc 900 caggataacc tcatccgtaa ggaagtttac
atcctcctaa attggatagg ctatgtcaat 960 tctggtttca atccccttat
ctactgccgg agcccagatt tcaggattgc cttccaggag 1020 cttctgtgcc
tgcgcaggtc ttctttgaag gcctatggca atggctactc cagcaacggc 1080
aacacagggg agcagagtgg atatcacgtg gaacaggaga aagaaaataa actgctgtgt
1140 gaagacctcc caggcacgga agactttgtg ggccatcaag gtactgtgcc
tagcgataac 1200 attgattcac aagggaggaa ttgtagtaca aatgactcac
tgctagagcg tggccagacg 1260 gtcaccaacc tgcagggaca caactcaaaa
gagatatcga tgagtcatat tggtactaaa 1320 ttcattcttg ctgaaaaatt
taccttcgat cccctaagca atactctgat tgacaaagaa 1380 gatagtgaag
agatcattcg attaggcagc aacgaaagcc gaattctttg gctgctggcc 1440
caacgtccaa acgaggtaat ttctcgcaat gatttgcatg actttgtttg gcgagagcaa
1500 ggttttgaag tcgatgattc cagcttaacc caagccattt cgactctgcg
caaaatgctc 1560 aaagattcga caaagtcccc acaatacgtc aaaacggttc
cgaagcgcgg ttaccaattg 1620 atcgcccgag tggaaacggt tgaagaagag
atggctcgcg aaaacgaagc tgctcatgac 1680 atctcttaat aatcaaggag
gccctcgaga tgggctgcct cgggaacagt aagaccgagg 1740 accagcgcaa
cgaggagaag gcgcagcgtg aggccaacaa aaagatcgag aagcagctgc 1800
agaaggacaa gcaggtctac cgggccacgc accgcctgct gctgctgggt gctggagaat
1860 ctggtaaaag caccattgtg aagcagatga ggatcctgca tgttaatggg
tttaatggag 1920 acagtgagaa ggcaaccaaa gtgcaggaca tcaaaaacaa
cctgaaagag gcgattgaaa 1980 ccattgtggc cgccatgagc aacctggtgc
cccccgtgga gctggccaac cccgagaacc 2040 agttcagagt ggactacatc
ctgagtgtga tgaacgtgcc tgactttgac ttccctcccg 2100 aattctatga
gcatgccaag gctctgtggg aggatgaagg agtgcgtgcc tgctacgaac 2160
gctccaacga gtaccagctg attgactgtg cccagtactt cctggacaag atcgacgtga
2220 tcaagcaggc tgactatgtg ccgagcgatc aggacctgct tcgctgccgt
gtcctgactt 2280 ctggaatctt tgagaccaag ttccaggtgg acaaagtcaa
cttccacatg tttgacgtgg 2340 gtggccagcg cgatgaacgc cgcaagtgga
tccagtgctt caacgatgtg actgccatca 2400 tcttcgtggt ggccagcagc
agctacaaca tggtcatccg ggaggacaac cagaccaacc 2460 gcctgcagga
ggctctgaac ctcttcaaga gcatctggaa caacagatgg ctgcgcacca 2520
tctctgtgat cctgttcctc aacaagcaag atctgctcgc tgagaaagtc cttgctggga
2580 aatcgaagat tgaggactac tttccagaat ttgctcgcta cactactcct
gaggatgcta 2640 ctcccgagcc cggagaggac ccacgcgtga cccgggccaa
gtacttcatt cgagatgagt 2700 ttctgaggat cagcactgcc agtggagatg
ggcgtcacta ctgctaccct catttcacct 2760 gcgctgtgga cactgagaac
atccgccgtg tgttcaacga ctgccgtgac atcattcagc 2820 gcatgcacct
tcgtcagtac gagctgctca tcgatggaca caactcaaaa gagatatcga 2880
tgagtcatat tggtactaaa ttcattcttg ctgaaaaatt taccttcgat cccctaagca
2940 atactctgat tgacaaagaa gatagtgaag agatcattcg attaggcagc
aacgaaagcc 3000 gaattctttg gctgctggcc caacgtccaa acgaggtaat
ttctcgcaat gatttgcatg 3060 actttgtttg gcgagagcaa ggttttgaag
tcgatgattc cagcttaacc caagccattt 3120 cgactctgcg caaaatgctc
aaagattcga caaagtcccc acaatacgtc aaaacggttc 3180 cgaagcgcgg
ttaccaattg atcgcccgag tggaaacggt tgaagaagag atggctcgcg 3240
aaaacgaagc tgctcatgac atctcttaat aatctagagg atccccgcgc cctcatccga
3300 aagggcg 3307 <210> SEQ ID NO 206 <400> SEQUENCE:
206 000 <210> SEQ ID NO 207 <400> SEQUENCE: 207 000
<210> SEQ ID NO 208 <211> LENGTH: 3284 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic nucleic acid
sequence <400> SEQUENCE: 208 tctagaggct gtgggtagaa gtgaaacggg
gtttaccgat aaaaacagaa aatgataaaa 60 aaggactaaa tagtatattt
tgatttttga tttttgattt caaataatac aaatttattt 120 acttatttaa
ttgttttgat caattatttt tctgttaaac aaagggagca ttatatggta 180
aagaccatga ttacggattc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
240 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg
gcgtaatagc 300 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
gcctgaatgg cgaatggcgc 360 tttgcctggt ttccggcacc agaagcggtg
ccggaaagct ggctggagtg cgatcttcct 420 gaggccgata ctgtcgtcgt
cccctcaaac tggcagatgc acggttacga tgcgcccatc 480 tacaccaacg
tgacctatcc cattacggtc aatccgccgt ttgttcccac ggagaatccg 540
acgggttgtt actcgctcac atttaatgtt gatgaaagct ggctacagga aggccagacg
600 cgaattattt ttgatggcgt taactcggcg tttcatctgt ggtgcaacgg
gcgctgggtc 660 ggttacggcc aggacagtcg tttgccgtct gaatttgacc
tgagcgcatt tttacgcgcc 720 ggagaaaacc gcctcgcggt gatggtgctg
cgctggagtg acggcagtta tctggaagat 780 caggatatgt ggcggatgag
cggcattttc cgtgacgtct cgttgctgca taaaccgact 840 acacaaatca
gcgatttcca tgttgccact cgctttaatg atgatttcag ccgcgctgta 900
ctggaggctg aagttcagat gtgcggcgag ttgcgtgact acctacgggt aacagtttct
960 ttatggcagg gtgaaacgca ggtcgccagc ggcaccgcgc ctttcggcgg
tgaaattatc 1020 gatgagcgtg gtggttatgc cgatcgcgtc acactacgtc
tgaacgtcga aaacccgaaa 1080 ctgtggagcg ccgaaatccc gaatctctat
cgtgcggtgg ttgaactgca caccgccgac 1140 ggcacgctga ttgaagcaga
agcctgcgat gtcggtttcc gcgaggtgcg gattgaaaat 1200 ggtctgctgc
tgctgaacgg caagccgttg ctgattcgag gcgttaaccg tcacgagcat 1260
catcctctgc atggtcaggt catggatgag cagacgatgg tgcaggatat cctgctgatg
1320 aagcagaaca actttaacgc cgtgcgctgt tcgcattatc cgaaccatcc
gctgtggtac 1380 acgctgtgcg accgctacgg cctgtatgtg gtggatgaag
ccaatattga aacccacggc 1440 atggtgccaa tgaatcgtct gaccgatgat
ccgcgctggc taccggcgat gagcgaacgc 1500 gtaacgcgaa tggtgcagcg
cgatcgtaat cacccgagtg tgatcatctg gtcgctgggg 1560 aatgaatcag
gccacggcgc taatcacgac gcgctgtatc gctggatcaa atctgtcgat 1620
ccttcccgcc cggtgcagta tgaaggcggc ggagccgaca ccacggccac cgatattatt
1680 tgcccgatgt acgcgcgcgt ggatgaagac cagcccttcc cggctgtgcc
gaaatggtcc 1740 atcaaaaaat ggctttcgct acctggagag acgcgcccgc
tgatcctttg cgaatacgcc 1800 cacgcgatgg gtaacagtct tggcggtttc
gctaaatact ggcaggcgtt tcgtcagtat 1860 ccccgtttac agggcggctt
cgtctgggac tgggtggatc agtcgctgat taaatatgat 1920 gaaaacggca
acccgtggtc ggcttacggc ggtgattttg gcgatacgcc gaacgatcgc 1980
cagttctgta tgaacggtct ggtctttgcc gaccgcacgc cgcatccagc gctgacggaa
2040 gcaaaacacc agcagcagtt tttccagttc cgtttatccg ggcaaaccat
cgaagtgacc 2100 agcgaatacc tgttccgtca tagcgataac gagctcctgc
actggatggt ggcgctggat 2160 ggtaagccgc tggcaagcgg tgaagtgcct
ctggatgtcg ctccacaagg taaacagttg 2220 attgaactgc ctgaactacc
gcagccggag agcgccgggc aactctggct cacagtacgc 2280 gtagtgcaac
cgaacgcgac cgcatggtca gaagccgggc acatcagcgc ctggcagcag 2340
tggcgtctgg cggaaaacct cagtgtgacg ctccccgccg cgtcccacgc catcccgcat
2400 ctgaccacca gcgaaatgga tttttgcatc gagctgggta ataagcgttg
gcaatttaac 2460 cgccagtcag gctttctttc acagatgtgg attggcgata
aaaaacaact gctgacgccg 2520 ctgcgcgatc agttcacccg tgcaccgctg
gataacgaca ttggcgtaag tgaagcgacc 2580 cgcattgacc ctaacgcctg
ggtcgaacgc tggaaggcgg cgggccatta ccaggccgaa 2640 gcagcgttgt
tgcagtgcac ggcagataca cttgctgatg cggtgctgat tacgaccgct 2700
cacgcgtggc agcatcaggg gaaaacctta tttatcagcc ggaaaaccta ccggattgat
2760 ggtagtggtc aaatggcgat taccgttgat gttgaagtgg cgagcgatac
accgcatccg 2820 gcgcggattg gcctgaactg ccagctggcg caggtagcag
agcgggtaaa ctggctcgga 2880 ttagggccgc aagaaaacta tcccgaccgc
cttactgccg cctgttttga ccgctgggat 2940 ctgccattgt cagacatgta
taccccgtac gtcttcccga gcgaaaacgg tctgcgctgc 3000 gggacgcgcg
aattgaatta tggcccacac cagtggcgcg gcgacttcca gttcaacatc 3060
agccgctaca gtcaacagca actgatggaa accagccatc gccatctgct gcacgcggaa
3120 gaaggcacat ggctgaatat cgacggtttc catatgggga ttggtggcga
cgactcctgg 3180 agcccgtcag tatcggcgga attccagctg agcgccggtc
gctaccatta ccagttggtc 3240 tggtgtcaaa aataataacg ccctcatccg
aaagggcgtc taga 3284 <210> SEQ ID NO 209 <211> LENGTH:
25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 209
ggggcaaccc gggaacggca gcgcc 25 <210> SEQ ID NO 210
<211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 210 gcagtgagtc atttgtacta caattcctcc 30 <210> SEQ
ID NO 211 <211> LENGTH: 37 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 211 cgcggtcgac atggggcaac ccgggaacgg cagcgcc
37 <210> SEQ ID NO 212 <211> LENGTH: 67 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 212 ggctcgagct gcaggttggt
gaccgtctgg ccacgctcta gcagtgagtc atttgtacta 60 caattcc 67
<210> SEQ ID NO 213 <211> LENGTH: 29 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 213 gggctgcctc gggaacagta agaccgagg
29 <210> SEQ ID NO 214 <211> LENGTH: 29 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 214 gagcagctcg tactgacgaa
ggtgcatgc 29 <210> SEQ ID NO 215 <211> LENGTH: 44
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 215
ggaggccctc gagatgggct gcctcgggaa cagtaagacc gagg 44 <210> SEQ
ID NO 216 <211> LENGTH: 48 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 216 cctctagatt attatcgatg agcagctcgt
actgacgaag gtgcatgc 48 <210> SEQ ID NO 217 <211>
LENGTH: 37 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 217
ccatcgatga gcagctcgta ctgacgaagg tgcatgc 37 <210> SEQ ID NO
218 <211> LENGTH: 26 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 218 ccggggtggt gcggaccctc agccgc 26
<210> SEQ ID NO 219 <211> LENGTH: 28 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 219 ctgcagcatg atgtccttca ggttctcc
28 <210> SEQ ID NO 220 <211> LENGTH: 41 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 220 gcgggctcga gatgtccggg
gtggtgcgga ccctcagccg c 41 <210> SEQ ID NO 221 <211>
LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 221
gcgccatcga tctgcagcat gatgtccttc aggttctcc 39 <210> SEQ ID NO
222 <211> LENGTH: 28 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 222 gactctggag tccatcatgg cgtgctgc 28
<210> SEQ ID NO 223 <211> LENGTH: 29 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 223 ccagattgta ctccttcagg ttcaactgg
29 <210> SEQ ID NO 224 <211> LENGTH: 30 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 224 atgactctgg agtccatcat
ggcgtgctgc 30 <210> SEQ ID NO 225 <211> LENGTH: 42
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 225
gcgccatcga tgaccagatt gtactccttc aggttcaact gg 42 <210> SEQ
ID NO 226 <211> LENGTH: 29 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 226 gggctgcacc gtgagcgccg aggacaagg 29
<210> SEQ ID NO 227 <211> LENGTH: 30 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 227 ccttcaggtt gttcttgatg atgacatcgg
30 <210> SEQ ID NO 228 <211> LENGTH: 31 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 228 atgggctgca ccgtgagcgc
cgaggacaag g 31 <210> SEQ ID NO 229 <211> LENGTH: 55
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 229
gcgccatcga tgaagaggcc gcagtccttc aggttgttct tgatgatgac atcgg 55
<210> SEQ ID NO 230 <211> LENGTH: 29 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 230 gggctgcctc gggaacagta agaccgagg
29 <210> SEQ ID NO 231 <211> LENGTH: 29 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 231 gagcagctcg tactgacgaa
ggtgcatgc 29 <210> SEQ ID NO 232 <211> LENGTH: 31
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 232
atgggctgcc tcgggaacag taagaccgag g 31 <210> SEQ ID NO 233
<211> LENGTH: 40 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 233 gcgccatcga tgagcagctc gtactgacga aggtgcatgc 40
<210> SEQ ID NO 234 <211> LENGTH: 107 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 234 ggctcgaggg cctccttgat tattactcga
gggcctcctt gattattact gcaggttggt 60 gaccgtctgg ccacgctcta
gcagtgagtc atttgtacta caattcc 107 <210> SEQ ID NO 235
<211> LENGTH: 61 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 235 ccctgcaggt tggtgaccgt ctggccacgc tctagcagtg
agtcatttgt actacaattc 60 c 61 <210> SEQ ID NO 236 <211>
LENGTH: 38 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 236
ggacacaact caaaagagat atcgatgagt catattgg 38 <210> SEQ ID NO
237 <211> LENGTH: 29 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 237 gagatgtcat gagcagcttc gttttcgcg 29
<210> SEQ ID NO 238 <211> LENGTH: 52 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 238 gcgtggccag acggtcacca acctgcaggg
acacaactca aaagagatat cg 52 <210> SEQ ID NO 239 <211>
LENGTH: 51 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 239
cggggatcct ctagattatt aagagatgtc atgagcagct tcgttttcgc g 51
<210> SEQ ID NO 240 <211> LENGTH: 32 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 240 ggctgtgggt agaagtgaaa cggggtttac
cg 32 <210> SEQ ID NO 241 <211> LENGTH: 32 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 241 ctttaccata taatgctccc
tttgtttaac ag 32 <210> SEQ ID NO 242 <211> LENGTH: 43
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 242
cgcggtctag aggctgtggg tagaagtgaa acggggttta ccg 43 <210> SEQ
ID NO 243 <211> LENGTH: 60 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 243 cgacggccag tgaatccgta atcatggtct
ttaccatata atgctccctt tgtttaacag 60 <210> SEQ ID NO 244
<211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 244 ccatgattac ggattcactg gccgtcg 27 <210> SEQ ID
NO 245 <211> LENGTH: 27 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 245 ccagaccaac tggtaatggt agcgacc 27
<210> SEQ ID NO 246 <211> LENGTH: 35 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 246 ggtaaagacc atgattacgg attcactggc
cgtcg 35 <210> SEQ ID NO 247 <211> LENGTH: 77
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 247
gcgcctctag aaatacgccc tttcggatga gggcgttatt atttttgaca ccagaccaac
60 tggtaatggt agcgacc 77 <210> SEQ ID NO 248 <400>
SEQUENCE: 248 000 <210> SEQ ID NO 249 <400> SEQUENCE:
249 000 <210> SEQ ID NO 250 <400> SEQUENCE: 250 000
<210> SEQ ID NO 251 <400> SEQUENCE: 251 000 <210>
SEQ ID NO 252 <400> SEQUENCE: 252 000 <210> SEQ ID NO
253 <211> LENGTH: 89 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 253 cgcggatgca tatgaaaata aaaacaggtg
cacgcatcct cgcattatcc gcattaacga 60 cgatgatgtt ttccgcctcg gctctcgcc
89 <210> SEQ ID NO 254 <211> LENGTH: 69 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 254 cgtcgaccga ggcctgcagg
cgggcttcga tgattttggc gagagccgag gcggaaaaca 60 tcatcgtcg 69
<210> SEQ ID NO 255 <211> LENGTH: 81 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 255 cgaagcccgc ctgcaggcct cggtcgacgc
cgaatctaga gattataaag atgacgatga 60 caaataataa gctagcggcg c 81
<210> SEQ ID NO 256 <211> LENGTH: 27 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 256 gcgccgctag cttattattt gtcatcg 27
<210> SEQ ID NO 257 <211> LENGTH: 26 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 257 ggtgcacgca tcctcgcatt atccgc 26
<210> SEQ ID NO 258 <211> LENGTH: 30 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 258 ggcgttttcc atggtggcgg caatacgtgg
30 <210> SEQ ID NO 259 <211> LENGTH: 52 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 259 cgcggatgca tatgaaaata
aaaacaggtg cacgcatcct cgcattatcc gc 52 <210> SEQ ID NO 260
<211> LENGTH: 82 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 260 ccgaggcctg caggcgggct tcgatacgca cggcatacca
gaaagcggac tgggcgtttt 60 ccatggtggc ggcaatacgt gg 82 <210>
SEQ ID NO 261 <211> LENGTH: 87 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 261 gcgccgctag cttattattt gtcatcgtca
tctttataat ctctagattc ggcgtcgacc 60 gaggcctgca ggcgggcttc gatacgc
87 <210> SEQ ID NO 262 <211> LENGTH: 26 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 262 cctgactgac gacagttttg
acacgg 26 <210> SEQ ID NO 263 <211> LENGTH: 32
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 263
cctttagaca gtgcacccac tttggttgcc gc 32 <210> SEQ ID NO 264
<211> LENGTH: 78 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 264 cgcggctgca ggcctcggtc gacgccgaat ctagaagcga
taaaattatt cacctgactg 60 acgacagttt tgacacgg 78 <210> SEQ ID
NO 265 <211> LENGTH: 95 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 265 gcgccgctag cttattattt gtcatcgtca
tctttataat ccgccaggtt ctctttcaac 60 tgacctttag acagtgcacc
cactttggtt gccgc 95 <210> SEQ ID NO 266 <211> LENGTH:
216 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleic acid sequence <400> SEQUENCE: 266 gaattcaggc
gctttttaga ctggtcgtaa tgaaattcag gaggttctgc atatgaaaat 60
aaaaacaggt gcacgcatcc tcgcattatc cgcattaacg acgatgatgt tttccgcctc
120 ggctctcgcc aaaatcatcg aagcccgcct gcaggcctcg gtcgacgccg
aatctagaga 180 ttataaagat gacgatgaca aataataagc tagagg 216
<210> SEQ ID NO 267 <211> LENGTH: 202 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleic acid sequence
<400> SEQUENCE: 267 ccatacccgt ttttttgggc tagcaggagg
ccctgcatat gaaaataaaa acaggtgcac 60 gcatcctcgc attatccgca
ttaacgacga tgatgttttc cgcctcggct ctcgccaaaa 120 tcatcgaagc
ccgcctgcag gcctcggtcg acgccgaatc tagagattat aaagatgacg 180
atgacaaata ataagctaga gg 202 <210> SEQ ID NO 268 <211>
LENGTH: 182 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleic acid sequence <400> SEQUENCE: 268
aggaggttct gcatatgaaa ataaaaacag gtgcacgcat cctcgcatta tccgcattaa
60 cgacgatgat gttttccgcc tcggctctcg ccaaaatcat cgaagcccgc
ctgcaggcct 120 cggtcgacgc cgaatctaga gattataaag atgacgatga
caaataataa gctagaggta 180 cc 182 <210> SEQ ID NO 269
<211> LENGTH: 182 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic nucleic acid sequence <400> SEQUENCE:
269 aggaggttct gcatatgaaa ataaaaacag gtgcacgcat cctcgcatta
tccgcattaa 60 cgacgatgat gttttccgcc tcggctctcg ccaaaatcat
cgaagcccgc ctgcaggcct 120 cggtcgacgc cgaatctaga gattataaag
atgacgatga caaataataa gctagaggta 180 cc 182 <210> SEQ ID NO
270 <211> LENGTH: 1080 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic nucleic acid sequence <400>
SEQUENCE: 270 gaattcaggc gctttttaga ctggtcgtaa tgaaattcag
gaggttctgc atatgaaaat 60 aaaaacaggt gcacgcatcc tcgcattatc
cgcattaacg acgatgatgt tttccgcctc 120 ggctctcgcc aaaatcgaag
aaggtaaact ggtaatctgg attaacggcg ataaaggcta 180 taacggtctc
gctgaagtcg gtaagaaatt cgagaaagat accggaatta aagtcaccgt 240
tgagcatccg gataaactgg aagagaaatt cccacaggtt gcggcaactg gcgatggccc
300 tgacattatc ttctgggcac acgaccgctt tggtggctac gctcaatctg
gcctgttggc 360 tgaaatcacc ccggacaaag cgttccagga caagctgtat
ccgtttacct gggatgccgt 420 acgttacaac ggcaagctga ttgcttaccc
gatcgctgtt gaagcgttat cgctgattta 480 taacaaagat ctgctgccga
acccgccaaa aacctgggaa gagatcccgg cgctggataa 540 agaactgaaa
gcgaaaggta agagcgcgct gatgttcaac ctgcaagaac cgtacttcac 600
ctggccgctg attgctgctg acgggggtta tgcgttcaag tatgaaaacg gcaagtacga
660 cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg ggtctgacct
tcctggttga 720 cctgattaaa aacaaacaca tgaatgcaga caccgattac
tccatcgcag aagctgcctt 780 taataaaggc gaaacagcga tgaccatcaa
cggcccgtgg gcatggtcca acatcgacac 840 cagcaaagtg aattatggtg
taacggtact gccgaccttc aagggtcaac catccaaacc 900 gttcgttggc
gtgctgagcg caggtattaa cgccgccagt ccgaacaaag agctggcgaa 960
agagttcctc gaaaactatc tgctgactga tgaaggtctg gaagcggtta ataaagacaa
1020 accgctgggt gccgtagcgc tgaagtctta cgaggaagag ttggcgaaag
atccacgtat 1080 <210> SEQ ID NO 271 <211> LENGTH: 1196
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleic acid sequence <400> SEQUENCE: 271 ccatacccgt
ttttttgggc tagcaggagg ccctgcatat gaaaataaaa acaggtgcac 60
gcatcctcgc attatccgca ttaacgacga tgatgttttc cgcctcggct ctcgccaaaa
120 tcgaagaagg taaactggta atctggatta acggcgataa aggctataac
ggtctcgctg 180 aagtcggtaa gaaattcgag aaagataccg gaattaaagt
caccgttgag catccggata 240 aactggaaga gaaattccca caggttgcgg
caactggcga tggccctgac attatcttct 300 gggcacacga ccgctttggt
ggctacgctc aatctggcct gttggctgaa atcaccccgg 360 acaaagcgtt
ccaggacaag ctgtatccgt ttacctggga tgccgtacgt tacaacggca 420
agctgattgc ttacccgatc gctgttgaag cgttatcgct gatttataac aaagatctgc
480 tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct ggataaagaa
ctgaaagcga 540 aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta
cttcacctgg ccgctgattg 600 ctgctgacgg gggttatgcg ttcaagtatg
aaaacggcaa gtacgacatt aaagacgtgg 660 gcgtggataa cgctggcgcg
aaagcgggtc tgaccttcct ggttgacctg attaaaaaca 720 aacacatgaa
tgcagacacc gattactcca tcgcagaagc tgcctttaat aaaggcgaaa 780
cagcgatgac catcaacggc ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt
840 atggtgtaac ggtactgccg accttcaagg gtcaaccatc caaaccgttc
gttggcgtgc 900 tgagcgcagg tattaacgcc gccagtccga acaaagagct
ggcgaaagag ttcctcgaaa 960 actatctgct gactgatgaa ggtctggaag
cggttaataa agacaaaccg ctgggtgccg 1020 tagcgctgaa gtcttacgag
gaagagttgg cgaaagatcc acgtattgcc gccaccatgg 1080 aaaacgccca
gtccgctttc tggtatgccg tgcgtatcga agcccgcctg caggcctcgg 1140
tcgacgccga atctagagat tataaagatg acgatgacaa ataataagct agagga 1196
<210> SEQ ID NO 272 <211> LENGTH: 1171 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic nucleic acid
sequence <400> SEQUENCE: 272 aggaggttct gcatatgaaa ataaaaacag
gtgcacgcat cctcgcatta tccgcattaa 60 cgacgatgat gttttccgcc
tcggctctcg ccaaaatcga agaaggtaaa ctggtaatct 120 ggattaacgg
cgataaaggc tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag 180
ataccggaat taaagtcacc gttgagcatc cggataaact ggaagagaaa ttcccacagg
240 ttgcggcaac tggcgatggc cctgacatta tcttctgggc acacgaccgc
tttggtggct 300 acgctcaatc tggcctgttg gctgaaatca ccccggacaa
agcgttccag gacaagctgt 360 atccgtttac ctgggatgcc gtacgttaca
acggcaagct gattgcttac ccgatcgctg 420 ttgaagcgtt atcgctgatt
tataacaaag atctgctgcc gaacccgcca aaaacctggg 480 aagagatccc
ggcgctggat aaagaactga aagcgaaagg taagagcgcg ctgatgttca 540
acctgcaaga accgtacttc acctggccgc tgattgctgc tgacgggggt tatgcgttca
600 agtatgaaaa cggcaagtac gacattaaag acgtgggcgt ggataacgct
ggcgcgaaag 660 cgggtctgac cttcctggtt gacctgatta aaaacaaaca
catgaatgca gacaccgatt 720 actccatcgc agaagctgcc tttaataaag
gcgaaacagc gatgaccatc aacggcccgt 780 gggcatggtc caacatcgac
accagcaaag tgaattatgg tgtaacggta ctgccgacct 840 tcaagggtca
accatccaaa ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca 900
gtccgaacaa agagctggcg aaagagttcc tcgaaaacta tctgctgact gatgaaggtc
960 tggaagcggt taataaagac aaaccgctgg gtgccgtagc gctgaagtct
tacgaggaag 1020 agttggcgaa agatccacgt attgccgcca ccatggaaaa
cgcccagtcc gctttctggt 1080 atgccgtgcg tatcgaagcc cgcctgcagg
cctcggtcga cgccgaatct agagattata 1140 aagatgacga tgacaaataa
taagctagag g 1171 <210> SEQ ID NO 273 <211> LENGTH:
1171 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleic acid sequence <400> SEQUENCE: 273
aggaggttct gcatatgaaa ataaaaacag gtgcacgcat cctcgcatta tccgcattaa
60 cgacgatgat gttttccgcc tcggctctcg ccaaaatcga agaaggtaaa
ctggtaatct 120 ggattaacgg cgataaaggc tataacggtc tcgctgaagt
cggtaagaaa ttcgagaaag 180 ataccggaat taaagtcacc gttgagcatc
cggataaact ggaagagaaa ttcccacagg 240 ttgcggcaac tggcgatggc
cctgacatta tcttctgggc acacgaccgc tttggtggct 300 acgctcaatc
tggcctgttg gctgaaatca ccccggacaa agcgttccag gacaagctgt 360
atccgtttac ctgggatgcc gtacgttaca acggcaagct gattgcttac ccgatcgctg
420 ttgaagcgtt atcgctgatt tataacaaag atctgctgcc gaacccgcca
aaaacctggg 480 aagagatccc ggcgctggat aaagaactga aagcgaaagg
taagagcgcg ctgatgttca 540 acctgcaaga accgtacttc acctggccgc
tgattgctgc tgacgggggt tatgcgttca 600 agtatgaaaa cggcaagtac
gacattaaag acgtgggcgt ggataacgct ggcgcgaaag 660 cgggtctgac
cttcctggtt gacctgatta aaaacaaaca catgaatgca gacaccgatt 720
actccatcgc agaagctgcc tttaataaag gcgaaacagc gatgaccatc aacggcccgt
780 gggcatggtc caacatcgac accagcaaag tgaattatgg tgtaacggta
ctgccgacct 840 tcaagggtca accatccaaa ccgttcgttg gcgtgctgag
cgcaggtatt aacgccgcca 900 gtccgaacaa agagctggcg aaagagttcc
tcgaaaacta tctgctgact gatgaaggtc 960 tggaagcggt taataaagac
aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag 1020 agttggcgaa
agatccacgt attgccgcca ccatggaaaa cgcccagtcc gctttctggt 1080
atgccgtgcg tatcgaagcc cgcctgcagg cctcggtcga cgccgaatct agagattata
1140 aagatgacga tgacaaataa taagctagag g 1171 <210> SEQ ID NO
274 <211> LENGTH: 392 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic nucleic acid sequence <400>
SEQUENCE: 274 tagcaggagg ccctgcaggc ctcggtcgac gccgaatcta
gaagcgataa aattattcac 60 ctgactgacg acagttttga cacggatgta
ctcaaagcgg acggggcgat cctcgtcgat 120 ttctgggcag agtggtgcgg
tccgtgcaaa atgatcgccc cgattctgga tgaaatcgct 180 gacgaatatc
agggcaaact gaccgttgca aaactgaaca tcgatcaaaa ccctggcact 240
gcgccgaaat atggcatccg tggtatcccg actctgctgc tgttcaaaaa cggtgaagtg
300 gcggcaacca aagtgggtgc actgtctaaa ggtcagttga aagagaacct
ggcggattat 360 aaagatgacg atgacaaata ataagctaga gg 392 <210>
SEQ ID NO 275 <211> LENGTH: 426 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleic acid sequence
<400> SEQUENCE: 275 gaattcaggc gctttttaga ctggtcgtaa
tgaaattcag gaggttctgc aggcctcggt 60 cgacgccgaa tctagaagcg
ataaaattat tcacctgact gacgacagtt ttgacacgga 120 tgtactcaaa
gcggacgggg cgatcctcgt cgatttctgg gcagagtggt gcggtccgtg 180
caaaatgatc gccccgattc tggatgaaat cgctgacgaa tatcagggca aactgaccgt
240 tgcaaaactg aacatcgatc aaaaccctgg cactgcgccg aaatatggca
tccgtggtat 300 cccgactctg ctgctgttca aaaacggtga agtggcggca
accaaagtgg gtgcactgtc 360 taaaggtcag ttgaaagaga acctggcgga
ttataaagat gacgatgaca aataataagc 420 tagagg 426 <210> SEQ ID
NO 276 <211> LENGTH: 392 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic nucleic acid sequence <400>
SEQUENCE: 276 aggaggttct gcaggcctcg gtcgacgccg aatctagaag
cgataaaatt attcacctga 60 ctgacgacag ttttgacacg gatgtactca
aagcggacgg ggcgatcctc gtcgatttct 120 gggcagagtg gtgcggtccg
tgcaaaatga tcgccccgat tctggatgaa atcgctgacg 180 aatatcaggg
caaactgacc gttgcaaaac tgaacatcga tcaaaaccct ggcactgcgc 240
cgaaatatgg catccgtggt atcccgactc tgctgctgtt caaaaacggt gaagtggcgg
300 caaccaaagt gggtgcactg tctaaaggtc agttgaaaga gaacctggcg
gattataaag 360 atgacgatga caaataataa gctagaggta cc 392 <210>
SEQ ID NO 277 <211> LENGTH: 392 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleic acid sequence
<400> SEQUENCE: 277 aggaggttct gcaggcctcg gtcgacgccg
aatctagaag cgataaaatt attcacctga 60 ctgacgacag ttttgacacg
gatgtactca aagcggacgg ggcgatcctc gtcgatttct 120 gggcagagtg
gtgcggtccg tgcaaaatga tcgccccgat tctggatgaa atcgctgacg 180
aatatcaggg caaactgacc gttgcaaaac tgaacatcga tcaaaaccct ggcactgcgc
240 cgaaatatgg catccgtggt atcccgactc tgctgctgtt caaaaacggt
gaagtggcgg 300 caaccaaagt gggtgcactg tctaaaggtc agttgaaaga
gaacctggcg gattataaag 360 atgacgatga caaataataa gctagaggta cc 392
<210> SEQ ID NO 278 <211> LENGTH: 528 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleic acid sequence
<400> SEQUENCE: 278 gaattcaggc gctttttaga ctggtcgtaa
tgaaattcag gaggttctgc atatgaaaat 60 aaaaacaggt gcacgcatcc
tcgcattatc cgcattaacg acgatgatgt tttccgcctc 120 ggctctcgcc
aaaatcatcg aagcccgcct gcaggcctcg gtcgacgccg aatctagaag 180
cgataaaatt attcacctga ctgacgacag ttttgacacg gatgtactca aagcggacgg
240 ggcgatcctc gtcgatttct gggcagagtg gtgcggtccg tgcaaaatga
tcgccccgat 300 tctggatgaa atcgctgacg aatatcaggg caaactgacc
gttgcaaaac tgaacatcga 360 tcaaaaccct ggcactgcgc cgaaatatgg
catccgtggt atcccgactc tgctgctgtt 420 caaaaacggt gaagtggcgg
caaccaaagt gggtgcactg tctaaaggtc agttgaaaga 480 gaacctggcg
gattataaag atgacgatga caaataataa gctagagg 528 <210> SEQ ID NO
279 <211> LENGTH: 514 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic nucleic acid sequence <400>
SEQUENCE: 279 ccatacccgt ttttttgggc tagcaggagg ccctgcatat
gaaaataaaa acaggtgcac 60 gcatcctcgc attatccgca ttaacgacga
tgatgttttc cgcctcggct ctcgccaaaa 120 tcatcgaagc ccgcctgcag
gcctcggtcg acgccgaatc tagaagcgat aaaattattc 180 acctgactga
cgacagtttt gacacggatg tactcaaagc ggacggggcg atcctcgtcg 240
atttctgggc agagtggtgc ggtccgtgca aaatgatcgc cccgattctg gatgaaatcg
300 ctgacgaata tcagggcaaa ctgaccgttg caaaactgaa catcgatcaa
aaccctggca 360 ctgcgccgaa atatggcatc cgtggtatcc cgactctgct
gctgttcaaa aacggtgaag 420 tggcggcaac caaagtgggt gcactgtcta
aaggtcagtt gaaagagaac ctggcggatt 480 ataaagatga cgatgacaaa
taataagcta gagg 514 <210> SEQ ID NO 280 <211> LENGTH:
494 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleic acid sequence <400> SEQUENCE: 280 aggaggttct
gcatatgaaa ataaaaacag gtgcacgcat cctcgcatta tccgcattaa 60
cgacgatgat gttttccgcc tcggctctcg ccaaaatcat cgaagcccgc ctgcaggcct
120 cggtcgacgc cgaatctaga agcgataaaa ttattcacct gactgacgac
agttttgaca 180 cggatgtact caaagcggac ggggcgatcc tcgtcgattt
ctgggcagag tggtgcggtc 240 cgtgcaaaat gatcgccccg attctggatg
aaatcgctga cgaatatcag ggcaaactga 300 ccgttgcaaa actgaacatc
gatcaaaacc ctggcactgc gccgaaatat ggcatccgtg 360 gtatcccgac
tctgctgctg ttcaaaaacg gtgaagtggc ggcaaccaaa gtgggtgcac 420
tgtctaaagg tcagttgaaa gagaacctgg cggattataa agatgacgat gacaaataat
480 aagctagagg tacc 494 <210> SEQ ID NO 281 <211>
LENGTH: 494 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleic acid sequence <400> SEQUENCE: 281
aggaggttct gcatatgaaa ataaaaacag gtgcacgcat cctcgcatta tccgcattaa
60 cgacgatgat gttttccgcc tcggctctcg ccaaaatcat cgaagcccgc
ctgcaggcct 120 cggtcgacgc cgaatctaga agcgataaaa ttattcacct
gactgacgac agttttgaca 180 cggatgtact caaagcggac ggggcgatcc
tcgtcgattt ctgggcagag tggtgcggtc 240 cgtgcaaaat gatcgccccg
attctggatg aaatcgctga cgaatatcag ggcaaactga 300 ccgttgcaaa
actgaacatc gatcaaaacc ctggcactgc gccgaaatat ggcatccgtg 360
gtatcccgac tctgctgctg ttcaaaaacg gtgaagtggc ggcaaccaaa gtgggtgcac
420 tgtctaaagg tcagttgaaa gagaacctgg cggattataa agatgacgat
gacaaataat 480 aagctagagg tacc 494 <210> SEQ ID NO 282
<211> LENGTH: 1521 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic nucleic acid sequence <400>
SEQUENCE: 282 gaattcaggc gctttttaga ctggtcgtaa tgaaattcag
gaggttctgc atatgaaaat 60 aaaaacaggt gcacgcatcc tcgcattatc
cgcattaacg acgatgatgt tttccgcctc 120 ggctctcgcc aaaatcgaag
aaggtaaact ggtaatctgg attaacggcg ataaaggcta 180 taacggtctc
gctgaagtcg gtaagaaatt cgagaaagat accggaatta aagtcaccgt 240
tgagcatccg gataaactgg aagagaaatt cccacaggtt gcggcaactg gcgatggccc
300 tgacattatc ttctgggcac acgaccgctt tggtggctac gctcaatctg
gcctgttggc 360 tgaaatcacc ccggacaaag cgttccagga caagctgtat
ccgtttacct gggatgccgt 420 acgttacaac ggcaagctga ttgcttaccc
gatcgctgtt gaagcgttat cgctgattta 480 taacaaagat ctgctgccga
acccgccaaa aacctgggaa gagatcccgg cgctggataa 540 agaactgaaa
gcgaaaggta agagcgcgct gatgttcaac ctgcaagaac cgtacttcac 600
ctggccgctg attgctgctg acgggggtta tgcgttcaag tatgaaaacg gcaagtacga
660 cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg ggtctgacct
tcctggttga 720 cctgattaaa aacaaacaca tgaatgcaga caccgattac
tccatcgcag aagctgcctt 780 taataaaggc gaaacagcga tgaccatcaa
cggcccgtgg gcatggtcca acatcgacac 840 cagcaaagtg aattatggtg
taacggtact gccgaccttc aagggtcaac catccaaacc 900 gttcgttggc
gtgctgagcg caggtattaa cgccgccagt ccgaacaaag agctggcgaa 960
agagttcctc gaaaactatc tgctgactga tgaaggtctg gaagcggtta ataaagacaa
1020 accgctgggt gccgtagcgc tgaagtctta cgaggaagag ttggcgaaag
atccacgtat 1080 tgccgccacc atggaaaacg cccagtccgc tttctggtat
gccgtgcgta tcgaagcccg 1140 cctgcaggcc tcggtcgacg ccgaatctag
aagcgataaa attattcacc tgactgacga 1200 cagttttgac acggatgtac
tcaaagcgga cggggcgatc ctcgtcgatt tctgggcaga 1260 gtggtgcggt
ccgtgcaaaa tgatcgcccc gattctggat gaaatcgctg acgaatatca 1320
gggcaaactg accgttgcaa aactgaacat cgatcaaaac cctggcactg cgccgaaata
1380 tggcatccgt ggtatcccga ctctgctgct gttcaaaaac ggtgaagtgg
cggcaaccaa 1440 agtgggtgca ctgtctaaag gtcagttgaa agagaacctg
gcggattata aagatgacga 1500 tgacaaataa taagctagag g 1521 <210>
SEQ ID NO 283 <211> LENGTH: 1507 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleic acid sequence
<400> SEQUENCE: 283 ccatacccgt ttttttgggc tagcaggagg
ccctgcatat gaaaataaaa acaggtgcac 60 gcatcctcgc attatccgca
ttaacgacga tgatgttttc cgcctcggct ctcgccaaaa 120 tcgaagaagg
taaactggta atctggatta acggcgataa aggctataac ggtctcgctg 180
aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag catccggata
240 aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac
attatcttct 300 gggcacacga ccgctttggt ggctacgctc aatctggcct
gttggctgaa atcaccccgg 360 acaaagcgtt ccaggacaag ctgtatccgt
ttacctggga tgccgtacgt tacaacggca 420 agctgattgc ttacccgatc
gctgttgaag cgttatcgct gatttataac aaagatctgc 480 tgccgaaccc
gccaaaaacc tgggaagaga tcccggcgct ggataaagaa ctgaaagcga 540
aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg ccgctgattg
600 ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt
aaagacgtgg 660 gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct
ggttgacctg attaaaaaca 720 aacacatgaa tgcagacacc gattactcca
tcgcagaagc tgcctttaat aaaggcgaaa 780 cagcgatgac catcaacggc
ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt 840 atggtgtaac
ggtactgccg accttcaagg gtcaaccatc caaaccgttc gttggcgtgc 900
tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcgaaagag ttcctcgaaa
960 actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg
ctgggtgccg 1020 tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc
acgtattgcc gccaccatgg 1080 aaaacgccca gtccgctttc tggtatgccg
tgcgtatcga agcccgcctg caggcctcgg 1140 tcgacgccga atctagaagc
gataaaatta ttcacctgac tgacgacagt tttgacacgg 1200 atgtactcaa
agcggacggg gcgatcctcg tcgatttctg ggcagagtgg tgcggtccgt 1260
gcaaaatgat cgccccgatt ctggatgaaa tcgctgacga atatcagggc aaactgaccg
1320 ttgcaaaact gaacatcgat caaaaccctg gcactgcgcc gaaatatggc
atccgtggta 1380 tcccgactct gctgctgttc aaaaacggtg aagtggcggc
aaccaaagtg ggtgcactgt 1440 ctaaaggtca gttgaaagag aacctggcgg
attataaaga tgacgatgac aaataataag 1500 ctagagg 1507 <210> SEQ
ID NO 284 <211> LENGTH: 1476 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleic acid sequence
<400> SEQUENCE: 284 aggaggttct gcatatgaaa ataaaaacag
gtgcacgcat cctcgcatta tccgcattaa 60 cgacgatgat gttttccgcc
tcggctctcg ccaaaatcga agaaggtaaa ctggtaatct 120 ggattaacgg
cgataaaggc tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag 180
ataccggaat taaagtcacc gttgagcatc cggataaact ggaagagaaa ttcccacagg
240 ttgcggcaac tggcgatggc cctgacatta tcttctgggc acacgaccgc
tttggtggct 300 acgctcaatc tggcctgttg gctgaaatca ccccggacaa
agcgttccag gacaagctgt 360 atccgtttac ctgggatgcc gtacgttaca
acggcaagct gattgcttac ccgatcgctg 420 ttgaagcgtt atcgctgatt
tataacaaag atctgctgcc gaacccgcca aaaacctggg 480 aagagatccc
ggcgctggat aaagaactga aagcgaaagg taagagcgcg ctgatgttca 540
acctgcaaga accgtacttc acctggccgc tgattgctgc tgacgggggt tatgcgttca
600 agtatgaaaa cggcaagtac gacattaaag acgtgggcgt ggataacgct
ggcgcgaaag 660 cgggtctgac cttcctggtt gacctgatta aaaacaaaca
catgaatgca gacaccgatt 720 actccatcgc agaagctgcc tttaataaag
gcgaaacagc gatgaccatc aacggcccgt 780 gggcatggtc caacatcgac
accagcaaag tgaattatgg tgtaacggta ctgccgacct 840 tcaagggtca
accatccaaa ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca 900
gtccgaacaa agagctggcg aaagagttcc tcgaaaacta tctgctgact gatgaaggtc
960 tggaagcggt taataaagac aaaccgctgg gtgccgtagc gctgaagtct
tacgaggaag 1020 agttggcgaa agatccacgt attgccgcca ccatggaaaa
cgcccagtcc gctttctggt 1080 atgccgtgcg tatcgaagcc cgcctgcagg
cctcggtcga cgccgaatct agaagcgata 1140 aaattattca cctgactgac
gacagttttg acacggatgt actcaaagcg gacggggcga 1200 tcctcgtcga
tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc ccgattctgg 1260
atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc aaaactgaac atcgatcaaa
1320 accctggcac tgcgccgaaa tatggcatcc gtggtatccc gactctgctg
ctgttcaaaa 1380 acggtgaagt ggcggcaacc aaagtgggtg cactgtctaa
aggtcagttg aaagagaacc 1440 tggcggatta taaagatgac gatgacaaat aataag
1476 <210> SEQ ID NO 285 <211> LENGTH: 1476 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic nucleic acid
sequence <400> SEQUENCE: 285 aggaggttct gcatatgaaa ataaaaacag
gtgcacgcat cctcgcatta tccgcattaa 60 cgacgatgat gttttccgcc
tcggctctcg ccaaaatcga agaaggtaaa ctggtaatct 120 ggattaacgg
cgataaaggc tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag 180
ataccggaat taaagtcacc gttgagcatc cggataaact ggaagagaaa ttcccacagg
240 ttgcggcaac tggcgatggc cctgacatta tcttctgggc acacgaccgc
tttggtggct 300 acgctcaatc tggcctgttg gctgaaatca ccccggacaa
agcgttccag gacaagctgt 360 atccgtttac ctgggatgcc gtacgttaca
acggcaagct gattgcttac ccgatcgctg 420 ttgaagcgtt atcgctgatt
tataacaaag atctgctgcc gaacccgcca aaaacctggg 480 aagagatccc
ggcgctggat aaagaactga aagcgaaagg taagagcgcg ctgatgttca 540
acctgcaaga accgtacttc acctggccgc tgattgctgc tgacgggggt tatgcgttca
600 agtatgaaaa cggcaagtac gacattaaag acgtgggcgt ggataacgct
ggcgcgaaag 660 cgggtctgac cttcctggtt gacctgatta aaaacaaaca
catgaatgca gacaccgatt 720 actccatcgc agaagctgcc tttaataaag
gcgaaacagc gatgaccatc aacggcccgt 780 gggcatggtc caacatcgac
accagcaaag tgaattatgg tgtaacggta ctgccgacct 840 tcaagggtca
accatccaaa ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca 900
gtccgaacaa agagctggcg aaagagttcc tcgaaaacta tctgctgact gatgaaggtc
960 tggaagcggt taataaagac aaaccgctgg gtgccgtagc gctgaagtct
tacgaggaag 1020 agttggcgaa agatccacgt attgccgcca ccatggaaaa
cgcccagtcc gctttctggt 1080 atgccgtgcg tatcgaagcc cgcctgcagg
cctcggtcga cgccgaatct agaagcgata 1140 aaattattca cctgactgac
gacagttttg acacggatgt actcaaagcg gacggggcga 1200 tcctcgtcga
tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc ccgattctgg 1260
atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc aaaactgaac atcgatcaaa
1320 accctggcac tgcgccgaaa tatggcatcc gtggtatccc gactctgctg
ctgttcaaaa 1380 acggtgaagt ggcggcaacc aaagtgggtg cactgtctaa
aggtcagttg aaagagaacc 1440 tggcggatta taaagatgac gatgacaaat aataag
1476 <210> SEQ ID NO 286 <211> LENGTH: 383 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic amino acid
sequence <400> SEQUENCE: 286 Met Phe Glu Pro Met Glu Leu Thr
Asn Asp Ala Val Ile Lys Val Ile 1 5 10 15 Gly Val Gly Gly Gly Gly
Gly Asn Ala Val Glu His Met Val Arg Glu 20 25 30 Arg Ile Glu Gly
Val Glu Phe Phe Ala Val Asn Thr Asp Ala Gln Ala 35 40 45 Leu Arg
Lys Thr Ala Val Gly Gln Thr Ile Gln Ile Gly Ser Gly Ile 50 55 60
Thr Lys Gly Leu Gly Ala Gly Ala Asn Pro Glu Val Gly Arg Asn Ala 65
70 75 80 Ala Asp Glu Asp Arg Asp Ala Leu Arg Ala Ala Leu Glu Gly
Ala Asp 85 90 95 Met Val Phe Ile Ala Ala Gly Met Gly Gly Gly Thr
Gly Thr Gly Ala 100 105 110 Ala Pro Val Val Ala Glu Val Ala Lys Asp
Leu Gly Ile Leu Thr Val 115 120 125 Ala Val Val Thr Lys Pro Phe Asn
Phe Glu Gly Lys Lys Arg Met Ala 130 135 140 Phe Ala Glu Gln Gly Ile
Thr Glu Leu Ser Lys His Val Asp Ser Leu 145 150 155 160 Ile Thr Ile
Pro Asn Asp Lys Leu Leu Lys Val Leu Gly Arg Gly Ile 165 170 175 Ser
Leu Leu Asp Ala Phe Gly Ala Ala Asn Asp Val Leu Lys Gly Ala 180 185
190 Val Gln Gly Ile Ala Glu Leu Ile Thr Arg Pro Gly Leu Met Asn Val
195 200 205 Asp Phe Ala Asp Val Arg Thr Val Met Ser Glu Met Gly Tyr
Ala Met 210 215 220 Met Gly Ser Gly Val Ala Ser Gly Glu Asp Arg Ala
Glu Glu Ala Ala 225 230 235 240 Glu Met Ala Ile Ser Ser Pro Leu Leu
Glu Asp Ile Asp Leu Ser Gly 245 250 255 Ala Arg Gly Val Leu Val Asn
Ile Thr Ala Gly Phe Asp Leu Arg Leu 260 265 270 Asp Glu Phe Glu Thr
Val Gly Asn Thr Ile Arg Ala Phe Ala Ser Asp 275 280 285 Asn Ala Thr
Val Val Ile Gly Thr Ser Leu Asp Pro Asp Met Asn Asp 290 295 300 Glu
Leu Arg Val Thr Val Val Ala Thr Gly Ile Gly Met Asp Lys Arg 305 310
315 320 Pro Glu Ile Thr Leu Val Thr Asn Lys Gln Val Gln Gln Pro Val
Met 325 330 335 Asp Arg Tyr Gln Gln His Gly Met Ala Pro Leu Thr Gln
Glu Gln Lys 340 345 350 Pro Val Ala Lys Val Val Asn Asp Asn Ala Pro
Gln Thr Ala Lys Glu 355 360 365 Pro Asp Tyr Leu Asp Ile Pro Ala Phe
Leu Arg Lys Gln Ala Asp 370 375 380 <210> SEQ ID NO 287
<211> LENGTH: 383 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic amino acid sequence <400> SEQUENCE:
287 Met Phe Glu Pro Met Glu Leu Thr Asn Asp Ala Val Ile Lys Val Ile
1 5 10 15 Gly Val Gly Gly Gly Gly Gly Asn Ala Val Glu His Met Val
Arg Glu 20 25 30 Arg Ile Glu Gly Val Glu Phe Phe Ala Val Asn Thr
Asp Ala Gln Ala 35 40 45 Leu Arg Lys Thr Ala Val Gly Gln Thr Ile
Gln Ile Gly Ser Gly Ile 50 55 60 Thr Lys Gly Leu Gly Ala Gly Ala
Asn Pro Glu Val Gly Arg Asn Ala 65 70 75 80 Ala Asp Glu Asp Arg Asp
Ala Leu Arg Ala Ala Leu Glu Gly Ala Asp 85 90 95 Met Val Phe Ile
Ala Ala Gly Met Gly Gly Gly Thr Gly Thr Gly Ala 100 105 110 Ala Pro
Val Val Ala Glu Val Ala Lys Asp Leu Gly Ile Leu Thr Val 115 120 125
Ala Val Val Thr Lys Pro Phe Asn Phe Glu Gly Lys Lys Arg Met Ala 130
135 140 Phe Ala Glu Gln Gly Ile Thr Glu Leu Ser Lys His Val Asp Ser
Leu 145 150 155 160 Ile Thr Ile Pro Asn Asp Lys Leu Leu Lys Val Leu
Gly Arg Gly Ile 165 170 175 Ser Leu Leu Asp Ala Phe Gly Ala Ala Asn
Asp Val Leu Lys Gly Ala 180 185 190 Val Gln Gly Ile Ala Glu Leu Ile
Thr Arg Pro Gly Leu Met Asn Val 195 200 205 Asp Phe Ala Asp Val Arg
Thr Val Met Ser Glu Met Gly Tyr Ala Met 210 215 220 Met Gly Ser Gly
Val Ala Ser Gly Glu Asp Arg Ala Glu Glu Ala Ala 225 230 235 240 Glu
Met Ala Ile Ser Ser Pro Leu Leu Glu Asp Ile Asp Leu Ser Gly 245 250
255 Ala Arg Gly Val Leu Val Asn Ile Thr Ala Gly Phe Asp Leu Arg Leu
260 265 270 Asp Glu Phe Glu Thr Val Gly Asn Thr Ile Arg Ala Phe Ala
Ser Asp 275 280 285 Asn Ala Thr Val Val Ile Gly Thr Ser Leu Asp Pro
Asp Met Asn Asp 290 295 300 Glu Leu Arg Val Thr Val Val Ala Thr Gly
Ile Gly Met Asp Lys Arg 305 310 315 320 Pro Glu Ile Thr Leu Val Thr
Asn Lys Gln Val Gln Gln Pro Val Met 325 330 335 Asp Arg Tyr Gln Gln
His Gly Met Ala Pro Leu Thr Gln Glu Gln Lys 340 345 350 Pro Val Ala
Lys Val Val Asn Asp Asn Ala Pro Gln Thr Ala Lys Glu 355 360 365 Pro
Asp Tyr Leu Asp Ile Pro Ala Phe Leu Arg Lys Gln Ala Asp 370 375 380
<210> SEQ ID NO 288 <211> LENGTH: 446 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 288 Met Pro Val Leu Glu Asn Arg Ala Ala Gln
Gly Asp Ile Thr Ala Pro 1 5 10 15 Gly Gly Ala Arg Arg Leu Thr Gly
Asp Gln Thr Ala Ala Leu Arg Asp 20 25 30 Ser Leu Ser Asp Lys Pro
Ala Lys Asn Ile Ile Leu Leu Ile Gly Asp 35 40 45 Gly Met Gly Asp
Ser Glu Ile Thr Ala Ala Arg Asn Tyr Ala Glu Gly 50 55 60 Ala Gly
Gly Phe Phe Lys Gly Ile Asp Ala Leu Pro Leu Thr Gly Gln 65 70 75 80
Tyr Thr His Tyr Ala Leu Asn Lys Lys Thr Gly Lys Pro Asp Tyr Val 85
90 95 Thr Asp Ser Ala Ala Ser Ala Thr Ala Trp Ser Thr Gly Val Lys
Thr 100 105 110 Tyr Asn Gly Ala Leu Gly Val Asp Ile His Glu Lys Asp
His Pro Thr 115 120 125 Ile Leu Glu Met Ala Lys Ala Ala Gly Leu Ala
Thr Gly Asn Val Ser 130 135 140 Thr Ala Glu Leu Gln Asp Ala Thr Pro
Ala Ala Leu Val Ala His Val 145 150 155 160 Thr Ser Arg Lys Cys Tyr
Gly Pro Ser Ala Thr Ser Glu Lys Cys Pro 165 170 175 Gly Asn Ala Leu
Glu Lys Gly Gly Lys Gly Ser Ile Thr Glu Gln Leu 180 185 190 Leu Asn
Ala Arg Ala Asp Val Thr Leu Gly Gly Gly Ala Lys Thr Phe 195 200 205
Ala Glu Thr Ala Thr Ala Gly Glu Trp Gln Gly Lys Thr Leu Arg Glu 210
215 220 Gln Ala Gln Ala Arg Gly Tyr Gln Leu Val Ser Asp Ala Ala Ser
Leu 225 230 235 240 Asn Ser Val Thr Glu Ala Asn Gln Gln Lys Pro Leu
Leu Gly Leu Phe 245 250 255 Ala Asp Gly Asn Met Pro Val Arg Trp Leu
Gly Pro Lys Ala Thr Tyr 260 265 270 His Gly Asn Ile Asp Lys Pro Ala
Val Thr Cys Thr Pro Asn Pro Gln 275 280 285 Arg Asn Asp Ser Val Pro
Thr Leu Ala Gln Met Thr Asp Lys Ala Ile 290 295 300 Glu Leu Leu Ser
Lys Asn Glu Lys Gly Phe Phe Leu Gln Val Glu Gly 305 310 315 320 Ala
Ser Ile Asp Lys Gln Asp His Ala Ala Asn Pro Cys Gly Gln Ile 325 330
335 Gly Glu Thr Val Asp Leu Asp Glu Ala Val Gln Arg Ala Leu Glu Phe
340 345 350 Ala Lys Lys Glu Gly Asn Thr Leu Val Ile Val Thr Ala Asp
His Ala 355 360 365 His Ala Ser Gln Ile Val Ala Pro Asp Thr Lys Ala
Pro Gly Leu Thr 370 375 380 Gln Ala Leu Asn Thr Lys Asp Gly Ala Val
Met Val Met Ser Tyr Gly 385 390 395 400 Asn Ser Glu Glu Asp Ser Gln
Glu His Thr Gly Ser Gln Leu Arg Ile 405 410 415 Ala Ala Tyr Gly Pro
His Ala Ala Asn Val Val Gly Leu Thr Asp Gln 420 425 430 Thr Asp Leu
Phe Tyr Thr Met Lys Ala Ala Leu Gly Leu Lys 435 440 445 <210>
SEQ ID NO 289 <211> LENGTH: 494 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 289 Met Ser Arg Pro Arg Leu Ile Val Ala Leu
Phe Leu Phe Phe Asn Val 1 5 10 15 Phe Val His Gly Glu Asn Lys Val
Lys Gln Ser Thr Ile Ala Leu Ala 20 25 30 Leu Leu Pro Leu Leu Phe
Thr Pro Val Thr Lys Ala Arg Thr Pro Glu 35 40 45 Met Pro Val Leu
Glu Asn Arg Ala Ala Gln Gly Asp Ile Thr Ala Pro 50 55 60 Gly Gly
Ala Arg Arg Leu Thr Gly Asp Gln Thr Ala Ala Leu Arg Asp 65 70 75 80
Ser Leu Ser Asp Lys Pro Ala Lys Asn Ile Ile Leu Leu Ile Gly Asp 85
90 95 Gly Met Gly Asp Ser Glu Ile Thr Ala Ala Arg Asn Tyr Ala Glu
Gly 100 105 110 Ala Gly Gly Phe Phe Lys Gly Ile Asp Ala Leu Pro Leu
Thr Gly Gln 115 120 125 Tyr Thr His Tyr Ala Leu Asn Lys Lys Thr Gly
Lys Pro Asp Tyr Val 130 135 140 Thr Asp Ser Ala Ala Ser Ala Thr Ala
Trp Ser Thr Gly Val Lys Thr 145 150 155 160 Tyr Asn Gly Ala Leu Gly
Val Asp Ile His Glu Lys Asp His Pro Thr 165 170 175 Ile Leu Glu Met
Ala Lys Ala Ala Gly Leu Ala Thr Gly Asn Val Ser 180 185 190 Thr Ala
Glu Leu Gln Asp Ala Thr Pro Ala Ala Leu Val Ala His Val 195 200 205
Thr Ser Arg Lys Cys Tyr Gly Pro Ser Ala Thr Ser Glu Lys Cys Pro 210
215 220 Gly Asn Ala Leu Glu Lys Gly Gly Lys Gly Ser Ile Thr Glu Gln
Leu 225 230 235 240 Leu Asn Ala Arg Ala Asp Val Thr Leu Gly Gly Gly
Ala Lys Thr Phe 245 250 255 Ala Glu Thr Ala Thr Ala Gly Glu Trp Gln
Gly Lys Thr Leu Arg Glu 260 265 270 Gln Ala Gln Ala Arg Gly Tyr Gln
Leu Val Ser Asp Ala Ala Ser Leu 275 280 285 Asn Ser Val Thr Glu Ala
Asn Gln Gln Lys Pro Leu Leu Gly Leu Phe 290 295 300 Ala Asp Gly Asn
Met Pro Val Arg Trp Leu Gly Pro Lys Ala Thr Tyr 305 310 315 320 His
Gly Asn Ile Asp Lys Pro Ala Val Thr Cys Thr Pro Asn Pro Gln 325 330
335 Arg Asn Asp Ser Val Pro Thr Leu Ala Gln Met Thr Asp Lys Ala Ile
340 345 350 Glu Leu Leu Ser Lys Asn Glu Lys Gly Phe Phe Leu Gln Val
Glu Gly 355 360 365 Ala Ser Ile Asp Lys Gln Asp His Ala Ala Asn Pro
Cys Gly Gln Ile 370 375 380 Gly Glu Thr Val Asp Leu Asp Glu Ala Val
Gln Arg Ala Leu Glu Phe 385 390 395 400 Ala Lys Lys Glu Gly Asn Thr
Leu Val Ile Val Thr Ala Asp His Ala 405 410 415 His Ala Ser Gln Ile
Val Ala Pro Asp Thr Lys Ala Pro Gly Leu Thr 420 425 430 Gln Ala Leu
Asn Thr Lys Asp Gly Ala Val Met Val Met Ser Tyr Gly 435 440 445 Asn
Ser Glu Glu Asp Ser Gln Glu His Thr Gly Ser Gln Leu Arg Ile 450 455
460 Ala Ala Tyr Gly Pro His Ala Ala Asn Val Val Gly Leu Thr Asp Gln
465 470 475 480 Thr Asp Leu Phe Tyr Thr Met Lys Ala Ala Leu Gly Leu
Lys 485 490 <210> SEQ ID NO 290 <211> LENGTH: 468
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
chimeric amino acid sequence <400> SEQUENCE: 290 Met Asn Leu
Gly Asn Arg Leu Phe Ile Leu Ile Ala Val Leu Leu Pro 1 5 10 15 Leu
Ala Val Leu Leu Leu Met Pro Val Leu Glu Asn Arg Ala Ala Gln 20 25
30 Gly Asp Ile Thr Ala Pro Gly Gly Ala Arg Arg Leu Thr Gly Asp Gln
35 40 45 Thr Ala Ala Leu Arg Asp Ser Leu Ser Asp Lys Pro Ala Lys
Asn Ile 50 55 60 Ile Leu Leu Ile Gly Asp Gly Met Gly Asp Ser Glu
Ile Thr Ala Ala 65 70 75 80 Arg Asn Tyr Ala Glu Gly Ala Gly Gly Phe
Phe Lys Gly Ile Asp Ala 85 90 95 Leu Pro Leu Thr Gly Gln Tyr Thr
His Tyr Ala Leu Asn Lys Lys Thr 100 105 110 Gly Lys Pro Asp Tyr Val
Thr Asp Ser Ala Ala Ser Ala Thr Ala Trp 115 120 125 Ser Thr Gly Val
Lys Thr Tyr Asn Gly Ala Leu Gly Val Asp Ile His 130 135 140 Glu Lys
Asp His Pro Thr Ile Leu Glu Met Ala Lys Ala Ala Gly Leu 145 150 155
160 Ala Thr Gly Asn Val Ser Thr Ala Glu Leu Gln Asp Ala Thr Pro Ala
165 170 175 Ala Leu Val Ala His Val Thr Ser Arg Lys Cys Tyr Gly Pro
Ser Ala 180 185 190 Thr Ser Glu Lys Cys Pro Gly Asn Ala Leu Glu Lys
Gly Gly Lys Gly 195 200 205 Ser Ile Thr Glu Gln Leu Leu Asn Ala Arg
Ala Asp Val Thr Leu Gly 210 215 220 Gly Gly Ala Lys Thr Phe Ala Glu
Thr Ala Thr Ala Gly Glu Trp Gln 225 230 235 240 Gly Lys Thr Leu Arg
Glu Gln Ala Gln Ala Arg Gly Tyr Gln Leu Val 245 250 255 Ser Asp Ala
Ala Ser Leu Asn Ser Val Thr Glu Ala Asn Gln Gln Lys 260 265 270 Pro
Leu Leu Gly Leu Phe Ala Asp Gly Asn Met Pro Val Arg Trp Leu 275 280
285 Gly Pro Lys Ala Thr Tyr His Gly Asn Ile Asp Lys Pro Ala Val Thr
290 295 300 Cys Thr Pro Asn Pro Gln Arg Asn Asp Ser Val Pro Thr Leu
Ala Gln 305 310 315 320 Met Thr Asp Lys Ala Ile Glu Leu Leu Ser Lys
Asn Glu Lys Gly Phe 325 330 335 Phe Leu Gln Val Glu Gly Ala Ser Ile
Asp Lys Gln Asp His Ala Ala 340 345 350 Asn Pro Cys Gly Gln Ile Gly
Glu Thr Val Asp Leu Asp Glu Ala Val 355 360 365 Gln Arg Ala Leu Glu
Phe Ala Lys Lys Glu Gly Asn Thr Leu Val Ile 370 375 380 Val Thr Ala
Asp His Ala His Ala Ser Gln Ile Val Ala Pro Asp Thr 385 390 395 400
Lys Ala Pro Gly Leu Thr Gln Ala Leu Asn Thr Lys Asp Gly Ala Val 405
410 415 Met Val Met Ser Tyr Gly Asn Ser Glu Glu Asp Ser Gln Glu His
Thr 420 425 430 Gly Ser Gln Leu Arg Ile Ala Ala Tyr Gly Pro His Ala
Ala Asn Val 435 440 445 Val Gly Leu Thr Asp Gln Thr Asp Leu Phe Tyr
Thr Met Lys Ala Ala 450 455 460 Leu Gly Leu Lys 465 <210> SEQ
ID NO 291 <211> LENGTH: 424 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 291 Met Ala Thr Thr His Ala Gln Gly His Pro Pro Val Leu
Gly Asn Asp 1 5 10 15 Thr Leu Arg Glu His Tyr Asp Tyr Val Gly Lys
Leu Ala Gly Arg Leu 20 25 30 Arg Asp Pro Pro Glu Gly Ser Thr Leu
Ile Thr Thr Ile Leu Phe Leu 35 40 45 Val Thr Cys Ser Phe Ile Val
Leu Glu Asn Leu Met Val Leu Ile Ala 50 55 60 Ile Trp Lys Asn Asn
Lys Phe His Asn Arg Met Tyr Phe Phe Ile Gly 65 70 75 80 Asn Leu Ala
Leu Cys Asp Leu Leu Ala Gly Ile Ala Tyr Lys Val Asn 85 90 95 Ile
Leu Met Ser Gly Arg Lys Thr Phe Ser Leu Ser Pro Thr Val Trp 100 105
110 Phe Leu Arg Glu Gly Ser Met Phe Val Ala Leu Gly Ala Ser Thr Cys
115 120 125 Ser Leu Leu Ala Ile Ala Ile Glu Arg His Leu Thr Met Ile
Lys Met 130 135 140 Arg Pro Tyr Asp Ala Asn Lys Lys His Arg Val Phe
Leu Leu Ile Gly 145 150 155 160 Met Cys Trp Leu Ile Ala Phe Ser Leu
Gly Ala Leu Pro Ile Leu Gly 165 170 175 Trp Asn Cys Leu Glu Asn Phe
Pro Asp Cys Ser Thr Ile Leu Pro Leu 180 185 190 Tyr Ser Lys Lys Tyr
Ile Ala Phe Leu Ile Ser Ile Phe Ile Ala Ile 195 200 205 Leu Val Thr
Ile Val Ile Leu Tyr Ala Arg Ile Tyr Phe Leu Val Lys 210 215 220 Ser
Ser Ser Arg Arg Val Ala Asn His Asn Ser Glu Arg Ser Met Ala 225 230
235 240 Leu Leu Arg Thr Val Val Ile Val Val Ser Val Phe Ile Ala Cys
Trp 245 250 255 Ser Pro Leu Phe Ile Leu Phe Leu Ile Asp Val Ala Cys
Arg Ala Lys 260 265 270 Glu Cys Ser Ile Leu Phe Lys Ser Gln Trp Phe
Ile Met Leu Ala Val 275 280 285 Leu Asn Ser Ala Met Asn Pro Val Ile
Tyr Thr Leu Ala Ser Lys Glu 290 295 300 Met Arg Arg Ala Phe Phe Arg
Leu Val Cys Gly Cys Leu Val Lys Gly 305 310 315 320 Lys Gly Thr Gln
Ala Ser Pro Met Gln Pro Ala Leu Asp Pro Ser Arg 325 330 335 Ser Lys
Ser Ser Ser Ser Asn Asn Ser Ser Ser His Ser Pro Lys Val 340 345 350
Lys Glu Asp Leu Pro His Val Ala Thr Ser Ser Cys Val Thr Asp Lys 355
360 365 Thr Arg Ser Leu Gln Asn Gly Val Leu Cys Lys Lys Gly Asn Ser
Ala 370 375 380 Asp Ile Gln His Ser Gly Gly Arg Ser Ser Leu Glu Gly
Pro Arg Phe 385 390 395 400 Glu Gly Lys Pro Ile Pro Asn Pro Leu Leu
Gly Leu Asp Ser Thr Arg 405 410 415 Thr Gly His His His His His His
420 <210> SEQ ID NO 292 <211> LENGTH: 413 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: UNSURE <222> LOCATION: 164 <223>
OTHER INFORMATION: Xaa = unknown amino acid <400> SEQUENCE:
292 Met Gly Gln Pro Gly Asn Gly Ser Ala Phe Leu Leu Ala Pro Asn Gly
1 5 10 15 Ser His Ala Pro Asp His Asp Val Thr Gln Gln Arg Asp Glu
Val Trp 20 25 30 Val Val Gly Met Gly Ile Val Met Ser Leu Ile Val
Leu Ala Ile Val 35 40 45 Phe Gly Asn Val Leu Val Ile Thr Ala Ile
Ala Lys Phe Glu Arg Leu 50 55 60 Gln Thr Val Thr Asn Tyr Phe Ile
Thr Ser Leu Ala Cys Ala Asp Leu 65 70 75 80 Val Met Gly Leu Ala Val
Val Pro Phe Gly Ala Ala His Ile Leu Met 85 90 95 Lys Met Trp Thr
Phe Gly Asn Phe Trp Cys Glu Phe Trp Thr Ser Ile 100 105 110 Asp Val
Leu Cys Val Thr Ala Ser Ile Glu Thr Leu Cys Val Ile Ala 115 120 125
Val Asp Arg Tyr Phe Ala Ile Thr Ser Pro Phe Lys Tyr Gln Ser Leu 130
135 140 Leu Thr Lys Asn Lys Ala Arg Val Ile Ile Leu Met Val Trp Ile
Val 145 150 155 160 Ser Gly Leu Xaa Ser Phe Leu Pro Ile Gln Met His
Trp Tyr Arg Ala 165 170 175 Thr His Gln Glu Ala Ile Asn Cys Tyr Ala
Asn Glu Thr Cys Cys Asp 180 185 190 Phe Phe Thr Asn Gln Ala Tyr Ala
Ile Ala Ser Ser Ile Val Ser Phe 195 200 205 Tyr Val Pro Leu Val Ile
Met Val Phe Val Tyr Ser Arg Val Phe Gln 210 215 220 Glu Ala Lys Arg
Gln Leu Gln Lys Ile Asp Lys Ser Glu Gly Arg Phe 225 230 235 240 His
Val Gln Asn Leu Ser Gln Val Glu Gln Asp Gly Arg Thr Gly His 245 250
255 Gly Leu Arg Arg Ser Ser Lys Phe Cys Leu Lys Glu His Lys Ala Leu
260 265 270 Lys Thr Leu Gly Ile Ile Met Gly Thr Phe Thr Leu Cys Trp
Leu Pro 275 280 285 Phe Phe Ile Val Asn Ile Val His Val Ile Gln Asp
Asn Leu Ile Arg 290 295 300 Lys Glu Val Tyr Ile Leu Leu Asn Trp Ile
Gly Tyr Val Asn Ser Gly 305 310 315 320 Phe Asn Pro Leu Ile Tyr Cys
Arg Ser Pro Asp Phe Arg Ile Ala Phe 325 330 335 Gln Glu Leu Leu Cys
Leu Arg Arg Ser Ser Leu Lys Ala Tyr Gly Asn 340 345 350 Gly Tyr Ser
Ser Asn Gly Asn Thr Gly Glu Gln Ser Gly Tyr His Val 355 360 365 Glu
Gln Glu Lys Glu Asn Lys Leu Leu Cys Glu Asp Leu Pro Gly Thr 370 375
380 Glu Asp Phe Val Gly His Gln Gly Thr Val Pro Ser Asp Asn Ile Asp
385 390 395 400 Ser Gln Gly Arg Asn Cys Ser Thr Asn Asp Ser Leu Leu
405 410 <210> SEQ ID NO 293 <211> LENGTH: 416
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 293 Met Asp Ser Val Cys Pro Gln
Gly Lys Tyr Ile His Pro Gln Asn Asn 1 5 10 15 Ser Ile Cys Cys Thr
Lys Cys His Lys Gly Thr Tyr Leu Tyr Asn Asp 20 25 30 Cys Pro Gly
Pro Gly Gln Asp Thr Asp Cys Arg Glu Cys Glu Ser Gly 35 40 45 Ser
Phe Thr Ala Ser Glu Asn His Leu Arg His Cys Leu Ser Cys Ser 50 55
60 Lys Cys Arg Lys Glu Met Gly Gln Val Glu Ile Ser Ser Cys Thr Val
65 70 75 80 Asp Arg Asp Thr Val Cys Gly Cys Arg Lys Asn Gln Tyr Arg
His Tyr 85 90 95 Trp Ser Glu Asn Leu Phe Gln Cys Phe Asn Cys Ser
Leu Cys Leu Asn 100 105 110 Gly Thr Val His Leu Ser Cys Gln Glu Lys
Gln Asn Thr Val Cys Thr 115 120 125 Cys His Ala Gly Phe Phe Leu Arg
Glu Asn Glu Cys Val Ser Cys Ser 130 135 140 Asn Cys Lys Lys Ser Leu
Glu Cys Thr Lys Leu Cys Leu Pro Gln Ile 145 150 155 160 Glu Asn Val
Lys Gly Thr Glu Asp Ser Gly Thr Thr Val Leu Leu Pro 165 170 175 Leu
Val Ile Phe Phe Gly Leu Cys Leu Leu Ser Leu Leu Phe Ile Gly 180 185
190 Leu Met Tyr Arg Tyr Gln Arg Trp Lys Ser Lys Leu Tyr Ser Ile Val
195 200 205 Cys Gly Lys Ser Thr Pro Glu Lys Glu Gly Glu Leu Glu Gly
Thr Thr 210 215 220 Thr Lys Pro Leu Ala Pro Asn Pro Ser Phe Ser Pro
Thr Pro Gly Phe 225 230 235 240 Thr Pro Thr Leu Gly Phe Ser Pro Val
Pro Ser Ser Thr Phe Thr Ser 245 250 255 Ser Ser Thr Tyr Thr Pro Gly
Asp Cys Pro Asn Phe Ala Ala Pro Arg 260 265 270 Arg Glu Val Ala Pro
Pro Tyr Gln Gly Ala Asp Pro Ile Leu Ala Thr 275 280 285 Ala Leu Ala
Ser Asp Pro Ile Pro Asn Pro Leu Gln Lys Trp Glu Asp 290 295 300 Ser
Ala His Lys Pro Gln Ser Leu Asp Thr Asp Asp Pro Ala Thr Leu 305 310
315 320 Tyr Ala Val Val Glu Asn Val Pro Pro Leu Arg Trp Lys Glu Phe
Val 325 330 335 Arg Arg Leu Gly Leu Ser Asp His Glu Ile Asp Arg Leu
Glu Leu Gln 340 345 350 Asn Gly Arg Cys Leu Arg Glu Ala Gln Tyr Ser
Met Leu Ala Thr Trp 355 360 365 Arg Arg Arg Thr Pro Arg Arg Glu Ala
Thr Leu Glu Leu Leu Gly Arg 370 375 380 Val Leu Arg Asp Met Asp Leu
Leu Gly Cys Leu Glu Asp Ile Glu Glu 385 390 395 400 Ala Leu Cys Gly
Pro Ala Ala Leu Pro Pro Ala Pro Ser Leu Leu Arg 405 410 415
<210> SEQ ID NO 294 <211> LENGTH: 428 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 294 Met Gly Leu Val Pro His Leu Gly Asp Arg
Glu Lys Arg Asp Ser Val 1 5 10 15 Cys Pro Gln Gly Lys Tyr Ile His
Pro Gln Asn Asn Ser Ile Cys Cys 20 25 30 Thr Lys Cys His Lys Gly
Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro 35 40 45 Gly Gln Asp Thr
Asp Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala 50 55 60 Ser Glu
Asn His Leu Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys 65 70 75 80
Glu Met Gly Gln Val Glu Ile Ser Ser Cys Thr Val Asp Arg Asp Thr 85
90 95 Val Cys Gly Cys Arg Lys Asn Gln Tyr Arg His Tyr Trp Ser Glu
Asn 100 105 110 Leu Phe Gln Cys Phe Asn Cys Ser Leu Cys Leu Asn Gly
Thr Val His 115 120 125 Leu Ser Cys Gln Glu Lys Gln Asn Thr Val Cys
Thr Cys His Ala Gly 130 135 140 Phe Phe Leu Arg Glu Asn Glu Cys Val
Ser Cys Ser Asn Cys Lys Lys 145 150 155 160 Ser Leu Glu Cys Thr Lys
Leu Cys Leu Pro Gln Ile Glu Asn Val Lys 165 170 175 Gly Thr Glu Asp
Ser Gly Thr Thr Val Leu Leu Pro Leu Val Ile Phe 180 185 190 Phe Gly
Leu Cys Leu Leu Ser Leu Leu Phe Ile Gly Leu Met Tyr Arg 195 200 205
Tyr Gln Arg Trp Lys Ser Lys Leu Tyr Ser Ile Val Cys Gly Lys Ser 210
215 220 Thr Pro Glu Lys Glu Gly Glu Leu Glu Gly Thr Thr Thr Lys Pro
Leu 225 230 235 240 Ala Pro Asn Pro Ser Phe Ser Pro Thr Pro Gly Phe
Thr Pro Thr Leu 245 250 255 Gly Phe Ser Pro Val Pro Ser Ser Thr Phe
Thr Ser Ser Ser Thr Tyr 260 265 270 Thr Pro Gly Asp Cys Pro Asn Phe
Ala Ala Pro Arg Arg Glu Val Ala 275 280 285 Pro Pro Tyr Gln Gly Ala
Asp Pro Ile Leu Ala Thr Ala Leu Ala Ser 290 295 300 Asp Pro Ile Pro
Asn Pro Leu Gln Lys Trp Glu Asp Ser Ala His Lys 305 310 315 320 Pro
Gln Ser Leu Asp Thr Asp Asp Pro Ala Thr Leu Tyr Ala Val Val 325 330
335 Glu Asn Val Pro Pro Leu Arg Trp Lys Glu Phe Val Arg Arg Leu Gly
340 345 350 Leu Ser Asp His Glu Ile Asp Arg Leu Glu Leu Gln Asn Gly
Arg Cys 355 360 365 Leu Arg Glu Ala Gln Tyr Ser Met Leu Ala Thr Trp
Arg Arg Arg Thr 370 375 380 Pro Arg Arg Glu Ala Thr Leu Glu Leu Leu
Gly Arg Val Leu Arg Asp 385 390 395 400 Met Asp Leu Leu Gly Cys Leu
Glu Asp Ile Glu Glu Ala Leu Cys Gly 405 410 415 Pro Ala Ala Leu Pro
Pro Ala Pro Ser Leu Leu Arg 420 425 <210> SEQ ID NO 295
<211> LENGTH: 233 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic amino acid sequence <400> SEQUENCE:
295 Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala
1 5 10 15 Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys
Leu Phe 20 25 30 Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala
Thr Thr Leu Phe 35 40 45 Cys Leu Leu His Phe Gly Val Ile Gly Pro
Gln Arg Glu Glu Phe Pro 50 55 60 Arg Asp Leu Ser Leu Ile Ser Pro
Leu Ala Gln Ala Val Arg Ser Ser 65 70 75 80 Ser Arg Thr Pro Ser Asp
Lys Pro Val Ala His Val Val Ala Asn Pro 85 90 95 Gln Ala Glu Gly
Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu 100 105 110 Leu Ala
Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser 115 120 125
Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly 130
135 140 Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile Ser Arg Ile
Ala 145 150 155 160 Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala
Ile Lys Ser Pro 165 170 175 Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu
Ala Lys Pro Trp Tyr Glu 180 185 190 Pro Ile Tyr Leu Gly Gly Val Phe
Gln Leu Glu Lys Gly Asp Arg Leu 195 200 205 Ser Ala Glu Ile Asn Arg
Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly 210 215 220 Gln Val Tyr Phe
Gly Ile Ile Ala Leu 225 230 <210> SEQ ID NO 296 <211>
LENGTH: 75 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic amino acid sequence <400> SEQUENCE: 296 Met Asn Leu
Gly Asn Arg Leu Phe Ile Leu Ile Ala Val Leu Leu Pro 1 5 10 15 Leu
Ala Val Leu Leu Leu Asn Ser Asp Ser Glu Cys Pro Leu Ser His 20 25
30 Asp Gly Tyr Cys Leu His Asp Gly Val Cys Met Tyr Ile Glu Ala Leu
35 40 45 Asp Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr Ile Gly Glu
Arg Cys 50 55 60 Gln Tyr Arg Asp Leu Lys Trp Trp Glu Leu Arg 65 70
75 <210> SEQ ID NO 297 <211> LENGTH: 518 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic amino acid
sequence <400> SEQUENCE: 297 Met Asn Leu Gly Asn Arg Leu Phe
Ile Leu Ile Ala Val Leu Leu Pro 1 5 10 15 Leu Ala Val Leu Leu Leu
Ser Phe Thr Leu Ser Val Thr Val Gln Gln 20 25 30 Pro Gln Leu Thr
Leu Thr Ala Ala Val Ile Gly Asp Gly Ala Pro Ala 35 40 45 Asn Gly
Lys Thr Ala Ile Thr Val Glu Phe Thr Val Ala Asp Phe Glu 50 55 60
Gly Lys Pro Leu Ala Gly Gln Glu Val Val Ile Thr Thr Asn Asn Gly 65
70 75 80 Ala Leu Pro Asn Lys Ile Thr Glu Lys Thr Asp Ala Asn Gly
Val Ala 85 90 95 Arg Ile Ala Leu Thr Asn Thr Thr Asp Gly Val Thr
Val Val Thr Ala 100 105 110 Glu Val Glu Gly Gln Arg Gln Ser Val Asp
Thr His Phe Val Lys Gly 115 120 125 Thr Ile Ala Ala Asp Lys Ser Thr
Leu Ala Ala Val Pro Thr Ser Ile 130 135 140 Ile Ala Asp Gly Leu Met
Ala Ser Thr Ile Thr Leu Glu Leu Lys Asp 145 150 155 160 Thr Tyr Gly
Asp Pro Gln Ala Gly Ala Asn Val Ala Phe Asp Thr Thr 165 170 175 Leu
Gly Asn Met Gly Val Ile Thr Asp His Asn Asp Gly Thr Tyr Ser 180 185
190 Ala Pro Leu Thr Ser Thr Thr Leu Gly Val Ala Thr Val Thr Val Lys
195 200 205 Val Asp Gly Ala Ala Phe Ser Val Pro Ser Val Thr Val Asn
Phe Thr 210 215 220 Ala Asp Pro Ile Pro Asp Ala Gly Arg Ser Ser Phe
Thr Val Ser Thr 225 230 235 240 Pro Asp Ile Leu Ala Asp Gly Thr Met
Ser Ser Thr Leu Ser Phe Val 245 250 255 Pro Val Asp Lys Asn Gly His
Phe Ile Ser Gly Met Gln Gly Leu Ser 260 265 270 Phe Thr Gln Asn Gly
Val Pro Val Ser Ile Ser Pro Ile Thr Glu Gln 275 280 285 Pro Asp Ser
Tyr Thr Ala Thr Val Val Gly Asn Ser Val Gly Asp Val 290 295 300 Thr
Ile Thr Pro Gln Val Asp Thr Leu Ile Leu Ser Thr Leu Gln Lys 305 310
315 320 Lys Ile Ser Leu Phe Pro Val Pro Thr Leu Thr Gly Ile Leu Val
Asn 325 330 335 Gly Gln Asn Phe Ala Thr Asp Lys Gly Phe Pro Lys Thr
Ile Phe Lys 340 345 350 Asn Ala Thr Phe Gln Leu Gln Met Asp Asn Asp
Val Ala Asn Asn Thr 355 360 365 Gln Tyr Glu Trp Ser Ser Ser Phe Thr
Pro Asn Val Ser Val Asn Asp 370 375 380 Gln Gly Gln Val Thr Ile Thr
Tyr Gln Thr Tyr Ser Glu Val Ala Val 385 390 395 400 Thr Ala Lys Ser
Lys Lys Phe Pro Ser Tyr Ser Val Ser Tyr Arg Phe 405 410 415 Tyr Pro
Asn Arg Trp Ile Tyr Asp Gly Gly Arg Ser Leu Val Ser Ser 420 425 430
Leu Glu Ala Ser Arg Gln Cys Gln Gly Ser Asp Met Ser Ala Val Leu 435
440 445 Glu Ser Ser Arg Ala Thr Asn Gly Thr Arg Ala Pro Asp Gly Thr
Leu 450 455 460 Trp Gly Glu Trp Gly Ser Leu Thr Ala Tyr Ser Ser Asp
Trp Gln Ser 465 470 475 480 Gly Glu Tyr Trp Val Lys Lys Thr Ser Thr
Asp Phe Glu Thr Met Asn 485 490 495 Met Asp Thr Gly Ala Leu Gln Pro
Gly Pro Ala Tyr Leu Ala Phe Pro 500 505 510 Leu Cys Ala Leu Ser Ile
515 <210> SEQ ID NO 298 <211> LENGTH: 50 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic amino acid
sequence <400> SEQUENCE: 298 Met Ser Arg Pro Arg Leu Ile Val
Ala Leu Phe Leu Phe Phe Asn Val 1 5 10 15 Phe Val His Gly Glu Asn
Lys Val Lys Gln Ser Thr Ile Ala Leu Ala 20 25 30 Leu Leu Pro Leu
Leu Phe Thr Pro Val Thr Lys Ala Arg Thr Pro Glu 35 40 45 Ser Arg 50
<210> SEQ ID NO 299 <211> LENGTH: 496 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 299 Met Ser Arg Pro Arg Leu Ile Val Ala Leu
Phe Leu Phe Phe Asn Val 1 5 10 15 Phe Val His Gly Glu Asn Lys Val
Lys Gln Ser Thr Ile Ala Leu Ala 20 25 30 Leu Leu Pro Leu Leu Phe
Thr Pro Val Thr Lys Ala Arg Thr Pro Glu 35 40 45 Met Pro Val Leu
Glu Asn Arg Ala Ala Gln Gly Asp Ile Thr Ala Pro 50 55 60 Gly Gly
Ala Arg Arg Leu Thr Gly Asp Gln Thr Ala Ala Leu Arg Asp 65 70 75 80
Ser Leu Ser Asp Lys Pro Ala Lys Asn Ile Ile Leu Leu Ile Gly Asp 85
90 95 Gly Met Gly Asp Ser Glu Ile Thr Ala Ala Arg Asn Tyr Ala Glu
Gly 100 105 110 Ala Gly Gly Phe Phe Lys Gly Ile Asp Ala Leu Pro Leu
Thr Gly Gln 115 120 125 Tyr Thr His Tyr Ala Leu Asn Lys Lys Thr Gly
Lys Pro Asp Tyr Val 130 135 140 Thr Asp Ser Ala Ala Ser Ala Thr Ala
Trp Ser Thr Gly Val Lys Thr 145 150 155 160 Tyr Asn Gly Ala Leu Gly
Val Asp Ile His Glu Lys Asp His Pro Thr 165 170 175 Ile Leu Glu Met
Ala Lys Ala Ala Gly Leu Ala Thr Gly Asn Val Ser 180 185 190 Thr Ala
Glu Leu Gln Asp Ala Thr Pro Ala Ala Leu Val Ala His Val 195 200 205
Thr Ser Arg Lys Cys Tyr Gly Pro Ser Ala Thr Ser Glu Lys Cys Pro 210
215 220 Gly Asn Ala Leu Glu Lys Gly Gly Lys Gly Ser Ile Thr Glu Gln
Leu 225 230 235 240 Leu Asn Ala Arg Ala Asp Val Thr Leu Gly Gly Gly
Ala Lys Thr Phe 245 250 255 Ala Glu Thr Ala Thr Ala Gly Glu Trp Gln
Gly Lys Thr Leu Arg Glu 260 265 270 Gln Ala Gln Ala Arg Gly Tyr Gln
Leu Val Ser Asp Ala Ala Ser Leu 275 280 285 Asn Ser Val Thr Glu Ala
Asn Gln Gln Lys Pro Leu Leu Gly Leu Phe 290 295 300 Ala Asp Gly Asn
Met Pro Val Arg Trp Leu Gly Pro Lys Ala Thr Tyr 305 310 315 320 His
Gly Asn Ile Asp Lys Pro Ala Val Thr Cys Thr Pro Asn Pro Gln 325 330
335 Arg Asn Asp Ser Val Pro Thr Leu Ala Gln Met Thr Asp Lys Ala Ile
340 345 350 Glu Leu Leu Ser Lys Asn Glu Lys Gly Phe Phe Leu Gln Val
Glu Gly 355 360 365 Ala Ser Ile Asp Lys Gln Asp His Ala Ala Asn Pro
Cys Gly Gln Ile 370 375 380 Gly Glu Thr Val Asp Leu Asp Glu Ala Val
Gln Arg Ala Leu Glu Phe 385 390 395 400 Ala Lys Lys Glu Gly Asn Thr
Leu Val Ile Val Thr Ala Asp His Ala 405 410 415 His Ala Ser Gln Ile
Val Ala Pro Asp Thr Lys Ala Pro Gly Leu Thr 420 425 430 Gln Ala Leu
Asn Thr Lys Asp Gly Ala Val Met Val Met Ser Tyr Gly 435 440 445 Asn
Ser Glu Glu Asp Ser Gln Glu His Thr Gly Ser Gln Leu Arg Ile 450 455
460 Ala Ala Tyr Gly Pro His Ala Ala Asn Val Val Gly Leu Thr Asp Gln
465 470 475 480 Thr Asp Leu Phe Tyr Thr Met Lys Ala Ala Leu Gly Leu
Lys Ser Arg 485 490 495 <210> SEQ ID NO 300 <211>
LENGTH: 30 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic amino acid sequence <400> SEQUENCE: 300 Met Lys Ile
Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr
Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Ser Arg 20 25 30
<210> SEQ ID NO 301 <211> LENGTH: 372 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 301 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu
Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala
Leu Ala Lys Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile Trp Ile Asn
Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val Gly Lys Lys
Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu 50 55 60 His Pro
Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly 65 70 75 80
Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85
90 95 Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe
Gln 100 105 110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr
Asn Gly Lys 115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu
Ser Leu Ile Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys
Thr Trp Glu Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys Glu Leu Lys
Ala Lys Gly Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu Gln Glu Pro
Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185 190 Tyr Ala
Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly 195 200 205
Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210
215 220 Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala
Glu 225 230 235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile
Asn Gly Pro Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr Ser Lys Val
Asn Tyr Gly Val Thr Val 260 265 270 Leu Pro Thr Phe Lys Gly Gln Pro
Ser Lys Pro Phe Val Gly Val Leu 275 280 285 Ser Ala Gly Ile Asn Ala
Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe Leu Glu Asn
Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310 315 320 Lys
Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 330
335 Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Lys
340 345 350 Gly Glu Ile Met Pro Asn Ile Pro Gln Met Ser Ala Phe Trp
Tyr Ala 355 360 365 Val Arg Ser Arg 370 <210> SEQ ID NO 302
<211> LENGTH: 133 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic amino acid sequence <400> SEQUENCE:
302 Met Ile Glu Ala Arg Ser Arg Leu Glu Ser Asp Lys Ile Ile His Leu
1 5 10 15 Thr Asp Asp Ser Phe Asp Thr Asp Val Leu Lys Ala Asp Gly
Ala Ile 20 25 30 Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Cys
Lys Met Ile Ala 35 40 45 Pro Ile Leu Asp Glu Ile Ala Asp Glu Tyr
Gln Gly Lys Leu Thr Val 50 55 60 Ala Lys Leu Asn Ile Asp Gln Asn
Pro Gly Thr Ala Pro Lys Tyr Gly 65 70 75 80 Ile Arg Gly Ile Pro Thr
Leu Leu Leu Phe Lys Asn Gly Glu Val Ala 85 90 95 Ala Thr Lys Val
Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Phe Leu 100 105 110 Asp Ala
Asn Leu Ala Leu Glu Asp Tyr Lys Asp His Asp Gly Asp Tyr 115 120 125
Lys Asp His Asp Asp 130 <210> SEQ ID NO 303 <211>
LENGTH: 767 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic amino acid sequence <400> SEQUENCE: 303 Met Lys Ile
Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr
Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Glu Glu Gly Lys 20 25
30 Leu Val Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu
35 40 45 Val Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr
Val Glu 50 55 60 His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val
Ala Ala Thr Gly 65 70 75 80 Asp Gly Pro Asp Ile Ile Phe Trp Ala His
Asp Arg Phe Gly Gly Tyr 85 90 95 Ala Gln Ser Gly Leu Leu Ala Glu
Ile Thr Pro Asp Lys Ala Phe Gln 100 105 110 Asp Lys Leu Tyr Pro Phe
Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys 115 120 125 Leu Ile Ala Tyr
Pro Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn 130 135 140 Lys Asp
Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala 145 150 155
160 Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn
165 170 175 Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp
Gly Gly 180 185 190 Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile
Lys Asp Val Gly 195 200 205 Val Asp Asn Ala Gly Ala Lys Ala Gly Leu
Thr Phe Leu Val Asp Leu 210 215 220 Ile Lys Asn Lys His Met Asn Ala
Asp Thr Asp Tyr Ser Ile Ala Glu 225 230 235 240 Ala Ala Phe Asn Lys
Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp 245 250 255 Ala Trp Ser
Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val 260 265 270 Leu
Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu 275 280
285 Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu
290 295 300 Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala
Val Asn 305 310 315 320 Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys
Ser Tyr Glu Glu Glu 325 330 335 Leu Ala Lys Asp Pro Arg Ile Ala Ala
Thr Met Glu Asn Ala Gln Lys 340 345 350 Gly Glu Ile Met Pro Asn Ile
Pro Gln Met Ser Ala Phe Trp Tyr Ala 355 360 365 Val Leu Ile Glu Ala
Arg Thr Ser Glu Ser Asp Thr Ala Gly Pro Asn 370 375 380 Ser Asp Leu
Asp Val Asn Thr Asp Ile Tyr Ser Lys Val Leu Val Thr 385 390 395 400
Ala Ile Tyr Leu Ala Leu Phe Val Val Gly Thr Val Gly Asn Ser Val 405
410 415 Thr Ala Phe Thr Leu Ala Arg Lys Lys Ser Leu Gln Ser Leu Gln
Ser 420 425 430 Thr Val His Tyr His Leu Gly Ser Leu Ala Leu Ser Asp
Leu Leu Ile 435 440 445 Leu Leu Leu Ala Met Pro Val Glu Leu Tyr Asn
Phe Ile Trp Val His 450 455 460 His Pro Trp Ala Phe Gly Asp Ala Gly
Cys Arg Gly Tyr Tyr Phe Leu 465 470 475 480 Arg Asp Ala Cys Thr Tyr
Ala Thr Ala Leu Asn Val Ala Ser Leu Ser 485 490 495 Val Glu Arg Tyr
Leu Ala Ile Cys His Pro Phe Lys Ala Lys Thr Leu 500 505 510 Met Ser
Arg Ser Arg Thr Lys Lys Phe Ile Ser Ala Ile Trp Leu Ala 515 520 525
Ser Ala Leu Leu Ala Ile Pro Met Leu Phe Thr Met Gly Leu Gln Asn 530
535 540 Arg Ser Gly Asp Gly Thr His Pro Gly Gly Leu Val Cys Thr Pro
Ile 545 550 555 560 Val Asp Thr Ala Thr Val Lys Val Val Ile Gln Val
Asn Thr Phe Met 565 570 575 Ser Phe Leu Phe Pro Met Leu Val Ile Ser
Ile Leu Asn Thr Val Ile 580 585 590 Ala Asn Lys Leu Thr Val Met Val
His Gln Ala Ala Glu Gln Gly Arg 595 600 605 Val Cys Thr Val Gly Thr
His Asn Gly Leu Glu His Ser Thr Phe Asn 610 615 620 Met Thr Ile Glu
Pro Gly Arg Val Gln Ala Leu Arg His Gly Val Leu 625 630 635 640 Val
Leu Arg Ala Val Val Ile Ala Phe Val Val Cys Trp Leu Pro Tyr 645 650
655 His Val Arg Arg Leu Met Phe Cys Tyr Ile Ser Asp Glu Gln Trp Thr
660 665 670 Thr Phe Leu Phe Asp Phe Tyr His Tyr Phe Tyr Met Leu Thr
Asn Ala 675 680 685 Leu Phe Tyr Val Ser Ser Ala Ile Asn Pro Ile Leu
Tyr Asn Leu Val 690 695 700 Ser Ala Asn Phe Arg Gln Val Phe Leu Ser
Thr Leu Ala Cys Leu Cys 705 710 715 720 Pro Gly Trp Arg His Arg Arg
Lys Lys Arg Pro Thr Phe Ser Arg Lys 725 730 735 Pro Asn Ser Met Ser
Ser Asn His Ala Phe Ser Thr Ser Ala Thr Arg 740 745 750 Glu Thr Leu
Tyr Ala Ala Ala Asp Tyr Lys Asp Asp Asp Asp Lys 755 760 765
<210> SEQ ID NO 304 <211> LENGTH: 425 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 304 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu
Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala
Leu Ala Lys Ile Ile Glu Ala Arg 20 25 30 Thr Ser Glu Ser Asp Thr
Ala Gly Pro Asn Ser Asp Leu Asp Val Asn 35 40 45 Thr Asp Ile Tyr
Ser Lys Val Leu Val Thr Ala Ile Tyr Leu Ala Leu 50 55 60 Phe Val
Val Gly Thr Val Gly Asn Ser Val Thr Ala Phe Thr Leu Ala 65 70 75 80
Arg Lys Lys Ser Leu Gln Ser Leu Gln Ser Thr Val His Tyr His Leu 85
90 95 Gly Ser Leu Ala Leu Ser Asp Leu Leu Ile Leu Leu Leu Ala Met
Pro 100 105 110 Val Glu Leu Tyr Asn Phe Ile Trp Val His His Pro Trp
Ala Phe Gly 115 120 125 Asp Ala Gly Cys Arg Gly Tyr Tyr Phe Leu Arg
Asp Ala Cys Thr Tyr 130 135 140 Ala Thr Ala Leu Asn Val Ala Ser Leu
Ser Val Glu Arg Tyr Leu Ala 145 150 155 160 Ile Cys His Pro Phe Lys
Ala Lys Thr Leu Met Ser Arg Ser Arg Thr 165 170 175 Lys Lys Phe Ile
Ser Ala Ile Trp Leu Ala Ser Ala Leu Leu Ala Ile 180 185 190 Pro Met
Leu Phe Thr Met Gly Leu Gln Asn Arg Ser Gly Asp Gly Thr 195 200 205
His Pro Gly Gly Leu Val Cys Thr Pro Ile Val Asp Thr Ala Thr Val 210
215 220 Lys Val Val Ile Gln Val Asn Thr Phe Met Ser Phe Leu Phe Pro
Met 225 230 235 240 Leu Val Ile Ser Ile Leu Asn Thr Val Ile Ala Asn
Lys Leu Thr Val 245 250 255 Met Val His Gln Ala Ala Glu Gln Gly Arg
Val Cys Thr Val Gly Thr 260 265 270 His Asn Gly Leu Glu His Ser Thr
Phe Asn Met Thr Ile Glu Pro Gly 275 280 285 Arg Val Gln Ala Leu Arg
His Gly Val Leu Val Leu Arg Ala Val Val 290 295 300 Ile Ala Phe Val
Val Cys Trp Leu Pro Tyr His Val Arg Arg Leu Met 305 310 315 320 Phe
Cys Tyr Ile Ser Asp Glu Gln Trp Thr Thr Phe Leu Phe Asp Phe 325 330
335 Tyr His Tyr Phe Tyr Met Leu Thr Asn Ala Leu Phe Tyr Val Ser Ser
340 345 350 Ala Ile Asn Pro Ile Leu Tyr Asn Leu Val Ser Ala Asn Phe
Arg Gln 355 360 365 Val Phe Leu Ser Thr Leu Ala Cys Leu Cys Pro Gly
Trp Arg His Arg 370 375 380 Arg Lys Lys Arg Pro Thr Phe Ser Arg Lys
Pro Asn Ser Met Ser Ser 385 390 395 400 Asn His Ala Phe Ser Thr Ser
Ala Thr Arg Glu Thr Leu Tyr Ala Ala 405 410 415 Ala Asp Tyr Lys Asp
Asp Asp Asp Lys 420 425 <210> SEQ ID NO 305 <211>
LENGTH: 878 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic amino acid sequence <400> SEQUENCE: 305 Met Lys Ile
Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr
Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Glu Glu Gly Lys 20 25
30 Leu Val Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu
35 40 45 Val Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr
Val Glu 50 55 60 His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val
Ala Ala Thr Gly 65 70 75 80 Asp Gly Pro Asp Ile Ile Phe Trp Ala His
Asp Arg Phe Gly Gly Tyr 85 90 95 Ala Gln Ser Gly Leu Leu Ala Glu
Ile Thr Pro Asp Lys Ala Phe Gln 100 105 110 Asp Lys Leu Tyr Pro Phe
Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys 115 120 125 Leu Ile Ala Tyr
Pro Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn 130 135 140 Lys Asp
Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala 145 150 155
160 Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn
165 170 175 Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp
Gly Gly 180 185 190 Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile
Lys Asp Val Gly 195 200 205 Val Asp Asn Ala Gly Ala Lys Ala Gly Leu
Thr Phe Leu Val Asp Leu 210 215 220 Ile Lys Asn Lys His Met Asn Ala
Asp Thr Asp Tyr Ser Ile Ala Glu 225 230 235 240 Ala Ala Phe Asn Lys
Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp 245 250 255 Ala Trp Ser
Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val 260 265 270 Leu
Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu 275 280
285 Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu
290 295 300 Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala
Val Asn 305 310 315 320 Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys
Ser Tyr Glu Glu Glu 325 330 335 Leu Ala Lys Asp Pro Arg Ile Ala Ala
Thr Met Glu Asn Ala Gln Lys 340 345 350 Gly Glu Ile Met Pro Asn Ile
Pro Gln Met Ser Ala Phe Trp Tyr Ala 355 360 365 Val Leu Ile Glu Ala
Arg Thr Ser Glu Ser Asp Thr Ala Gly Pro Asn 370 375 380 Ser Asp Leu
Asp Val Asn Thr Asp Ile Tyr Ser Lys Val Leu Val Thr 385 390 395 400
Ala Ile Tyr Leu Ala Leu Phe Val Val Gly Thr Val Gly Asn Ser Val 405
410 415 Thr Ala Phe Thr Leu Ala Arg Lys Lys Ser Leu Gln Ser Leu Gln
Ser 420 425 430 Thr Val His Tyr His Leu Gly Ser Leu Ala Leu Ser Asp
Leu Leu Ile 435 440 445 Leu Leu Leu Ala Met Pro Val Glu Leu Tyr Asn
Phe Ile Trp Val His 450 455 460 His Pro Trp Ala Phe Gly Asp Ala Gly
Cys Arg Gly Tyr Tyr Phe Leu 465 470 475 480 Arg Asp Ala Cys Thr Tyr
Ala Thr Ala Leu Asn Val Ala Ser Leu Ser 485 490 495 Val Glu Arg Tyr
Leu Ala Ile Cys His Pro Phe Lys Ala Lys Thr Leu 500 505 510 Met Ser
Arg Ser Arg Thr Lys Lys Phe Ile Ser Ala Ile Trp Leu Ala 515 520 525
Ser Ala Leu Leu Ala Ile Pro Met Leu Phe Thr Met Gly Leu Gln Asn 530
535 540 Arg Ser Gly Asp Gly Thr His Pro Gly Gly Leu Val Cys Thr Pro
Ile 545 550 555 560 Val Asp Thr Ala Thr Val Lys Val Val Ile Gln Val
Asn Thr Phe Met 565 570 575 Ser Phe Leu Phe Pro Met Leu Val Ile Ser
Ile Leu Asn Thr Val Ile 580 585 590 Ala Asn Lys Leu Thr Val Met Val
His Gln Ala Ala Glu Gln Gly Arg 595 600 605 Val Cys Thr Val Gly Thr
His Asn Gly Leu Glu His Ser Thr Phe Asn 610 615 620 Met Thr Ile Glu
Pro Gly Arg Val Gln Ala Leu Arg His Gly Val Leu 625 630 635 640 Val
Leu Arg Ala Val Val Ile Ala Phe Val Val Cys Trp Leu Pro Tyr 645 650
655 His Val Arg Arg Leu Met Phe Cys Tyr Ile Ser Asp Glu Gln Trp Thr
660 665 670 Thr Phe Leu Phe Asp Phe Tyr His Tyr Phe Tyr Met Leu Thr
Asn Ala 675 680 685 Leu Phe Tyr Val Ser Ser Ala Ile Asn Pro Ile Leu
Tyr Asn Leu Val 690 695 700 Ser Ala Asn Phe Arg Gln Val Phe Leu Ser
Thr Leu Ala Cys Leu Cys 705 710 715 720 Pro Gly Trp Arg His Arg Arg
Lys Lys Arg Pro Thr Phe Ser Arg Lys 725 730 735 Pro Asn Ser Met Ser
Ser Asn His Ala Phe Ser Thr Ser Ala Thr Arg 740 745 750 Glu Thr Leu
Tyr Ala Ala Ala Ser Asp Lys Ile Ile His Leu Thr Asp 755 760 765 Asp
Ser Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala Ile Leu Val 770 775
780 Asp Phe Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile Ala Pro Ile
785 790 795 800 Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr
Val Ala Lys 805 810 815 Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro
Lys Tyr Gly Ile Arg 820 825 830 Gly Ile Pro Thr Leu Leu Leu Phe Lys
Asn Gly Glu Val Ala Ala Thr 835 840 845 Lys Val Gly Ala Leu Ser Lys
Gly Gln Leu Lys Glu Phe Leu Asp Ala 850 855 860 Asn Leu Ala Ala Ala
Ala Asp Tyr Lys Asp Asp Asp Asp Lys 865 870 875 <210> SEQ ID
NO 306 <211> LENGTH: 536 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 306 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser
Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys
Ile Ile Glu Ala Arg 20 25 30 Thr Ser Glu Ser Asp Thr Ala Gly Pro
Asn Ser Asp Leu Asp Val Asn 35 40 45 Thr Asp Ile Tyr Ser Lys Val
Leu Val Thr Ala Ile Tyr Leu Ala Leu 50 55 60 Phe Val Val Gly Thr
Val Gly Asn Ser Val Thr Ala Phe Thr Leu Ala 65 70 75 80 Arg Lys Lys
Ser Leu Gln Ser Leu Gln Ser Thr Val His Tyr His Leu 85 90 95 Gly
Ser Leu Ala Leu Ser Asp Leu Leu Ile Leu Leu Leu Ala Met Pro 100 105
110 Val Glu Leu Tyr Asn Phe Ile Trp Val His His Pro Trp Ala Phe Gly
115 120 125 Asp Ala Gly Cys Arg Gly Tyr Tyr Phe Leu Arg Asp Ala Cys
Thr Tyr 130 135 140 Ala Thr Ala Leu Asn Val Ala Ser Leu Ser Val Glu
Arg Tyr Leu Ala 145 150 155 160 Ile Cys His Pro Phe Lys Ala Lys Thr
Leu Met Ser Arg Ser Arg Thr 165 170 175 Lys Lys Phe Ile Ser Ala Ile
Trp Leu Ala Ser Ala Leu Leu Ala Ile 180 185 190 Pro Met Leu Phe Thr
Met Gly Leu Gln Asn Arg Ser Gly Asp Gly Thr 195 200 205 His Pro Gly
Gly Leu Val Cys Thr Pro Ile Val Asp Thr Ala Thr Val 210 215 220 Lys
Val Val Ile Gln Val Asn Thr Phe Met Ser Phe Leu Phe Pro Met 225 230
235 240 Leu Val Ile Ser Ile Leu Asn Thr Val Ile Ala Asn Lys Leu Thr
Val 245 250 255 Met Val His Gln Ala Ala Glu Gln Gly Arg Val Cys Thr
Val Gly Thr 260 265 270 His Asn Gly Leu Glu His Ser Thr Phe Asn Met
Thr Ile Glu Pro Gly 275 280 285 Arg Val Gln Ala Leu Arg His Gly Val
Leu Val Leu Arg Ala Val Val 290 295 300 Ile Ala Phe Val Val Cys Trp
Leu Pro Tyr His Val Arg Arg Leu Met 305 310 315 320 Phe Cys Tyr Ile
Ser Asp Glu Gln Trp Thr Thr Phe Leu Phe Asp Phe 325 330 335 Tyr His
Tyr Phe Tyr Met Leu Thr Asn Ala Leu Phe Tyr Val Ser Ser 340 345 350
Ala Ile Asn Pro Ile Leu Tyr Asn Leu Val Ser Ala Asn Phe Arg Gln 355
360 365 Val Phe Leu Ser Thr Leu Ala Cys Leu Cys Pro Gly Trp Arg His
Arg 370 375 380 Arg Lys Lys Arg Pro Thr Phe Ser Arg Lys Pro Asn Ser
Met Ser Ser 385 390 395 400 Asn His Ala Phe Ser Thr Ser Ala Thr Arg
Glu Thr Leu Tyr Ala Ala 405 410 415 Ala Ser Asp Lys Ile Ile His Leu
Thr Asp Asp Ser Phe Asp Thr Asp 420 425 430 Val Leu Lys Ala Asp Gly
Ala Ile Leu Val Asp Phe Trp Ala Glu Trp 435 440 445 Cys Gly Pro Cys
Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp 450 455 460 Glu Tyr
Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn 465 470 475
480 Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu
485 490 495 Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala
Leu Ser 500 505 510 Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu
Ala Ala Ala Ala 515 520 525 Asp Tyr Lys Asp Asp Asp Asp Lys 530 535
<210> SEQ ID NO 307 <211> LENGTH: 380 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 307 Met Gly Cys Leu Gly Asn Ser Lys Thr Glu
Asp Gln Arg Asn Glu Glu 1 5 10 15 Lys Ala Gln Arg Glu Ala Asn Lys
Lys Ile Glu Lys Gln Leu Gln Lys 20 25 30 Asp Lys Gln Val Tyr Arg
Ala Thr His Arg Leu Leu Leu Leu Gly Ala 35 40 45 Gly Glu Ser Gly
Lys Ser Thr Ile Val Lys Gln Met Arg Ile Leu His 50 55 60 Val Asn
Gly Phe Asn Gly Asp Ser Glu Lys Ala Thr Lys Val Gln Asp 65 70 75 80
Ile Lys Asn Asn Leu Lys Glu Ala Ile Glu Thr Ile Val Ala Ala Met 85
90 95 Ser Asn Leu Val Pro Pro Val Glu Leu Ala Asn Pro Glu Asn Gln
Phe 100 105 110 Arg Val Asp Tyr Ile Leu Ser Val Met Asn Val Pro Asp
Phe Asp Phe 115 120 125 Pro Pro Glu Phe Tyr Glu His Ala Lys Ala Leu
Trp Glu Asp Glu Gly 130 135 140 Val Arg Ala Cys Tyr Glu Arg Ser Asn
Glu Tyr Gln Leu Ile Asp Cys 145 150 155 160 Ala Gln Tyr Phe Leu Asp
Lys Ile Asp Val Ile Lys Gln Ala Asp Tyr 165 170 175 Val Pro Ser Asp
Gln Asp Leu Leu Arg Cys Arg Val Leu Thr Ser Gly 180 185 190 Ile Phe
Glu Thr Lys Phe Gln Val Asp Lys Val Asn Phe His Met Phe 195 200 205
Asp Val Gly Gly Gln Arg Asp Glu Arg Arg Lys Trp Ile Gln Cys Phe 210
215 220 Asn Asp Val Thr Ala Ile Ile Phe Val Val Ala Ser Ser Ser Tyr
Asn 225 230 235 240 Met Val Ile Arg Glu Asp Asn Gln Thr Asn Arg Leu
Gln Glu Ala Leu 245 250 255 Asn Leu Phe Lys Ser Ile Trp Asn Asn Arg
Trp Leu Arg Thr Ile Ser 260 265 270 Val Ile Leu Phe Leu Asn Lys Gln
Asp Leu Leu Ala Glu Lys Val Leu 275 280 285 Ala Gly Lys Ser Lys Ile
Glu Asp Tyr Phe Pro Glu Phe Ala Arg Tyr 290 295 300 Thr Thr Pro Glu
Asp Ala Thr Pro Glu Pro Gly Glu Asp Pro Arg Val 305 310 315 320 Thr
Arg Ala Lys Tyr Phe Ile Arg Asp Glu Phe Leu Arg Ile Ser Thr 325 330
335 Ala Ser Gly Asp Gly Arg His Tyr Cys Tyr Pro His Phe Thr Cys Ala
340 345 350 Val Asp Thr Glu Asn Ile Arg Arg Val Phe Asn Asp Cys Arg
Asp Ile 355 360 365 Ile Gln Arg Met His Leu Arg Gln Tyr Glu Leu Leu
370 375 380 <210> SEQ ID NO 308 <211> LENGTH: 394
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 308 Met Gly Cys Leu Gly Asn Ser
Lys Thr Glu Asp Gln Arg Asn Glu Glu 1 5 10 15 Lys Ala Gln Arg Glu
Ala Asn Lys Lys Ile Glu Lys Gln Leu Gln Lys 20 25 30 Asp Lys Gln
Val Tyr Arg Ala Thr His Arg Leu Leu Leu Leu Gly Ala 35 40 45 Gly
Glu Ser Gly Lys Ser Thr Ile Val Lys Gln Met Arg Ile Leu His 50 55
60 Val Asn Gly Phe Asn Gly Glu Gly Gly Glu Glu Asp Pro Gln Ala Ala
65 70 75 80 Arg Ser Asn Ser Asp Gly Glu Lys Ala Thr Lys Val Gln Asp
Ile Lys 85 90 95 Asn Asn Leu Lys Glu Ala Ile Glu Thr Ile Val Ala
Ala Met Ser Asn 100 105 110 Leu Val Pro Pro Val Glu Leu Ala Asn Pro
Glu Asn Gln Phe Arg Val 115 120 125 Asp Tyr Ile Leu Ser Val Met Asn
Val Pro Asp Phe Asp Phe Pro Pro 130 135 140 Glu Phe Tyr Glu His Ala
Lys Ala Leu Trp Glu Asp Glu Gly Val Arg 145 150 155 160 Ala Cys Tyr
Glu Arg Ser Asn Glu Tyr Gln Leu Ile Asp Cys Ala Gln 165 170 175 Tyr
Phe Leu Asp Lys Ile Asp Val Ile Lys Gln Ala Asp Tyr Val Pro 180 185
190 Ser Asp Gln Asp Leu Leu Arg Cys Arg Val Leu Thr Ser Gly Ile Phe
195 200 205 Glu Thr Lys Phe Gln Val Asp Lys Val Asn Phe His Met Phe
Asp Val 210 215 220 Gly Gly Gln Arg Asp Glu Arg Arg Lys Trp Ile Gln
Cys Phe Asn Asp 225 230 235 240 Val Thr Ala Ile Ile Phe Val Val Ala
Ser Ser Ser Tyr Asn Met Val 245 250 255 Ile Arg Glu Asp Asn Gln Thr
Asn Arg Leu Gln Glu Ala Leu Asn Leu 260 265 270 Phe Lys Ser Ile Trp
Asn Asn Arg Trp Leu Arg Thr Ile Ser Val Ile 275 280 285 Leu Phe Leu
Asn Lys Gln Asp Leu Leu Ala Glu Lys Val Leu Ala Gly 290 295 300 Lys
Ser Lys Ile Glu Asp Tyr Phe Pro Glu Phe Ala Arg Tyr Thr Thr 305 310
315 320 Pro Glu Asp Ala Thr Pro Glu Pro Gly Glu Asp Pro Arg Val Thr
Arg 325 330 335 Ala Lys Tyr Phe Ile Arg Asp Glu Phe Leu Arg Ile Ser
Thr Ala Ser 340 345 350 Gly Asp Gly Arg His Tyr Cys Tyr Pro His Phe
Thr Cys Ala Val Asp 355 360 365 Thr Glu Asn Ile Arg Arg Val Phe Asn
Asp Cys Arg Asp Ile Ile Gln 370 375 380 Arg Met His Leu Arg Gln Tyr
Glu Leu Leu 385 390 <210> SEQ ID NO 309 <211> LENGTH:
359 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 309 Met Thr Leu Glu Ser Ile Met
Ala Cys Cys Leu Ser Glu Glu Ala Lys 1 5 10 15 Glu Ala Arg Arg Ile
Asn Asp Glu Ile Glu Arg Gln Leu Arg Arg Asp 20 25 30 Lys Arg Asp
Ala Arg Arg Glu Leu Lys Leu Leu Leu Leu Gly Thr Gly 35 40 45 Glu
Ser Gly Lys Ser Thr Phe Ile Lys Gln Met Arg Ile Ile His Gly 50 55
60 Ser Gly Tyr Ser Asp Glu Asp Lys Arg Gly Phe Thr Lys Leu Val Tyr
65 70 75 80 Gln Asn Ile Phe Thr Ala Met Gln Ala Met Ile Arg Ala Met
Asp Thr 85 90 95 Leu Lys Ile Pro Tyr Lys Tyr Glu His Asn Lys Ala
His Ala Gln Leu 100 105 110 Val Arg Glu Val Asp Val Glu Lys Val Ser
Ala Phe Glu Asn Pro Tyr 115 120 125 Val Asp Ala Ile Lys Ser Leu Trp
Asn Asp Pro Gly Ile Gln Glu Cys 130 135 140 Tyr Asp Arg Arg Arg Glu
Tyr Gln Leu Ser Asp Ser Thr Lys Tyr Tyr 145 150 155 160 Leu Asn Asp
Leu Asp Arg Val Ala Asp Pro Ala Tyr Leu Pro Thr Gln 165 170 175 Gln
Asp Val Leu Arg Val Arg Val Pro Thr Thr Gly Ile Ile Glu Tyr 180 185
190 Pro Phe Asp Leu Gln Ser Val Ile Phe Arg Met Val Asp Val Gly Gly
195 200 205 Gln Arg Ser Glu Arg Arg Lys Trp Ile His Cys Phe Glu Asn
Val Thr 210 215 220 Ser Ile Met Phe Leu Val Ala Leu Ser Glu Tyr Asp
Gln Val Leu Val 225 230 235 240 Glu Ser Asp Asn Glu Asn Arg Met Glu
Glu Ser Lys Ala Leu Phe Arg 245 250 255 Thr Ile Ile Thr Tyr Pro Trp
Phe Gln Asn Ser Ser Val Ile Leu Phe 260 265 270 Leu Asn Lys Lys Asp
Leu Leu Glu Glu Lys Ile Met Tyr Ser His Leu 275 280 285 Val Asp Tyr
Phe Pro Glu Tyr Asp Gly Pro Gln Arg Asp Ala Gln Ala 290 295 300 Ala
Arg Glu Phe Ile Leu Lys Met Phe Val Asp Leu Asn Pro Asp Ser 305 310
315 320 Asp Lys Ile Ile Tyr Ser His Phe Thr Cys Ala Thr Asp Thr Glu
Asn 325 330 335 Ile Arg Phe Val Phe Ala Ala Val Lys Asp Thr Ile Leu
Gln Leu Asn 340 345 350 Leu Lys Glu Tyr Asn Leu Val 355 <210>
SEQ ID NO 310 <211> LENGTH: 355 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 310 Met Gly Cys Thr Val Ser Ala Glu Asp Lys
Ala Ala Ala Glu Arg Ser 1 5 10 15 Lys Met Ile Asp Lys Asn Leu Arg
Glu Asp Gly Glu Lys Ala Ala Arg 20 25 30 Glu Val Lys Leu Leu Leu
Leu Gly Ala Gly Glu Ser Gly Lys Ser Thr 35 40 45 Ile Val Lys Gln
Met Lys Ile Ile His Glu Asp Gly Tyr Ser Glu Glu 50 55 60 Glu Cys
Arg Gln Tyr Arg Ala Val Val Tyr Ser Asn Thr Ile Gln Ser 65 70 75 80
Ile Met Ala Ile Val Lys Ala Met Gly Asn Leu Gln Ile Asp Phe Ala 85
90 95 Asp Pro Ser Arg Ala Asp Asp Ala Arg Gln Leu Phe Ala Leu Ser
Cys 100 105 110 Thr Ala Glu Glu Gln Gly Val Leu Pro Asp Asp Leu Ser
Gly Val Ile 115 120 125 Arg Arg Leu Trp Ala Asp His Gly Val Gln Ala
Cys Phe Gly Arg Ser 130 135 140 Arg Glu Tyr Gln Leu Asn Asp Ser Ala
Ala Tyr Tyr Leu Asn Asp Leu 145 150 155 160 Glu Arg Ile Ala Gln Ser
Asp Tyr Ile Pro Thr Gln Gln Asp Val Leu 165 170 175 Arg Thr Arg Val
Lys Thr Thr Gly Ile Val Glu Thr His Phe Thr Phe 180 185 190 Lys Asp
Leu His Phe Lys Met Phe Asp Val Gly Gly Gln Arg Ser Glu 195 200 205
Arg Lys Lys Trp Ile His Cys Phe Glu Gly Val Thr Ala Ile Ile Phe 210
215 220 Cys Val Ala Leu Ser Ala Tyr Asp Leu Val Leu Ala Glu Asp Glu
Glu 225 230 235 240 Met Asn Arg Met His Glu Ser Met Lys Leu Phe Asp
Ser Ile Cys Asn 245 250 255 Asn Lys Trp Phe Thr Asp Thr Ser Ile Ile
Leu Phe Leu Asn Lys Lys 260 265 270 Asp Leu Phe Glu Glu Lys Ile Thr
His Ser Pro Leu Thr Ile Cys Phe 275 280 285 Pro Glu Tyr Thr Gly Ala
Asn Lys Tyr Asp Glu Ala Ala Ser Tyr Ile 290 295 300 Gln Ser Lys Phe
Glu Asp Leu Asn Lys Arg Lys Asp Thr Lys Glu Ile 305 310 315 320 Tyr
Thr His Phe Thr Cys Ala Thr Asp Thr Lys Asn Val Gln Phe Val 325 330
335 Phe Asp Ala Val Thr Asp Val Ile Ile Lys Asn Asn Leu Lys Asp Cys
340 345 350 Gly Leu Phe 355 <210> SEQ ID NO 311 <211>
LENGTH: 381 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic amino acid sequence <400> SEQUENCE: 311 Met Ser Gly
Val Val Arg Thr Leu Ser Arg Cys Leu Leu Pro Ala Glu 1 5 10 15 Ala
Gly Gly Ala Arg Glu Arg Arg Ala Gly Ser Gly Ala Arg Asp Ala 20 25
30 Glu Arg Glu Ala Arg Arg Arg Ser Arg Asp Ile Asp Ala Leu Leu Ala
35 40 45 Arg Glu Arg Arg Ala Val Arg Arg Leu Val Lys Ile Leu Leu
Leu Gly 50 55 60 Ala Gly Glu Ser Gly Lys Ser Thr Phe Leu Lys Gln
Met Arg Ile Ile 65 70 75 80 His Gly Arg Glu Phe Asp Gln Lys Ala Leu
Leu Glu Phe Arg Asp Thr 85 90 95 Ile Phe Asp Asn Ile Leu Lys Gly
Ser Arg Val Leu Val Asp Ala Arg 100 105 110 Asp Lys Leu Gly Ile Pro
Trp Gln Tyr Ser Glu Asn Glu Lys His Gly 115 120 125 Met Phe Leu Met
Ala Phe Glu Asn Lys Ala Gly Leu Pro Val Glu Pro 130 135 140 Ala Thr
Phe Gln Leu Tyr Val Pro Ala Leu Ser Ala Leu Trp Arg Asp 145 150 155
160 Ser Gly Ile Arg Glu Ala Phe Ser Arg Arg Ser Glu Phe Gln Leu Gly
165 170 175 Glu Ser Val Lys Tyr Phe Leu Asp Asn Leu Asp Arg Ile Gly
Gln Leu 180 185 190 Asn Tyr Phe Pro Ser Lys Gln Asp Ile Leu Leu Ala
Arg Lys Ala Thr 195 200 205 Lys Gly Ile Val Glu His Asp Phe Val Ile
Lys Lys Ile Pro Phe Lys 210 215 220 Met Val Asp Val Gly Gly Gln Arg
Ser Gln Arg Gln Lys Trp Phe Gln 225 230 235 240 Cys Phe Asp Gly Ile
Thr Ser Ile Leu Phe Met Val Ser Ser Ser Glu 245 250 255 Tyr Asp Gln
Val Leu Met Glu Asp Arg Arg Thr Asn Arg Leu Val Glu 260 265 270 Ser
Met Asn Ile Phe Glu Thr Ile Val Asn Asn Lys Leu Phe Phe Asn 275 280
285 Val Ser Ile Ile Leu Phe Leu Asn Lys Met Asp Leu Leu Val Glu Lys
290 295 300 Val Lys Thr Val Ser Ile Lys Lys His Phe Pro Asp Phe Arg
Gly Asp 305 310 315 320 Pro His Gln Leu Glu Asp Val Gln Arg Tyr Leu
Val Gln Cys Phe Asp 325 330 335 Arg Lys Arg Arg Asn Arg Ser Lys Pro
Leu Phe His His Phe Thr Thr 340 345 350 Ala Ile Asp Thr Glu Asn Val
Arg Phe Val Phe His Ala Val Lys Asp 355 360 365 Thr Ile Leu Gln Glu
Asn Leu Lys Asp Ile Met Leu Gln 370 375 380 <210> SEQ ID NO
312 <211> LENGTH: 50 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 312 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser
Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys
Ile Ile Glu Ala Arg 20 25 30 Leu Gln Ala Ser Val Asp Ala Glu Ser
Arg Asp Tyr Lys Asp Asp Asp 35 40 45 Asp Lys 50 <210> SEQ ID
NO 313 <211> LENGTH: 27 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 313 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser
Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys
20 25 <210> SEQ ID NO 314 <211> LENGTH: 50 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic amino acid
sequence <400> SEQUENCE: 314 Met Lys Ile Lys Thr Gly Ala Arg
Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala
Ser Ala Leu Ala Lys Ile Ile Glu Ala Arg 20 25 30 Leu Gln Ala Ser
Val Asp Ala Glu Ser Arg Asp Tyr Lys Asp Asp Asp 35 40 45 Asp Lys 50
<210> SEQ ID NO 315 <211> LENGTH: 50 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 315 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu
Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala
Leu Ala Lys Ile Ile Glu Ala Arg 20 25 30 Leu Gln Ala Ser Val Asp
Ala Glu Ser Arg Asp Tyr Lys Asp Asp Asp 35 40 45 Asp Lys 50
<210> SEQ ID NO 316 <211> LENGTH: 343 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 316 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu
Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala
Leu Ala Lys Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile Trp Ile Asn
Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val Gly Lys Lys
Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu 50 55 60 His Pro
Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly 65 70 75 80
Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85
90 95 Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe
Gln 100 105 110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr
Asn Gly Lys 115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu
Ser Leu Ile Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys
Thr Trp Glu Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys Glu Leu Lys
Ala Lys Gly Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu Gln Glu Pro
Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185 190 Tyr Ala
Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly 195 200 205
Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210
215 220 Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala
Glu 225 230 235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile
Asn Gly Pro Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr Ser Lys Val
Asn Tyr Gly Val Thr Val 260 265 270 Leu Pro Thr Phe Lys Gly Gln Pro
Ser Lys Pro Phe Val Gly Val Leu 275 280 285 Ser Ala Gly Ile Asn Ala
Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe Leu Glu Asn
Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310 315 320 Lys
Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 330
335 Leu Ala Lys Asp Pro Arg Ile 340 <210> SEQ ID NO 317
<211> LENGTH: 381 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic amino acid sequence <400> SEQUENCE:
317 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr
1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Glu Glu
Gly Lys 20 25 30 Leu Val Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn
Gly Leu Ala Glu 35 40 45 Val Gly Lys Lys Phe Glu Lys Asp Thr Gly
Ile Lys Val Thr Val Glu 50 55 60 His Pro Asp Lys Leu Glu Glu Lys
Phe Pro Gln Val Ala Ala Thr Gly 65 70 75 80 Asp Gly Pro Asp Ile Ile
Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85 90 95 Ala Gln Ser Gly
Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln 100 105 110 Asp Lys
Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys 115 120 125
Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn 130
135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro
Ala 145 150 155 160 Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala
Leu Met Phe Asn 165 170 175 Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu
Ile Ala Ala Asp Gly Gly 180 185 190 Tyr Ala Phe Lys Tyr Glu Asn Gly
Lys Tyr Asp Ile Lys Asp Val Gly 195 200 205 Val Asp Asn Ala Gly Ala
Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210 215 220 Ile Lys Asn Lys
His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu 225 230 235 240 Ala
Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp 245 250
255 Ala Trp Ser Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val
260 265 270 Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly
Val Leu 275 280 285 Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu
Leu Ala Lys Glu 290 295 300 Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu
Gly Leu Glu Ala Val Asn 305 310 315 320 Lys Asp Lys Pro Leu Gly Ala
Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 330 335 Leu Ala Lys Asp Pro
Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Ser 340 345 350 Ala Phe Trp
Tyr Ala Val Arg Ile Glu Ala Arg Leu Gln Ala Ser Val 355 360 365 Asp
Ala Glu Ser Arg Asp Tyr Lys Asp Asp Asp Asp Lys 370 375 380
<210> SEQ ID NO 318 <211> LENGTH: 381 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 318 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu
Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala
Leu Ala Lys Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile Trp Ile Asn
Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val Gly Lys Lys
Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu 50 55 60 His Pro
Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly 65 70 75 80
Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85
90 95 Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe
Gln 100 105 110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr
Asn Gly Lys 115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu
Ser Leu Ile Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys
Thr Trp Glu Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys Glu Leu Lys
Ala Lys Gly Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu Gln Glu Pro
Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185 190 Tyr Ala
Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly 195 200 205
Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210
215 220 Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala
Glu 225 230 235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile
Asn Gly Pro Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr Ser Lys Val
Asn Tyr Gly Val Thr Val 260 265 270 Leu Pro Thr Phe Lys Gly Gln Pro
Ser Lys Pro Phe Val Gly Val Leu 275 280 285 Ser Ala Gly Ile Asn Ala
Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe Leu Glu Asn
Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310 315 320 Lys
Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 330
335 Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Ser
340 345 350 Ala Phe Trp Tyr Ala Val Arg Ile Glu Ala Arg Leu Gln Ala
Ser Val 355 360 365 Asp Ala Glu Ser Arg Asp Tyr Lys Asp Asp Asp Asp
Lys 370 375 380 <210> SEQ ID NO 319 <211> LENGTH: 381
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 319 Met Lys Ile Lys Thr Gly Ala
Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser
Ala Ser Ala Leu Ala Lys Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile
Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val
Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu 50 55
60 His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly
65 70 75 80 Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly
Gly Tyr 85 90 95 Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp
Lys Ala Phe Gln 100 105 110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala
Val Arg Tyr Asn Gly Lys 115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val
Glu Ala Leu Ser Leu Ile Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn
Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys
Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu
Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185
190 Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly
195 200 205 Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val
Asp Leu 210 215 220 Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr
Ser Ile Ala Glu 225 230 235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala
Met Thr Ile Asn Gly Pro Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr
Ser Lys Val Asn Tyr Gly Val Thr Val 260 265 270 Leu Pro Thr Phe Lys
Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu 275 280 285 Ser Ala Gly
Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe
Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310
315 320 Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu
Glu 325 330 335 Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn
Ala Gln Ser 340 345 350 Ala Phe Trp Tyr Ala Val Arg Ile Glu Ala Arg
Leu Gln Ala Ser Val 355 360 365 Asp Ala Glu Ser Arg Asp Tyr Lys Asp
Asp Asp Asp Lys 370 375 380 <210> SEQ ID NO 320 <211>
LENGTH: 120 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic amino acid sequence <400> SEQUENCE: 320 Ala Ser Val
Asp Ala Glu Ser Arg Ser Asp Lys Ile Ile His Leu Thr 1 5 10 15 Asp
Asp Ser Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala Ile Leu 20 25
30 Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile Ala Pro
35 40 45 Ile Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr
Val Ala 50 55 60 Lys Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro
Lys Tyr Gly Ile 65 70 75 80 Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys
Asn Gly Glu Val Ala Ala 85 90 95 Thr Lys Val Gly Ala Leu Ser Lys
Gly Gln Leu Lys Glu Asn Leu Ala 100 105 110 Asp Tyr Lys Asp Asp Asp
Asp Lys 115 120 <210> SEQ ID NO 321 <211> LENGTH: 120
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 321 Ala Ser Val Asp Ala Glu Ser
Arg Ser Asp Lys Ile Ile His Leu Thr 1 5 10 15 Asp Asp Ser Phe Asp
Thr Asp Val Leu Lys Ala Asp Gly Ala Ile Leu 20 25 30 Val Asp Phe
Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile Ala Pro 35 40 45 Ile
Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala 50 55
60 Lys Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile
65 70 75 80 Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val
Ala Ala 85 90 95 Thr Lys Val Gly Ala Leu Ser Lys Gly Gln Leu Lys
Glu Asn Leu Ala 100 105 110 Asp Tyr Lys Asp Asp Asp Asp Lys 115 120
<210> SEQ ID NO 322 <211> LENGTH: 120 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 322 Ala Ser Val Asp Ala Glu Ser Arg Ser Asp
Lys Ile Ile His Leu Thr 1 5 10 15 Asp Asp Ser Phe Asp Thr Asp Val
Leu Lys Ala Asp Gly Ala Ile Leu 20 25 30 Val Asp Phe Trp Ala Glu
Trp Cys Gly Pro Cys Lys Met Ile Ala Pro 35 40 45 Ile Leu Asp Glu
Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala 50 55 60 Lys Leu
Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile 65 70 75 80
Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala 85
90 95 Thr Lys Val Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Asn Leu
Ala 100 105 110 Asp Tyr Lys Asp Asp Asp Asp Lys 115 120 <210>
SEQ ID NO 323 <211> LENGTH: 120 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 323 Ala Ser Val Asp Ala Glu Ser Arg Ser Asp
Lys Ile Ile His Leu Thr 1 5 10 15 Asp Asp Ser Phe Asp Thr Asp Val
Leu Lys Ala Asp Gly Ala Ile Leu 20 25 30 Val Asp Phe Trp Ala Glu
Trp Cys Gly Pro Cys Lys Met Ile Ala Pro 35 40 45 Ile Leu Asp Glu
Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala 50 55 60 Lys Leu
Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile 65 70 75 80
Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala 85
90 95 Thr Lys Val Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Asn Leu
Ala 100 105 110 Asp Tyr Lys Asp Asp Asp Asp Lys 115 120 <210>
SEQ ID NO 324 <211> LENGTH: 154 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 324 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu
Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala
Leu Ala Lys Ile Ile Glu Ala Arg 20 25 30 Leu Gln Ala Ser Val Asp
Ala Glu Ser Arg Ser Asp Lys Ile Ile His 35 40 45 Leu Thr Asp Asp
Ser Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala 50 55 60 Ile Leu
Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile 65 70 75 80
Ala Pro Ile Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr 85
90 95 Val Ala Lys Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys
Tyr 100 105 110 Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys Asn
Gly Glu Val 115 120 125 Ala Ala Thr Lys Val Gly Ala Leu Ser Lys Gly
Gln Leu Lys Glu Asn 130 135 140 Leu Ala Asp Tyr Lys Asp Asp Asp Asp
Lys 145 150 <210> SEQ ID NO 325 <211> LENGTH: 154
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 325 Met Lys Ile Lys Thr Gly Ala
Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser
Ala Ser Ala Leu Ala Lys Ile Ile Glu Ala Arg 20 25 30 Leu Gln Ala
Ser Val Asp Ala Glu Ser Arg Ser Asp Lys Ile Ile His 35 40 45 Leu
Thr Asp Asp Ser Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala 50 55
60 Ile Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile
65 70 75 80 Ala Pro Ile Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys
Leu Thr 85 90 95 Val Ala Lys Leu Asn Ile Asp Gln Asn Pro Gly Thr
Ala Pro Lys Tyr 100 105 110 Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu
Phe Lys Asn Gly Glu Val 115 120 125 Ala Ala Thr Lys Val Gly Ala Leu
Ser Lys Gly Gln Leu Lys Glu Asn 130 135 140 Leu Ala Asp Tyr Lys Asp
Asp Asp Asp Lys 145 150 <210> SEQ ID NO 326 <211>
LENGTH: 154 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic amino acid sequence <400> SEQUENCE: 326 Met Lys Ile
Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr
Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Ile Glu Ala Arg 20 25
30 Leu Gln Ala Ser Val Asp Ala Glu Ser Arg Ser Asp Lys Ile Ile His
35 40 45 Leu Thr Asp Asp Ser Phe Asp Thr Asp Val Leu Lys Ala Asp
Gly Ala 50 55 60 Ile Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro
Cys Lys Met Ile 65 70 75 80 Ala Pro Ile Leu Asp Glu Ile Ala Asp Glu
Tyr Gln Gly Lys Leu Thr 85 90 95 Val Ala Lys Leu Asn Ile Asp Gln
Asn Pro Gly Thr Ala Pro Lys Tyr 100 105 110 Gly Ile Arg Gly Ile Pro
Thr Leu Leu Leu Phe Lys Asn Gly Glu Val 115 120 125 Ala Ala Thr Lys
Val Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Asn 130 135 140 Leu Ala
Asp Tyr Lys Asp Asp Asp Asp Lys 145 150 <210> SEQ ID NO 327
<211> LENGTH: 154 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic amino acid sequence <400> SEQUENCE:
327 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr
1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Ile Glu
Ala Arg 20 25 30 Leu Gln Ala Ser Val Asp Ala Glu Ser Arg Ser Asp
Lys Ile Ile His 35 40 45 Leu Thr Asp Asp Ser Phe Asp Thr Asp Val
Leu Lys Ala Asp Gly Ala 50 55 60 Ile Leu Val Asp Phe Trp Ala Glu
Trp Cys Gly Pro Cys Lys Met Ile 65 70 75 80 Ala Pro Ile Leu Asp Glu
Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr 85 90 95 Val Ala Lys Leu
Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr 100 105 110 Gly Ile
Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val 115 120 125
Ala Ala Thr Lys Val Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Asn 130
135 140 Leu Ala Asp Tyr Lys Asp Asp Asp Asp Lys 145 150 <210>
SEQ ID NO 328 <211> LENGTH: 485 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 328 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu
Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala
Leu Ala Lys Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile Trp Ile Asn
Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val Gly Lys Lys
Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu 50 55 60 His Pro
Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly 65 70 75 80
Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85
90 95 Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe
Gln 100 105 110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr
Asn Gly Lys 115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu
Ser Leu Ile Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys
Thr Trp Glu Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys Glu Leu Lys
Ala Lys Gly Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu Gln Glu Pro
Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185 190 Tyr Ala
Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly 195 200 205
Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210
215 220 Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala
Glu 225 230 235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile
Asn Gly Pro Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr Ser Lys Val
Asn Tyr Gly Val Thr Val 260 265 270 Leu Pro Thr Phe Lys Gly Gln Pro
Ser Lys Pro Phe Val Gly Val Leu 275 280 285 Ser Ala Gly Ile Asn Ala
Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe Leu Glu Asn
Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310 315 320 Lys
Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 330
335 Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Ser
340 345 350 Ala Phe Trp Tyr Ala Val Arg Ile Glu Ala Arg Leu Gln Ala
Ser Val 355 360 365 Asp Ala Glu Ser Arg Ser Asp Lys Ile Ile His Leu
Thr Asp Asp Ser 370 375 380 Phe Asp Thr Asp Val Leu Lys Ala Asp Gly
Ala Ile Leu Val Asp Phe 385 390 395 400 Trp Ala Glu Trp Cys Gly Pro
Cys Lys Met Ile Ala Pro Ile Leu Asp 405 410 415 Glu Ile Ala Asp Glu
Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn 420 425 430 Ile Asp Gln
Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile 435 440 445 Pro
Thr Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val 450 455
460 Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Asn Leu Ala Asp Tyr Lys
465 470 475 480 Asp Asp Asp Asp Lys 485 <210> SEQ ID NO 329
<211> LENGTH: 485 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic amino acid sequence <400> SEQUENCE:
329 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr
1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Glu Glu
Gly Lys 20 25 30 Leu Val Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn
Gly Leu Ala Glu 35 40 45 Val Gly Lys Lys Phe Glu Lys Asp Thr Gly
Ile Lys Val Thr Val Glu 50 55 60 His Pro Asp Lys Leu Glu Glu Lys
Phe Pro Gln Val Ala Ala Thr Gly 65 70 75 80 Asp Gly Pro Asp Ile Ile
Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85 90 95 Ala Gln Ser Gly
Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln 100 105 110 Asp Lys
Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys 115 120 125
Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn 130
135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro
Ala 145 150 155 160 Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala
Leu Met Phe Asn 165 170 175 Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu
Ile Ala Ala Asp Gly Gly 180 185 190 Tyr Ala Phe Lys Tyr Glu Asn Gly
Lys Tyr Asp Ile Lys Asp Val Gly 195 200 205 Val Asp Asn Ala Gly Ala
Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210 215 220 Ile Lys Asn Lys
His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu 225 230 235 240 Ala
Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp 245 250
255 Ala Trp Ser Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val
260 265 270 Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly
Val Leu 275 280 285 Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu
Leu Ala Lys Glu 290 295 300 Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu
Gly Leu Glu Ala Val Asn 305 310 315 320 Lys Asp Lys Pro Leu Gly Ala
Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 330 335 Leu Ala Lys Asp Pro
Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Ser 340 345 350 Ala Phe Trp
Tyr Ala Val Arg Ile Glu Ala Arg Leu Gln Ala Ser Val 355 360 365 Asp
Ala Glu Ser Arg Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser 370 375
380 Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe
385 390 395 400 Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile Ala Pro
Ile Leu Asp 405 410 415 Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr
Val Ala Lys Leu Asn 420 425 430 Ile Asp Gln Asn Pro Gly Thr Ala Pro
Lys Tyr Gly Ile Arg Gly Ile 435 440 445 Pro Thr Leu Leu Leu Phe Lys
Asn Gly Glu Val Ala Ala Thr Lys Val 450 455 460 Gly Ala Leu Ser Lys
Gly Gln Leu Lys Glu Asn Leu Ala Asp Tyr Lys 465 470 475 480 Asp Asp
Asp Asp Lys 485 <210> SEQ ID NO 330 <211> LENGTH: 485
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 330 Met Lys Ile Lys Thr Gly Ala
Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser
Ala Ser Ala Leu Ala Lys Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile
Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val
Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu 50 55
60 His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly
65 70 75 80 Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly
Gly Tyr 85 90 95 Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp
Lys Ala Phe Gln 100 105 110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala
Val Arg Tyr Asn Gly Lys 115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val
Glu Ala Leu Ser Leu Ile Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn
Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys
Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu
Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185
190 Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly
195 200 205 Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val
Asp Leu 210 215 220 Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr
Ser Ile Ala Glu 225 230 235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala
Met Thr Ile Asn Gly Pro Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr
Ser Lys Val Asn Tyr Gly Val Thr Val 260 265 270 Leu Pro Thr Phe Lys
Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu 275 280 285 Ser Ala Gly
Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe
Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310
315 320 Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu
Glu 325 330 335 Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn
Ala Gln Ser 340 345 350 Ala Phe Trp Tyr Ala Val Arg Ile Glu Ala Arg
Leu Gln Ala Ser Val 355 360 365 Asp Ala Glu Ser Arg Ser Asp Lys Ile
Ile His Leu Thr Asp Asp Ser 370 375 380 Phe Asp Thr Asp Val Leu Lys
Ala Asp Gly Ala Ile Leu Val Asp Phe 385 390 395 400 Trp Ala Glu Trp
Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp 405 410 415 Glu Ile
Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn 420 425 430
Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile 435
440 445 Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys
Val 450 455 460 Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Asn Leu Ala
Asp Tyr Lys 465 470 475 480 Asp Asp Asp Asp Lys 485 <210> SEQ
ID NO 331 <211> LENGTH: 485 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 331 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser
Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys
Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile Trp Ile Asn Gly Asp Lys
Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val Gly Lys Lys Phe Glu Lys
Asp Thr Gly Ile Lys Val Thr Val Glu 50 55 60 His Pro Asp Lys Leu
Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly 65 70 75 80 Asp Gly Pro
Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85 90 95 Ala
Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln 100 105
110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys
115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile
Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu
Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys Glu Leu Lys Ala Lys Gly
Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu Gln Glu Pro Tyr Phe Thr
Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185 190 Tyr Ala Phe Lys Tyr
Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly 195 200 205 Val Asp Asn
Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210 215 220 Ile
Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu 225 230
235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile Asn Gly Pro
Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly
Val Thr Val 260 265 270 Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro
Phe Val Gly Val Leu 275 280 285 Ser Ala Gly Ile Asn Ala Ala Ser Pro
Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe Leu Glu Asn Tyr Leu Leu
Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310 315 320 Lys Asp Lys Pro
Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 330 335 Leu Ala
Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Ser 340 345 350
Ala Phe Trp Tyr Ala Val Arg Ile Glu Ala Arg Leu Gln Ala Ser Val 355
360 365 Asp Ala Glu Ser Arg Ser Asp Lys Ile Ile His Leu Thr Asp Asp
Ser 370 375 380 Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala Ile Leu
Val Asp Phe 385 390 395 400 Trp Ala Glu Trp Cys Gly Pro Cys Lys Met
Ile Ala Pro Ile Leu Asp 405 410 415 Glu Ile Ala Asp Glu Tyr Gln Gly
Lys Leu Thr Val Ala Lys Leu Asn 420 425 430 Ile Asp Gln Asn Pro Gly
Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile 435 440 445 Pro Thr Leu Leu
Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val 450 455 460 Gly Ala
Leu Ser Lys Gly Gln Leu Lys Glu Asn Leu Ala Asp Tyr Lys 465 470 475
480 Asp Asp Asp Asp Lys 485 <210> SEQ ID NO 332 <211>
LENGTH: 767 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic amino acid sequence <400> SEQUENCE: 332 Met Lys Ile
Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr
Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Glu Glu Gly Lys 20 25
30 Leu Val Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu
35 40 45 Val Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr
Val Glu 50 55 60 His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val
Ala Ala Thr Gly 65 70 75 80 Asp Gly Pro Asp Ile Ile Phe Trp Ala His
Asp Arg Phe Gly Gly Tyr 85 90 95 Ala Gln Ser Gly Leu Leu Ala Glu
Ile Thr Pro Asp Lys Ala Phe Gln 100 105 110 Asp Lys Leu Tyr Pro Phe
Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys 115 120 125 Leu Ile Ala Tyr
Pro Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn 130 135 140 Lys Asp
Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala 145 150 155
160 Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn
165 170 175 Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp
Gly Gly 180 185 190 Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile
Lys Asp Val Gly 195 200 205 Val Asp Asn Ala Gly Ala Lys Ala Gly Leu
Thr Phe Leu Val Asp Leu 210 215 220 Ile Lys Asn Lys His Met Asn Ala
Asp Thr Asp Tyr Ser Ile Ala Glu 225 230 235 240 Ala Ala Phe Asn Lys
Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp 245 250 255 Ala Trp Ser
Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val 260 265 270 Leu
Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu 275 280
285 Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu
290 295 300 Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala
Val Asn 305 310 315 320 Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys
Ser Tyr Glu Glu Glu 325 330 335 Leu Ala Lys Asp Pro Arg Ile Ala Ala
Thr Met Glu Asn Ala Gln Lys 340 345 350 Gly Glu Ile Met Pro Asn Ile
Pro Gln Met Ser Ala Phe Trp Tyr Ala 355 360 365 Val Leu Ile Glu Ala
Arg Thr Ser Glu Ser Asp Thr Ala Gly Pro Asn 370 375 380 Ser Asp Leu
Asp Val Asn Thr Asp Ile Tyr Ser Lys Val Leu Val Thr 385 390 395 400
Ala Ile Tyr Leu Ala Leu Phe Val Val Gly Thr Val Gly Asn Ser Val 405
410 415 Thr Ala Phe Thr Leu Ala Arg Lys Lys Ser Leu Gln Ser Leu Gln
Ser 420 425 430 Thr Val His Tyr His Leu Gly Ser Leu Ala Leu Ser Asp
Leu Leu Ile 435 440 445 Leu Leu Leu Ala Met Pro Val Glu Leu Tyr Asn
Phe Ile Trp Val His 450 455 460 His Pro Trp Ala Phe Gly Asp Ala Gly
Cys Arg Gly Tyr Tyr Phe Leu 465 470 475 480 Arg Asp Ala Cys Thr Tyr
Ala Thr Ala Leu Asn Val Ala Ser Leu Ser 485 490 495 Val Glu Arg Tyr
Leu Ala Ile Cys His Pro Phe Lys Ala Lys Thr Leu 500 505 510 Met Ser
Arg Ser Arg Thr Lys Lys Phe Ile Ser Ala Ile Trp Leu Ala 515 520 525
Ser Ala Leu Leu Ala Ile Pro Met Leu Phe Thr Met Gly Leu Gln Asn 530
535 540 Arg Ser Gly Asp Gly Thr His Pro Gly Gly Leu Val Cys Thr Pro
Ile 545 550 555 560 Val Asp Thr Ala Thr Val Lys Val Val Ile Gln Val
Asn Thr Phe Met 565 570 575 Ser Phe Leu Phe Pro Met Leu Val Ile Ser
Ile Leu Asn Thr Val Ile 580 585 590 Ala Asn Lys Leu Thr Val Met Val
His Gln Ala Ala Glu Gln Gly Arg 595 600 605 Val Cys Thr Val Gly Thr
His Asn Gly Leu Glu His Ser Thr Phe Asn 610 615 620 Met Thr Ile Glu
Pro Gly Arg Val Gln Ala Leu Arg His Gly Val Leu 625 630 635 640 Val
Leu Arg Ala Val Val Ile Ala Phe Val Val Cys Trp Leu Pro Tyr 645 650
655 His Val Arg Arg Leu Met Phe Cys Tyr Ile Ser Asp Glu Gln Trp Thr
660 665 670 Thr Phe Leu Phe Asp Phe Tyr His Tyr Phe Tyr Met Leu Thr
Asn Ala 675 680 685 Leu Phe Tyr Val Ser Ser Ala Ile Asn Pro Ile Leu
Tyr Asn Leu Val 690 695 700 Ser Ala Asn Phe Arg Gln Val Phe Leu Ser
Thr Leu Ala Cys Leu Cys 705 710 715 720 Pro Gly Trp Arg His Arg Arg
Lys Lys Arg Pro Thr Phe Ser Arg Lys 725 730 735 Pro Asn Ser Met Ser
Ser Asn His Ala Phe Ser Thr Ser Ala Thr Arg 740 745 750 Glu Thr Leu
Tyr Ala Ala Ala Asp Tyr Lys Asp Asp Asp Asp Lys 755 760 765
<210> SEQ ID NO 333 <211> LENGTH: 425 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 333 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu
Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala
Leu Ala Lys Ile Ile Glu Ala Arg 20 25 30 Thr Ser Glu Ser Asp Thr
Ala Gly Pro Asn Ser Asp Leu Asp Val Asn 35 40 45 Thr Asp Ile Tyr
Ser Lys Val Leu Val Thr Ala Ile Tyr Leu Ala Leu 50 55 60 Phe Val
Val Gly Thr Val Gly Asn Ser Val Thr Ala Phe Thr Leu Ala 65 70 75 80
Arg Lys Lys Ser Leu Gln Ser Leu Gln Ser Thr Val His Tyr His Leu 85
90 95 Gly Ser Leu Ala Leu Ser Asp Leu Leu Ile Leu Leu Leu Ala Met
Pro 100 105 110 Val Glu Leu Tyr Asn Phe Ile Trp Val His His Pro Trp
Ala Phe Gly 115 120 125 Asp Ala Gly Cys Arg Gly Tyr Tyr Phe Leu Arg
Asp Ala Cys Thr Tyr 130 135 140 Ala Thr Ala Leu Asn Val Ala Ser Leu
Ser Val Glu Arg Tyr Leu Ala 145 150 155 160 Ile Cys His Pro Phe Lys
Ala Lys Thr Leu Met Ser Arg Ser Arg Thr 165 170 175 Lys Lys Phe Ile
Ser Ala Ile Trp Leu Ala Ser Ala Leu Leu Ala Ile 180 185 190 Pro Met
Leu Phe Thr Met Gly Leu Gln Asn Arg Ser Gly Asp Gly Thr 195 200 205
His Pro Gly Gly Leu Val Cys Thr Pro Ile Val Asp Thr Ala Thr Val 210
215 220 Lys Val Val Ile Gln Val Asn Thr Phe Met Ser Phe Leu Phe Pro
Met 225 230 235 240 Leu Val Ile Ser Ile Leu Asn Thr Val Ile Ala Asn
Lys Leu Thr Val 245 250 255 Met Val His Gln Ala Ala Glu Gln Gly Arg
Val Cys Thr Val Gly Thr 260 265 270 His Asn Gly Leu Glu His Ser Thr
Phe Asn Met Thr Ile Glu Pro Gly 275 280 285 Arg Val Gln Ala Leu Arg
His Gly Val Leu Val Leu Arg Ala Val Val 290 295 300 Ile Ala Phe Val
Val Cys Trp Leu Pro Tyr His Val Arg Arg Leu Met 305 310 315 320 Phe
Cys Tyr Ile Ser Asp Glu Gln Trp Thr Thr Phe Leu Phe Asp Phe 325 330
335 Tyr His Tyr Phe Tyr Met Leu Thr Asn Ala Leu Phe Tyr Val Ser Ser
340 345 350 Ala Ile Asn Pro Ile Leu Tyr Asn Leu Val Ser Ala Asn Phe
Arg Gln 355 360 365 Val Phe Leu Ser Thr Leu Ala Cys Leu Cys Pro Gly
Trp Arg His Arg 370 375 380 Arg Lys Lys Arg Pro Thr Phe Ser Arg Lys
Pro Asn Ser Met Ser Ser 385 390 395 400 Asn His Ala Phe Ser Thr Ser
Ala Thr Arg Glu Thr Leu Tyr Ala Ala 405 410 415 Ala Asp Tyr Lys Asp
Asp Asp Asp Lys 420 425 <210> SEQ ID NO 334 <211>
LENGTH: 878 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic amino acid sequence <400> SEQUENCE: 334 Met Lys Ile
Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr
Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Glu Glu Gly Lys 20 25
30 Leu Val Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu
35 40 45 Val Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr
Val Glu 50 55 60 His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val
Ala Ala Thr Gly 65 70 75 80 Asp Gly Pro Asp Ile Ile Phe Trp Ala His
Asp Arg Phe Gly Gly Tyr 85 90 95 Ala Gln Ser Gly Leu Leu Ala Glu
Ile Thr Pro Asp Lys Ala Phe Gln 100 105 110 Asp Lys Leu Tyr Pro Phe
Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys 115 120 125 Leu Ile Ala Tyr
Pro Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn 130 135 140 Lys Asp
Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala 145 150 155
160 Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn
165 170 175 Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp
Gly Gly 180 185 190 Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile
Lys Asp Val Gly 195 200 205 Val Asp Asn Ala Gly Ala Lys Ala Gly Leu
Thr Phe Leu Val Asp Leu 210 215 220 Ile Lys Asn Lys His Met Asn Ala
Asp Thr Asp Tyr Ser Ile Ala Glu 225 230 235 240 Ala Ala Phe Asn Lys
Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp 245 250 255 Ala Trp Ser
Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val 260 265 270 Leu
Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu 275 280
285 Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu
290 295 300 Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala
Val Asn 305 310 315 320 Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys
Ser Tyr Glu Glu Glu 325 330 335 Leu Ala Lys Asp Pro Arg Ile Ala Ala
Thr Met Glu Asn Ala Gln Lys 340 345 350 Gly Glu Ile Met Pro Asn Ile
Pro Gln Met Ser Ala Phe Trp Tyr Ala 355 360 365 Val Leu Ile Glu Ala
Arg Thr Ser Glu Ser Asp Thr Ala Gly Pro Asn 370 375 380 Ser Asp Leu
Asp Val Asn Thr Asp Ile Tyr Ser Lys Val Leu Val Thr 385 390 395 400
Ala Ile Tyr Leu Ala Leu Phe Val Val Gly Thr Val Gly Asn Ser Val 405
410 415 Thr Ala Phe Thr Leu Ala Arg Lys Lys Ser Leu Gln Ser Leu Gln
Ser 420 425 430 Thr Val His Tyr His Leu Gly Ser Leu Ala Leu Ser Asp
Leu Leu Ile 435 440 445 Leu Leu Leu Ala Met Pro Val Glu Leu Tyr Asn
Phe Ile Trp Val His 450 455 460 His Pro Trp Ala Phe Gly Asp Ala Gly
Cys Arg Gly Tyr Tyr Phe Leu 465 470 475 480 Arg Asp Ala Cys Thr Tyr
Ala Thr Ala Leu Asn Val Ala Ser Leu Ser 485 490 495 Val Glu Arg Tyr
Leu Ala Ile Cys His Pro Phe Lys Ala Lys Thr Leu 500 505 510 Met Ser
Arg Ser Arg Thr Lys Lys Phe Ile Ser Ala Ile Trp Leu Ala 515 520 525
Ser Ala Leu Leu Ala Ile Pro Met Leu Phe Thr Met Gly Leu Gln Asn 530
535 540 Arg Ser Gly Asp Gly Thr His Pro Gly Gly Leu Val Cys Thr Pro
Ile 545 550 555 560 Val Asp Thr Ala Thr Val Lys Val Val Ile Gln Val
Asn Thr Phe Met 565 570 575 Ser Phe Leu Phe Pro Met Leu Val Ile Ser
Ile Leu Asn Thr Val Ile 580 585 590 Ala Asn Lys Leu Thr Val Met Val
His Gln Ala Ala Glu Gln Gly Arg 595 600 605 Val Cys Thr Val Gly Thr
His Asn Gly Leu Glu His Ser Thr Phe Asn 610 615 620 Met Thr Ile Glu
Pro Gly Arg Val Gln Ala Leu Arg His Gly Val Leu 625 630 635 640 Val
Leu Arg Ala Val Val Ile Ala Phe Val Val Cys Trp Leu Pro Tyr 645 650
655 His Val Arg Arg Leu Met Phe Cys Tyr Ile Ser Asp Glu Gln Trp Thr
660 665 670 Thr Phe Leu Phe Asp Phe Tyr His Tyr Phe Tyr Met Leu Thr
Asn Ala 675 680 685 Leu Phe Tyr Val Ser Ser Ala Ile Asn Pro Ile Leu
Tyr Asn Leu Val 690 695 700 Ser Ala Asn Phe Arg Gln Val Phe Leu Ser
Thr Leu Ala Cys Leu Cys 705 710 715 720 Pro Gly Trp Arg His Arg Arg
Lys Lys Arg Pro Thr Phe Ser Arg Lys 725 730 735 Pro Asn Ser Met Ser
Ser Asn His Ala Phe Ser Thr Ser Ala Thr Arg 740 745 750 Glu Thr Leu
Tyr Ala Ala Ala Ser Asp Lys Ile Ile His Leu Thr Asp 755 760 765 Asp
Ser Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala Ile Leu Val 770 775
780 Asp Phe Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile Ala Pro Ile
785 790 795 800 Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr
Val Ala Lys 805 810 815 Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro
Lys Tyr Gly Ile Arg 820 825 830 Gly Ile Pro Thr Leu Leu Leu Phe Lys
Asn Gly Glu Val Ala Ala Thr 835 840 845 Lys Val Gly Ala Leu Ser Lys
Gly Gln Leu Lys Glu Phe Leu Asp Ala 850 855 860 Asn Leu Ala Ala Ala
Ala Asp Tyr Lys Asp Asp Asp Asp Lys 865 870 875 <210> SEQ ID
NO 335 <211> LENGTH: 536 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 335 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser
Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys
Ile Ile Glu Ala Arg 20 25 30 Thr Ser Glu Ser Asp Thr Ala Gly Pro
Asn Ser Asp Leu Asp Val Asn 35 40 45 Thr Asp Ile Tyr Ser Lys Val
Leu Val Thr Ala Ile Tyr Leu Ala Leu 50 55 60 Phe Val Val Gly Thr
Val Gly Asn Ser Val Thr Ala Phe Thr Leu Ala 65 70 75 80 Arg Lys Lys
Ser Leu Gln Ser Leu Gln Ser Thr Val His Tyr His Leu 85 90 95 Gly
Ser Leu Ala Leu Ser Asp Leu Leu Ile Leu Leu Leu Ala Met Pro 100 105
110 Val Glu Leu Tyr Asn Phe Ile Trp Val His His Pro Trp Ala Phe Gly
115 120 125 Asp Ala Gly Cys Arg Gly Tyr Tyr Phe Leu Arg Asp Ala Cys
Thr Tyr 130 135 140 Ala Thr Ala Leu Asn Val Ala Ser Leu Ser Val Glu
Arg Tyr Leu Ala 145 150 155 160 Ile Cys His Pro Phe Lys Ala Lys Thr
Leu Met Ser Arg Ser Arg Thr 165 170 175 Lys Lys Phe Ile Ser Ala Ile
Trp Leu Ala Ser Ala Leu Leu Ala Ile 180 185 190 Pro Met Leu Phe Thr
Met Gly Leu Gln Asn Arg Ser Gly Asp Gly Thr 195 200 205 His Pro Gly
Gly Leu Val Cys Thr Pro Ile Val Asp Thr Ala Thr Val 210 215 220 Lys
Val Val Ile Gln Val Asn Thr Phe Met Ser Phe Leu Phe Pro Met 225 230
235 240 Leu Val Ile Ser Ile Leu Asn Thr Val Ile Ala Asn Lys Leu Thr
Val 245 250 255 Met Val His Gln Ala Ala Glu Gln Gly Arg Val Cys Thr
Val Gly Thr 260 265 270 His Asn Gly Leu Glu His Ser Thr Phe Asn Met
Thr Ile Glu Pro Gly 275 280 285 Arg Val Gln Ala Leu Arg His Gly Val
Leu Val Leu Arg Ala Val Val 290 295 300 Ile Ala Phe Val Val Cys Trp
Leu Pro Tyr His Val Arg Arg Leu Met 305 310 315 320 Phe Cys Tyr Ile
Ser Asp Glu Gln Trp Thr Thr Phe Leu Phe Asp Phe 325 330 335 Tyr His
Tyr Phe Tyr Met Leu Thr Asn Ala Leu Phe Tyr Val Ser Ser 340 345 350
Ala Ile Asn Pro Ile Leu Tyr Asn Leu Val Ser Ala Asn Phe Arg Gln 355
360 365 Val Phe Leu Ser Thr Leu Ala Cys Leu Cys Pro Gly Trp Arg His
Arg 370 375 380 Arg Lys Lys Arg Pro Thr Phe Ser Arg Lys Pro Asn Ser
Met Ser Ser 385 390 395 400 Asn His Ala Phe Ser Thr Ser Ala Thr Arg
Glu Thr Leu Tyr Ala Ala 405 410 415 Ala Ser Asp Lys Ile Ile His Leu
Thr Asp Asp Ser Phe Asp Thr Asp 420 425 430 Val Leu Lys Ala Asp Gly
Ala Ile Leu Val Asp Phe Trp Ala Glu Trp 435 440 445 Cys Gly Pro Cys
Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp 450 455 460 Glu Tyr
Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn 465 470 475
480 Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu
485 490 495 Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala
Leu Ser 500 505 510 Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu
Ala Ala Ala Ala 515 520 525 Asp Tyr Lys Asp Asp Asp Asp Lys 530 535
<210> SEQ ID NO 336 <211> LENGTH: 380 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 336 Met Gly Cys Leu Gly Asn Ser Lys Thr Glu
Asp Gln Arg Asn Glu Glu 1 5 10 15 Lys Ala Gln Arg Glu Ala Asn Lys
Lys Ile Glu Lys Gln Leu Gln Lys 20 25 30 Asp Lys Gln Val Tyr Arg
Ala Thr His Arg Leu Leu Leu Leu Gly Ala 35 40 45 Gly Glu Ser Gly
Lys Ser Thr Ile Val Lys Gln Met Arg Ile Leu His 50 55 60 Val Asn
Gly Phe Asn Gly Asp Ser Glu Lys Ala Thr Lys Val Gln Asp 65 70 75 80
Ile Lys Asn Asn Leu Lys Glu Ala Ile Glu Thr Ile Val Ala Ala Met 85
90 95 Ser Asn Leu Val Pro Pro Val Glu Leu Ala Asn Pro Glu Asn Gln
Phe 100 105 110 Arg Val Asp Tyr Ile Leu Ser Val Met Asn Val Pro Asp
Phe Asp Phe 115 120 125 Pro Pro Glu Phe Tyr Glu His Ala Lys Ala Leu
Trp Glu Asp Glu Gly 130 135 140 Val Arg Ala Cys Tyr Glu Arg Ser Asn
Glu Tyr Gln Leu Ile Asp Cys 145 150 155 160 Ala Gln Tyr Phe Leu Asp
Lys Ile Asp Val Ile Lys Gln Ala Asp Tyr 165 170 175 Val Pro Ser Asp
Gln Asp Leu Leu Arg Cys Arg Val Leu Thr Ser Gly 180 185 190 Ile Phe
Glu Thr Lys Phe Gln Val Asp Lys Val Asn Phe His Met Phe 195 200 205
Asp Val Gly Gly Gln Arg Asp Glu Arg Arg Lys Trp Ile Gln Cys Phe 210
215 220 Asn Asp Val Thr Ala Ile Ile Phe Val Val Ala Ser Ser Ser Tyr
Asn 225 230 235 240 Met Val Ile Arg Glu Asp Asn Gln Thr Asn Arg Leu
Gln Glu Ala Leu 245 250 255 Asn Leu Phe Lys Ser Ile Trp Asn Asn Arg
Trp Leu Arg Thr Ile Ser 260 265 270 Val Ile Leu Phe Leu Asn Lys Gln
Asp Leu Leu Ala Glu Lys Val Leu 275 280 285 Ala Gly Lys Ser Lys Ile
Glu Asp Tyr Phe Pro Glu Phe Ala Arg Tyr 290 295 300 Thr Thr Pro Glu
Asp Ala Thr Pro Glu Pro Gly Glu Asp Pro Arg Val 305 310 315 320 Thr
Arg Ala Lys Tyr Phe Ile Arg Asp Glu Phe Leu Arg Ile Ser Thr 325 330
335 Ala Ser Gly Asp Gly Arg His Tyr Cys Tyr Pro His Phe Thr Cys Ala
340 345 350 Val Asp Thr Glu Asn Ile Arg Arg Val Phe Asn Asp Cys Arg
Asp Ile 355 360 365 Ile Gln Arg Met His Leu Arg Gln Tyr Glu Leu Leu
370 375 380 <210> SEQ ID NO 337 <211> LENGTH: 394
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 337 Met Gly Cys Leu Gly Asn Ser
Lys Thr Glu Asp Gln Arg Asn Glu Glu 1 5 10 15 Lys Ala Gln Arg Glu
Ala Asn Lys Lys Ile Glu Lys Gln Leu Gln Lys 20 25 30 Asp Lys Gln
Val Tyr Arg Ala Thr His Arg Leu Leu Leu Leu Gly Ala 35 40 45 Gly
Glu Ser Gly Lys Ser Thr Ile Val Lys Gln Met Arg Ile Leu His 50 55
60 Val Asn Gly Phe Asn Gly Glu Gly Gly Glu Glu Asp Pro Gln Ala Ala
65 70 75 80 Arg Ser Asn Ser Asp Gly Glu Lys Ala Thr Lys Val Gln Asp
Ile Lys 85 90 95 Asn Asn Leu Lys Glu Ala Ile Glu Thr Ile Val Ala
Ala Met Ser Asn 100 105 110 Leu Val Pro Pro Val Glu Leu Ala Asn Pro
Glu Asn Gln Phe Arg Val 115 120 125 Asp Tyr Ile Leu Ser Val Met Asn
Val Pro Asp Phe Asp Phe Pro Pro 130 135 140 Glu Phe Tyr Glu His Ala
Lys Ala Leu Trp Glu Asp Glu Gly Val Arg 145 150 155 160 Ala Cys Tyr
Glu Arg Ser Asn Glu Tyr Gln Leu Ile Asp Cys Ala Gln 165 170 175 Tyr
Phe Leu Asp Lys Ile Asp Val Ile Lys Gln Ala Asp Tyr Val Pro 180 185
190 Ser Asp Gln Asp Leu Leu Arg Cys Arg Val Leu Thr Ser Gly Ile Phe
195 200 205 Glu Thr Lys Phe Gln Val Asp Lys Val Asn Phe His Met Phe
Asp Val 210 215 220 Gly Gly Gln Arg Asp Glu Arg Arg Lys Trp Ile Gln
Cys Phe Asn Asp 225 230 235 240 Val Thr Ala Ile Ile Phe Val Val Ala
Ser Ser Ser Tyr Asn Met Val 245 250 255 Ile Arg Glu Asp Asn Gln Thr
Asn Arg Leu Gln Glu Ala Leu Asn Leu 260 265 270 Phe Lys Ser Ile Trp
Asn Asn Arg Trp Leu Arg Thr Ile Ser Val Ile 275 280 285 Leu Phe Leu
Asn Lys Gln Asp Leu Leu Ala Glu Lys Val Leu Ala Gly 290 295 300 Lys
Ser Lys Ile Glu Asp Tyr Phe Pro Glu Phe Ala Arg Tyr Thr Thr 305 310
315 320 Pro Glu Asp Ala Thr Pro Glu Pro Gly Glu Asp Pro Arg Val Thr
Arg 325 330 335 Ala Lys Tyr Phe Ile Arg Asp Glu Phe Leu Arg Ile Ser
Thr Ala Ser 340 345 350 Gly Asp Gly Arg His Tyr Cys Tyr Pro His Phe
Thr Cys Ala Val Asp 355 360 365 Thr Glu Asn Ile Arg Arg Val Phe Asn
Asp Cys Arg Asp Ile Ile Gln 370 375 380 Arg Met His Leu Arg Gln Tyr
Glu Leu Leu 385 390 <210> SEQ ID NO 338 <211> LENGTH:
359 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 338 Met Thr Leu Glu Ser Ile Met
Ala Cys Cys Leu Ser Glu Glu Ala Lys 1 5 10 15 Glu Ala Arg Arg Ile
Asn Asp Glu Ile Glu Arg Gln Leu Arg Arg Asp 20 25 30 Lys Arg Asp
Ala Arg Arg Glu Leu Lys Leu Leu Leu Leu Gly Thr Gly 35 40 45 Glu
Ser Gly Lys Ser Thr Phe Ile Lys Gln Met Arg Ile Ile His Gly 50 55
60 Ser Gly Tyr Ser Asp Glu Asp Lys Arg Gly Phe Thr Lys Leu Val Tyr
65 70 75 80 Gln Asn Ile Phe Thr Ala Met Gln Ala Met Ile Arg Ala Met
Asp Thr 85 90 95 Leu Lys Ile Pro Tyr Lys Tyr Glu His Asn Lys Ala
His Ala Gln Leu 100 105 110 Val Arg Glu Val Asp Val Glu Lys Val Ser
Ala Phe Glu Asn Pro Tyr 115 120 125 Val Asp Ala Ile Lys Ser Leu Trp
Asn Asp Pro Gly Ile Gln Glu Cys 130 135 140 Tyr Asp Arg Arg Arg Glu
Tyr Gln Leu Ser Asp Ser Thr Lys Tyr Tyr 145 150 155 160 Leu Asn Asp
Leu Asp Arg Val Ala Asp Pro Ala Tyr Leu Pro Thr Gln 165 170 175 Gln
Asp Val Leu Arg Val Arg Val Pro Thr Thr Gly Ile Ile Glu Tyr 180 185
190 Pro Phe Asp Leu Gln Ser Val Ile Phe Arg Met Val Asp Val Gly Gly
195 200 205 Gln Arg Ser Glu Arg Arg Lys Trp Ile His Cys Phe Glu Asn
Val Thr 210 215 220 Ser Ile Met Phe Leu Val Ala Leu Ser Glu Tyr Asp
Gln Val Leu Val 225 230 235 240 Glu Ser Asp Asn Glu Asn Arg Met Glu
Glu Ser Lys Ala Leu Phe Arg 245 250 255 Thr Ile Ile Thr Tyr Pro Trp
Phe Gln Asn Ser Ser Val Ile Leu Phe 260 265 270 Leu Asn Lys Lys Asp
Leu Leu Glu Glu Lys Ile Met Tyr Ser His Leu 275 280 285 Val Asp Tyr
Phe Pro Glu Tyr Asp Gly Pro Gln Arg Asp Ala Gln Ala 290 295 300 Ala
Arg Glu Phe Ile Leu Lys Met Phe Val Asp Leu Asn Pro Asp Ser 305 310
315 320 Asp Lys Ile Ile Tyr Ser His Phe Thr Cys Ala Thr Asp Thr Glu
Asn 325 330 335 Ile Arg Phe Val Phe Ala Ala Val Lys Asp Thr Ile Leu
Gln Leu Asn 340 345 350 Leu Lys Glu Tyr Asn Leu Val 355 <210>
SEQ ID NO 339 <211> LENGTH: 355 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 339 Met Gly Cys Thr Val Ser Ala Glu Asp Lys
Ala Ala Ala Glu Arg Ser 1 5 10 15 Lys Met Ile Asp Lys Asn Leu Arg
Glu Asp Gly Glu Lys Ala Ala Arg 20 25 30 Glu Val Lys Leu Leu Leu
Leu Gly Ala Gly Glu Ser Gly Lys Ser Thr 35 40 45 Ile Val Lys Gln
Met Lys Ile Ile His Glu Asp Gly Tyr Ser Glu Glu 50 55 60 Glu Cys
Arg Gln Tyr Arg Ala Val Val Tyr Ser Asn Thr Ile Gln Ser 65 70 75 80
Ile Met Ala Ile Val Lys Ala Met Gly Asn Leu Gln Ile Asp Phe Ala 85
90 95 Asp Pro Ser Arg Ala Asp Asp Ala Arg Gln Leu Phe Ala Leu Ser
Cys 100 105 110 Thr Ala Glu Glu Gln Gly Val Leu Pro Asp Asp Leu Ser
Gly Val Ile 115 120 125 Arg Arg Leu Trp Ala Asp His Gly Val Gln Ala
Cys Phe Gly Arg Ser 130 135 140 Arg Glu Tyr Gln Leu Asn Asp Ser Ala
Ala Tyr Tyr Leu Asn Asp Leu 145 150 155 160 Glu Arg Ile Ala Gln Ser
Asp Tyr Ile Pro Thr Gln Gln Asp Val Leu 165 170 175 Arg Thr Arg Val
Lys Thr Thr Gly Ile Val Glu Thr His Phe Thr Phe 180 185 190 Lys Asp
Leu His Phe Lys Met Phe Asp Val Gly Gly Gln Arg Ser Glu 195 200 205
Arg Lys Lys Trp Ile His Cys Phe Glu Gly Val Thr Ala Ile Ile Phe 210
215 220 Cys Val Ala Leu Ser Ala Tyr Asp Leu Val Leu Ala Glu Asp Glu
Glu 225 230 235 240 Met Asn Arg Met His Glu Ser Met Lys Leu Phe Asp
Ser Ile Cys Asn 245 250 255 Asn Lys Trp Phe Thr Asp Thr Ser Ile Ile
Leu Phe Leu Asn Lys Lys 260 265 270 Asp Leu Phe Glu Glu Lys Ile Thr
His Ser Pro Leu Thr Ile Cys Phe 275 280 285 Pro Glu Tyr Thr Gly Ala
Asn Lys Tyr Asp Glu Ala Ala Ser Tyr Ile 290 295 300 Gln Ser Lys Phe
Glu Asp Leu Asn Lys Arg Lys Asp Thr Lys Glu Ile 305 310 315 320 Tyr
Thr His Phe Thr Cys Ala Thr Asp Thr Lys Asn Val Gln Phe Val 325 330
335 Phe Asp Ala Val Thr Asp Val Ile Ile Lys Asn Asn Leu Lys Asp Cys
340 345 350 Gly Leu Phe 355 <210> SEQ ID NO 340 <211>
LENGTH: 381 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic amino acid sequence <400> SEQUENCE: 340 Met Ser Gly
Val Val Arg Thr Leu Ser Arg Cys Leu Leu Pro Ala Glu 1 5 10 15 Ala
Gly Gly Ala Arg Glu Arg Arg Ala Gly Ser Gly Ala Arg Asp Ala 20 25
30 Glu Arg Glu Ala Arg Arg Arg Ser Arg Asp Ile Asp Ala Leu Leu Ala
35 40 45 Arg Glu Arg Arg Ala Val Arg Arg Leu Val Lys Ile Leu Leu
Leu Gly 50 55 60 Ala Gly Glu Ser Gly Lys Ser Thr Phe Leu Lys Gln
Met Arg Ile Ile 65 70 75 80 His Gly Arg Glu Phe Asp Gln Lys Ala Leu
Leu Glu Phe Arg Asp Thr 85 90 95 Ile Phe Asp Asn Ile Leu Lys Gly
Ser Arg Val Leu Val Asp Ala Arg 100 105 110 Asp Lys Leu Gly Ile Pro
Trp Gln Tyr Ser Glu Asn Glu Lys His Gly 115 120 125 Met Phe Leu Met
Ala Phe Glu Asn Lys Ala Gly Leu Pro Val Glu Pro 130 135 140 Ala Thr
Phe Gln Leu Tyr Val Pro Ala Leu Ser Ala Leu Trp Arg Asp 145 150 155
160 Ser Gly Ile Arg Glu Ala Phe Ser Arg Arg Ser Glu Phe Gln Leu Gly
165 170 175 Glu Ser Val Lys Tyr Phe Leu Asp Asn Leu Asp Arg Ile Gly
Gln Leu 180 185 190 Asn Tyr Phe Pro Ser Lys Gln Asp Ile Leu Leu Ala
Arg Lys Ala Thr 195 200 205 Lys Gly Ile Val Glu His Asp Phe Val Ile
Lys Lys Ile Pro Phe Lys 210 215 220 Met Val Asp Val Gly Gly Gln Arg
Ser Gln Arg Gln Lys Trp Phe Gln 225 230 235 240 Cys Phe Asp Gly Ile
Thr Ser Ile Leu Phe Met Val Ser Ser Ser Glu 245 250 255 Tyr Asp Gln
Val Leu Met Glu Asp Arg Arg Thr Asn Arg Leu Val Glu 260 265 270 Ser
Met Asn Ile Phe Glu Thr Ile Val Asn Asn Lys Leu Phe Phe Asn 275 280
285 Val Ser Ile Ile Leu Phe Leu Asn Lys Met Asp Leu Leu Val Glu Lys
290 295 300 Val Lys Thr Val Ser Ile Lys Lys His Phe Pro Asp Phe Arg
Gly Asp 305 310 315 320 Pro His Gln Leu Glu Asp Val Gln Arg Tyr Leu
Val Gln Cys Phe Asp 325 330 335 Arg Lys Arg Arg Asn Arg Ser Lys Pro
Leu Phe His His Phe Thr Thr 340 345 350 Ala Ile Asp Thr Glu Asn Val
Arg Phe Val Phe His Ala Val Lys Asp 355 360 365 Thr Ile Leu Gln Glu
Asn Leu Lys Asp Ile Met Leu Gln 370 375 380 <210> SEQ ID NO
341 <211> LENGTH: 50 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 341 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser
Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys
Ile Ile Glu Ala Arg 20 25 30 Leu Gln Ala Ser Val Asp Ala Glu Ser
Arg Asp Tyr Lys Asp Asp Asp 35 40 45 Asp Lys 50 <210> SEQ ID
NO 342 <211> LENGTH: 27 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 342 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser
Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys
20 25 <210> SEQ ID NO 343 <211> LENGTH: 50 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic amino acid
sequence <400> SEQUENCE: 343 Met Lys Ile Lys Thr Gly Ala Arg
Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala
Ser Ala Leu Ala Lys Ile Ile Glu Ala Arg 20 25 30 Leu Gln Ala Ser
Val Asp Ala Glu Ser Arg Asp Tyr Lys Asp Asp Asp 35 40 45 Asp Lys 50
<210> SEQ ID NO 344 <211> LENGTH: 50 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 344 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu
Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala
Leu Ala Lys Ile Ile Glu Ala Arg 20 25 30 Leu Gln Ala Ser Val Asp
Ala Glu Ser Arg Asp Tyr Lys Asp Asp Asp 35 40 45 Asp Lys 50
<210> SEQ ID NO 345 <211> LENGTH: 343 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 345 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu
Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala
Leu Ala Lys Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile Trp Ile Asn
Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val Gly Lys Lys
Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu 50 55 60 His Pro
Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly 65 70 75 80
Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85
90 95 Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe
Gln 100 105 110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr
Asn Gly Lys 115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu
Ser Leu Ile Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys
Thr Trp Glu Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys Glu Leu Lys
Ala Lys Gly Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu Gln Glu Pro
Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185 190 Tyr Ala
Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly 195 200 205
Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210
215 220 Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala
Glu 225 230 235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile
Asn Gly Pro Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr Ser Lys Val
Asn Tyr Gly Val Thr Val 260 265 270 Leu Pro Thr Phe Lys Gly Gln Pro
Ser Lys Pro Phe Val Gly Val Leu 275 280 285 Ser Ala Gly Ile Asn Ala
Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe Leu Glu Asn
Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310 315 320 Lys
Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 330
335 Leu Ala Lys Asp Pro Arg Ile 340 <210> SEQ ID NO 346
<211> LENGTH: 381 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic amino acid sequence <400> SEQUENCE:
346 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr
1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Glu Glu
Gly Lys 20 25 30 Leu Val Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn
Gly Leu Ala Glu 35 40 45 Val Gly Lys Lys Phe Glu Lys Asp Thr Gly
Ile Lys Val Thr Val Glu 50 55 60 His Pro Asp Lys Leu Glu Glu Lys
Phe Pro Gln Val Ala Ala Thr Gly 65 70 75 80 Asp Gly Pro Asp Ile Ile
Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85 90 95 Ala Gln Ser Gly
Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln 100 105 110 Asp Lys
Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys 115 120 125
Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn 130
135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro
Ala 145 150 155 160 Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala
Leu Met Phe Asn 165 170 175 Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu
Ile Ala Ala Asp Gly Gly 180 185 190 Tyr Ala Phe Lys Tyr Glu Asn Gly
Lys Tyr Asp Ile Lys Asp Val Gly 195 200 205 Val Asp Asn Ala Gly Ala
Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210 215 220 Ile Lys Asn Lys
His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu 225 230 235 240 Ala
Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp 245 250
255 Ala Trp Ser Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val
260 265 270 Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly
Val Leu 275 280 285 Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu
Leu Ala Lys Glu 290 295 300 Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu
Gly Leu Glu Ala Val Asn 305 310 315 320 Lys Asp Lys Pro Leu Gly Ala
Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 330 335 Leu Ala Lys Asp Pro
Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Ser 340 345 350 Ala Phe Trp
Tyr Ala Val Arg Ile Glu Ala Arg Leu Gln Ala Ser Val 355 360 365 Asp
Ala Glu Ser Arg Asp Tyr Lys Asp Asp Asp Asp Lys 370 375 380
<210> SEQ ID NO 347 <211> LENGTH: 381 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 347 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu
Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala
Leu Ala Lys Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile Trp Ile Asn
Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val Gly Lys Lys
Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu 50 55 60 His Pro
Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly 65 70 75 80
Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85
90 95 Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe
Gln 100 105 110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr
Asn Gly Lys 115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu
Ser Leu Ile Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys
Thr Trp Glu Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys Glu Leu Lys
Ala Lys Gly Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu Gln Glu Pro
Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185 190 Tyr Ala
Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly 195 200 205
Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210
215 220 Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala
Glu 225 230 235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile
Asn Gly Pro Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr Ser Lys Val
Asn Tyr Gly Val Thr Val 260 265 270 Leu Pro Thr Phe Lys Gly Gln Pro
Ser Lys Pro Phe Val Gly Val Leu 275 280 285 Ser Ala Gly Ile Asn Ala
Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe Leu Glu Asn
Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310 315 320 Lys
Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 330
335 Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Ser
340 345 350 Ala Phe Trp Tyr Ala Val Arg Ile Glu Ala Arg Leu Gln Ala
Ser Val 355 360 365 Asp Ala Glu Ser Arg Asp Tyr Lys Asp Asp Asp Asp
Lys 370 375 380 <210> SEQ ID NO 348 <211> LENGTH: 381
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 348 Met Lys Ile Lys Thr Gly Ala
Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser
Ala Ser Ala Leu Ala Lys Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile
Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val
Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu 50 55
60 His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly
65 70 75 80 Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly
Gly Tyr 85 90 95 Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp
Lys Ala Phe Gln 100 105 110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala
Val Arg Tyr Asn Gly Lys 115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val
Glu Ala Leu Ser Leu Ile Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn
Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys
Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu
Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185
190 Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly
195 200 205 Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val
Asp Leu 210 215 220 Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr
Ser Ile Ala Glu 225 230 235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala
Met Thr Ile Asn Gly Pro Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr
Ser Lys Val Asn Tyr Gly Val Thr Val 260 265 270 Leu Pro Thr Phe Lys
Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu 275 280 285 Ser Ala Gly
Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe
Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310
315 320 Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu
Glu 325 330 335 Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn
Ala Gln Ser 340 345 350 Ala Phe Trp Tyr Ala Val Arg Ile Glu Ala Arg
Leu Gln Ala Ser Val 355 360 365 Asp Ala Glu Ser Arg Asp Tyr Lys Asp
Asp Asp Asp Lys 370 375 380 <210> SEQ ID NO 349 <211>
LENGTH: 120 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic amino acid sequence <400> SEQUENCE: 349 Ala Ser Val
Asp Ala Glu Ser Arg Ser Asp Lys Ile Ile His Leu Thr 1 5 10 15 Asp
Asp Ser Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala Ile Leu 20 25
30 Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile Ala Pro
35 40 45 Ile Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr
Val Ala 50 55 60 Lys Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro
Lys Tyr Gly Ile 65 70 75 80 Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys
Asn Gly Glu Val Ala Ala 85 90 95 Thr Lys Val Gly Ala Leu Ser Lys
Gly Gln Leu Lys Glu Asn Leu Ala 100 105 110 Asp Tyr Lys Asp Asp Asp
Asp Lys 115 120 <210> SEQ ID NO 350 <211> LENGTH: 120
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 350 Ala Ser Val Asp Ala Glu Ser
Arg Ser Asp Lys Ile Ile His Leu Thr 1 5 10 15 Asp Asp Ser Phe Asp
Thr Asp Val Leu Lys Ala Asp Gly Ala Ile Leu 20 25 30 Val Asp Phe
Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile Ala Pro 35 40 45 Ile
Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala 50 55
60 Lys Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile
65 70 75 80 Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val
Ala Ala 85 90 95 Thr Lys Val Gly Ala Leu Ser Lys Gly Gln Leu Lys
Glu Asn Leu Ala 100 105 110 Asp Tyr Lys Asp Asp Asp Asp Lys 115 120
<210> SEQ ID NO 351 <211> LENGTH: 120 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 351 Ala Ser Val Asp Ala Glu Ser Arg Ser Asp
Lys Ile Ile His Leu Thr 1 5 10 15 Asp Asp Ser Phe Asp Thr Asp Val
Leu Lys Ala Asp Gly Ala Ile Leu 20 25 30 Val Asp Phe Trp Ala Glu
Trp Cys Gly Pro Cys Lys Met Ile Ala Pro 35 40 45 Ile Leu Asp Glu
Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala 50 55 60 Lys Leu
Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile 65 70 75 80
Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala 85
90 95 Thr Lys Val Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Asn Leu
Ala 100 105 110 Asp Tyr Lys Asp Asp Asp Asp Lys 115 120 <210>
SEQ ID NO 352 <211> LENGTH: 120 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 352 Ala Ser Val Asp Ala Glu Ser Arg Ser Asp
Lys Ile Ile His Leu Thr 1 5 10 15 Asp Asp Ser Phe Asp Thr Asp Val
Leu Lys Ala Asp Gly Ala Ile Leu 20 25 30 Val Asp Phe Trp Ala Glu
Trp Cys Gly Pro Cys Lys Met Ile Ala Pro 35 40 45 Ile Leu Asp Glu
Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala 50 55 60 Lys Leu
Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile 65 70 75 80
Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala 85
90 95 Thr Lys Val Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Asn Leu
Ala 100 105 110 Asp Tyr Lys Asp Asp Asp Asp Lys 115 120 <210>
SEQ ID NO 353 <211> LENGTH: 154 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 353 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu
Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala
Leu Ala Lys Ile Ile Glu Ala Arg 20 25 30 Leu Gln Ala Ser Val Asp
Ala Glu Ser Arg Ser Asp Lys Ile Ile His 35 40 45 Leu Thr Asp Asp
Ser Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala 50 55 60 Ile Leu
Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile 65 70 75 80
Ala Pro Ile Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr 85
90 95 Val Ala Lys Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys
Tyr 100 105 110 Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys Asn
Gly Glu Val 115 120 125 Ala Ala Thr Lys Val Gly Ala Leu Ser Lys Gly
Gln Leu Lys Glu Asn 130 135 140 Leu Ala Asp Tyr Lys Asp Asp Asp Asp
Lys 145 150 <210> SEQ ID NO 354 <211> LENGTH: 154
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 354 Met Lys Ile Lys Thr Gly Ala
Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser
Ala Ser Ala Leu Ala Lys Ile Ile Glu Ala Arg 20 25 30 Leu Gln Ala
Ser Val Asp Ala Glu Ser Arg Ser Asp Lys Ile Ile His 35 40 45 Leu
Thr Asp Asp Ser Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala 50 55
60 Ile Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile
65 70 75 80 Ala Pro Ile Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys
Leu Thr 85 90 95 Val Ala Lys Leu Asn Ile Asp Gln Asn Pro Gly Thr
Ala Pro Lys Tyr 100 105 110 Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu
Phe Lys Asn Gly Glu Val 115 120 125 Ala Ala Thr Lys Val Gly Ala Leu
Ser Lys Gly Gln Leu Lys Glu Asn 130 135 140 Leu Ala Asp Tyr Lys Asp
Asp Asp Asp Lys 145 150 <210> SEQ ID NO 355 <211>
LENGTH: 154 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic amino acid sequence <400> SEQUENCE: 355 Met Lys Ile
Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr
Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Ile Glu Ala Arg 20 25
30 Leu Gln Ala Ser Val Asp Ala Glu Ser Arg Ser Asp Lys Ile Ile His
35 40 45 Leu Thr Asp Asp Ser Phe Asp Thr Asp Val Leu Lys Ala Asp
Gly Ala 50 55 60 Ile Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro
Cys Lys Met Ile 65 70 75 80 Ala Pro Ile Leu Asp Glu Ile Ala Asp Glu
Tyr Gln Gly Lys Leu Thr 85 90 95 Val Ala Lys Leu Asn Ile Asp Gln
Asn Pro Gly Thr Ala Pro Lys Tyr 100 105 110 Gly Ile Arg Gly Ile Pro
Thr Leu Leu Leu Phe Lys Asn Gly Glu Val 115 120 125 Ala Ala Thr Lys
Val Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Asn 130 135 140 Leu Ala
Asp Tyr Lys Asp Asp Asp Asp Lys 145 150 <210> SEQ ID NO 356
<211> LENGTH: 154 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic amino acid sequence <400> SEQUENCE:
356 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr
1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Ile Glu
Ala Arg 20 25 30 Leu Gln Ala Ser Val Asp Ala Glu Ser Arg Ser Asp
Lys Ile Ile His 35 40 45 Leu Thr Asp Asp Ser Phe Asp Thr Asp Val
Leu Lys Ala Asp Gly Ala 50 55 60 Ile Leu Val Asp Phe Trp Ala Glu
Trp Cys Gly Pro Cys Lys Met Ile 65 70 75 80 Ala Pro Ile Leu Asp Glu
Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr 85 90 95 Val Ala Lys Leu
Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr 100 105 110 Gly Ile
Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val 115 120 125
Ala Ala Thr Lys Val Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Asn 130
135 140 Leu Ala Asp Tyr Lys Asp Asp Asp Asp Lys 145 150 <210>
SEQ ID NO 357 <211> LENGTH: 485 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 357 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu
Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala
Leu Ala Lys Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile Trp Ile Asn
Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val Gly Lys Lys
Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu 50 55 60 His Pro
Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly 65 70 75 80
Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85
90 95 Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe
Gln 100 105 110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr
Asn Gly Lys 115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu
Ser Leu Ile Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys
Thr Trp Glu Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys Glu Leu Lys
Ala Lys Gly Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu Gln Glu Pro
Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185 190 Tyr Ala
Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly 195 200 205
Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210
215 220 Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala
Glu 225 230 235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile
Asn Gly Pro Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr Ser Lys Val
Asn Tyr Gly Val Thr Val 260 265 270 Leu Pro Thr Phe Lys Gly Gln Pro
Ser Lys Pro Phe Val Gly Val Leu 275 280 285 Ser Ala Gly Ile Asn Ala
Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe Leu Glu Asn
Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310 315 320 Lys
Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 330
335 Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Ser
340 345 350 Ala Phe Trp Tyr Ala Val Arg Ile Glu Ala Arg Leu Gln Ala
Ser Val 355 360 365 Asp Ala Glu Ser Arg Ser Asp Lys Ile Ile His Leu
Thr Asp Asp Ser 370 375 380 Phe Asp Thr Asp Val Leu Lys Ala Asp Gly
Ala Ile Leu Val Asp Phe 385 390 395 400 Trp Ala Glu Trp Cys Gly Pro
Cys Lys Met Ile Ala Pro Ile Leu Asp 405 410 415 Glu Ile Ala Asp Glu
Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn 420 425 430 Ile Asp Gln
Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile 435 440 445 Pro
Thr Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val 450 455
460 Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Asn Leu Ala Asp Tyr Lys
465 470 475 480 Asp Asp Asp Asp Lys 485 <210> SEQ ID NO 358
<211> LENGTH: 485 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic amino acid sequence <400> SEQUENCE:
358 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr
1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Glu Glu
Gly Lys 20 25 30 Leu Val Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn
Gly Leu Ala Glu 35 40 45 Val Gly Lys Lys Phe Glu Lys Asp Thr Gly
Ile Lys Val Thr Val Glu 50 55 60 His Pro Asp Lys Leu Glu Glu Lys
Phe Pro Gln Val Ala Ala Thr Gly 65 70 75 80 Asp Gly Pro Asp Ile Ile
Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85 90 95 Ala Gln Ser Gly
Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln 100 105 110 Asp Lys
Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys 115 120 125
Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn 130
135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro
Ala 145 150 155 160 Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala
Leu Met Phe Asn 165 170 175 Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu
Ile Ala Ala Asp Gly Gly 180 185 190 Tyr Ala Phe Lys Tyr Glu Asn Gly
Lys Tyr Asp Ile Lys Asp Val Gly 195 200 205 Val Asp Asn Ala Gly Ala
Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210 215 220 Ile Lys Asn Lys
His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu 225 230 235 240 Ala
Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp 245 250
255 Ala Trp Ser Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val
260 265 270 Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly
Val Leu 275 280 285 Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu
Leu Ala Lys Glu 290 295 300 Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu
Gly Leu Glu Ala Val Asn 305 310 315 320 Lys Asp Lys Pro Leu Gly Ala
Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 330 335 Leu Ala Lys Asp Pro
Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Ser 340 345 350 Ala Phe Trp
Tyr Ala Val Arg Ile Glu Ala Arg Leu Gln Ala Ser Val 355 360 365 Asp
Ala Glu Ser Arg Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser 370 375
380 Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe
385 390 395 400 Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile Ala Pro
Ile Leu Asp 405 410 415 Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr
Val Ala Lys Leu Asn 420 425 430 Ile Asp Gln Asn Pro Gly Thr Ala Pro
Lys Tyr Gly Ile Arg Gly Ile 435 440 445 Pro Thr Leu Leu Leu Phe Lys
Asn Gly Glu Val Ala Ala Thr Lys Val 450 455 460 Gly Ala Leu Ser Lys
Gly Gln Leu Lys Glu Asn Leu Ala Asp Tyr Lys 465 470 475 480 Asp Asp
Asp Asp Lys 485 <210> SEQ ID NO 359 <211> LENGTH: 485
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 359 Met Lys Ile Lys Thr Gly Ala
Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser
Ala Ser Ala Leu Ala Lys Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile
Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val
Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu 50 55
60 His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly
65 70 75 80 Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly
Gly Tyr 85 90 95 Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp
Lys Ala Phe Gln 100 105 110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala
Val Arg Tyr Asn Gly Lys 115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val
Glu Ala Leu Ser Leu Ile Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn
Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys
Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu
Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185
190 Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly
195 200 205 Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val
Asp Leu 210 215 220 Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr
Ser Ile Ala Glu 225 230 235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala
Met Thr Ile Asn Gly Pro Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr
Ser Lys Val Asn Tyr Gly Val Thr Val 260 265 270 Leu Pro Thr Phe Lys
Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu 275 280 285 Ser Ala Gly
Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe
Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310
315 320 Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu
Glu 325 330 335 Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn
Ala Gln Ser 340 345 350 Ala Phe Trp Tyr Ala Val Arg Ile Glu Ala Arg
Leu Gln Ala Ser Val 355 360 365 Asp Ala Glu Ser Arg Ser Asp Lys Ile
Ile His Leu Thr Asp Asp Ser 370 375 380 Phe Asp Thr Asp Val Leu Lys
Ala Asp Gly Ala Ile Leu Val Asp Phe 385 390 395 400 Trp Ala Glu Trp
Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp 405 410 415 Glu Ile
Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn 420 425 430
Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile 435
440 445 Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys
Val 450 455 460 Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Asn Leu Ala
Asp Tyr Lys 465 470 475 480 Asp Asp Asp Asp Lys 485 <210> SEQ
ID NO 360 <211> LENGTH: 485 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 360 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser
Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys
Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile Trp Ile Asn Gly Asp Lys
Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val Gly Lys Lys Phe Glu Lys
Asp Thr Gly Ile Lys Val Thr Val Glu 50 55 60 His Pro Asp Lys Leu
Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly 65 70 75 80 Asp Gly Pro
Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85 90 95 Ala
Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln 100 105
110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys
115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile
Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu
Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys Glu Leu Lys Ala Lys Gly
Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu Gln Glu Pro Tyr Phe Thr
Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185 190 Tyr Ala Phe Lys Tyr
Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly 195 200 205 Val Asp Asn
Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210 215 220 Ile
Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu 225 230
235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile Asn Gly Pro
Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly
Val Thr Val 260 265 270 Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro
Phe Val Gly Val Leu 275 280 285 Ser Ala Gly Ile Asn Ala Ala Ser Pro
Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe Leu Glu Asn Tyr Leu Leu
Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310 315 320 Lys Asp Lys Pro
Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 330 335 Leu Ala
Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Ser 340 345 350
Ala Phe Trp Tyr Ala Val Arg Ile Glu Ala Arg Leu Gln Ala Ser Val 355
360 365 Asp Ala Glu Ser Arg Ser Asp Lys Ile Ile His Leu Thr Asp Asp
Ser 370 375 380 Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala Ile Leu
Val Asp Phe 385 390 395 400 Trp Ala Glu Trp Cys Gly Pro Cys Lys Met
Ile Ala Pro Ile Leu Asp 405 410 415 Glu Ile Ala Asp Glu Tyr Gln Gly
Lys Leu Thr Val Ala Lys Leu Asn 420 425 430 Ile Asp Gln Asn Pro Gly
Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile 435 440 445 Pro Thr Leu Leu
Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val 450 455 460 Gly Ala
Leu Ser Lys Gly Gln Leu Lys Glu Asn Leu Ala Asp Tyr Lys 465 470 475
480 Asp Asp Asp Asp Lys 485 <210> SEQ ID NO 361 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleic acid sequence <400> SEQUENCE: 361
atgcctaaag ccgctccctc a 21 <210> SEQ ID NO 362 <211>
LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleic acid sequence <400> SEQUENCE: 362
atgaagccgc tccct 15 <210> SEQ ID NO 363 <211> LENGTH:
15 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleic acid sequence <400> SEQUENCE: 363 augaagccgc ucccu 15
<210> SEQ ID NO 364 <211> LENGTH: 5 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 364 Met Pro Lys Ala Ala 1 5 <210> SEQ
ID NO 365 <211> LENGTH: 15 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic nucleic acid sequence <400>
SEQUENCE: 365 tgggttactc actca 15 <210> SEQ ID NO 366
<211> LENGTH: 15 <212> TYPE: RNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic nucleic acid sequence <400> SEQUENCE:
366 uggguuacuc acuca 15 <210> SEQ ID NO 367 <211>
LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic amino acid sequence <400> SEQUENCE: 367 Trp Val Thr
His Ser 1 5 <210> SEQ ID NO 368 <211> LENGTH: 15
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleic acid sequence <400> SEQUENCE: 368 atgaagccgc actca 15
<210> SEQ ID NO 369 <211> LENGTH: 15 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleic acid sequence
<400> SEQUENCE: 369 augaagccgc acuca 15 <210> SEQ ID NO
370 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 370 Met Pro Lys His Ser 1 5
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 370
<210> SEQ ID NO 1 <211> LENGTH: 1260 <212> TYPE:
DNA <213> ORGANISM: E. coli <400> SEQUENCE: 1
ccatacccgt ttttttgggc tagcaggagg aattcaccct gcagatgttt gaaccaatgg
60 aacttaccaa tgacgcggtg attaaagtca tcggcgtcgg cggcggcggc
ggtaatgctg 120 ttgaacacat ggtgcgcgag cgcattgaag gtgttgaatt
cttcgcggta aataccgatg 180 cacaagcgct gcgtaaaaca gcggttggac
agacgattca aatcggtagc ggtatcacca 240 aaggactggg cgctggcgct
aatccagaag ttggccgcaa tgcggctgat gaggatcgcg 300 atgcattgcg
tgcggcgctg gaaggtgcag acatggtctt tattgctgcg ggtatgggtg 360
gtggtaccgg tacaggtgca gcaccagtcg tcgctgaagt ggcaaaagat ttgggtatcc
420 tgaccgttgc tgtcgtcact aagcctttca actttgaagg caagaagcgt
atggcattcg 480 cggagcaggg gatcactgaa ctgtccaagc atgtggactc
tctgatcact atcccgaacg 540 acaaactgct gaaagttctg ggccgcggta
tctccctgct ggatgcgttt ggcgcagcga 600 acgatgtact gaaaggcgct
gtgcaaggta tcgctgaact gattactcgt ccgggtttga 660 tgaacgtgga
ctttgcagac gtacgcaccg taatgtctga gatgggctac gcaatgatgg 720
gttctggcgt ggcgagcggt gaagaccgtg cggaagaagc tgctgaaatg gctatctctt
780 ctccgctgct ggaagatatc gacctgtctg gcgcgcgcgg cgtgctggtt
aacatcacgg 840 cgggcttcga cctgcgtctg gatgagttcg aaacggtagg
taacaccatc cgtgcatttg 900 cttccgacaa cgcgactgtg gttatcggta
cttctcttga cccggatatg aatgacgagc 960 tgcgcgtaac cgttgttgcg
acaggtatcg gcatggacaa acgtcctgaa atcactctgg 1020 tgaccaataa
gcaggttcag cagccagtga tggatcgcta ccagcagcat gggatggctc 1080
cgctgaccca ggagcagaag ccggttgcta aagtcgtgaa tgacaatgcg ccgcaaactg
1140 cgaaagagcc ggattatctg gatatcccag cattcctgcg taagcaagct
gattaataat 1200 ctagaggatc cccgggtacc gagctcgaat tcgtaatcat
ggtcatagct gtttcctgtg 1260 <210> SEQ ID NO 2 <211>
LENGTH: 1260 <212> TYPE: DNA <213> ORGANISM: E. coli
<400> SEQUENCE: 2 gaattcaggc gctttttaga ctggtcgtaa tgaaattcag
caggatcaca ttctgcagat 60 gtttgaacca atggaactta ccaatgacgc
ggtgattaaa gtcatcggcg tcggcggcgg 120 cggcggtaat gctgttgaac
acatggtgcg cgagcgcatt gaaggtgttg aattcttcgc 180 ggtaaatacc
gatgcacaag cgctgcgtaa aacagcggtt ggacagacga ttcaaatcgg 240
tagcggtatc accaaaggac tgggcgctgg cgctaatcca gaagttggcc gcaatgcggc
300 tgatgaggat cgcgatgcat tgcgtgcggc gctggaaggt gcagacatgg
tctttattgc 360 tgcgggtatg ggtggtggta ccggtacagg tgcagcacca
gtcgtcgctg aagtggcaaa 420 agatttgggt atcctgaccg ttgctgtcgt
cactaagcct ttcaactttg aaggcaagaa 480 gcgtatggca ttcgcggagc
aggggatcac tgaactgtcc aagcatgtgg actctctgat 540 cactatcccg
aacgacaaac tgctgaaagt tctgggccgc ggtatctccc tgctggatgc 600
gtttggcgca gcgaacgatg tactgaaagg cgctgtgcaa ggtatcgctg aactgattac
660 tcgtccgggt ttgatgaacg tggactttgc agacgtacgc accgtaatgt
ctgagatggg 720 ctacgcaatg atgggttctg gcgtggcgag cggtgaagac
cgtgcggaag aagctgctga 780 aatggctatc tcttctccgc tgctggaaga
tatcgacctg tctggcgcgc gcggcgtgct 840 ggttaacatc acggcgggct
tcgacctgcg tctggatgag ttcgaaacgg taggtaacac 900 catccgtgca
tttgcttccg acaacgcgac tgtggttatc ggtacttctc ttgacccgga 960
tatgaatgac gagctgcgcg taaccgttgt tgcgacaggt atcggcatgg acaaacgtcc
1020 tgaaatcact ctggtgacca ataagcaggt tcagcagcca gtgatggatc
gctaccagca 1080 gcatgggatg gctccgctga cccaggagca gaagccggtt
gctaaagtcg tgaatgacaa 1140 tgcgccgcaa actgcgaaag agccggatta
tctggatatc ccagcattcc tgcgtaagca 1200 agctgattaa taatctagag
gatccccggg taccgagctc gaattcgtaa tcatggtcat 1260 <210> SEQ ID
NO 3 <211> LENGTH: 2544 <212> TYPE: DNA <213>
ORGANISM: E. coli <400> SEQUENCE: 3 aagcctgcat tgcggcgctt
cagtctccgc tgcatactgt cccgttacca attatgacaa 60 cttgacggct
acatcattca ctttttcttc acaaccggca cggaactcgc tcgggctggc 120
cccggtgcat tttttaaata cccgcgagaa atagagttga tcgtcaaaac caacattgcg
180 accgacggtg gcgataggca tccgggtggt gctcaaaagc agcttcgcct
ggctgatacg 240 ttggtcctcg cgccagctta agacgctaat ccctaactgc
tggcggaaaa gatgtgacag 300 acgcgacggc gacaagcaaa catgctgtgc
gacgctggcg atatcaaaat tgctgtctgc 360 caggtgatcg ctgatgtact
gacaagcctc gcgtacccga ttatccatcg gtggatggag 420 cgactcgtta
atcgcttcca tgcgccgcag taacaattgc tcaagcagat ttatcgccag 480
cagctccgaa tagcgccctt ccccttgccc ggcgttaatg atttgcccaa acaggtcgct
540 gaaatgcggc tggtgcgctt catccgggcg aaagaacccc gtattggcaa
atattgacgg 600 ccagttaagc cattcatgcc agtaggcgcg cggacgaaag
taaacccact ggtgatacca 660 ttcgcgagcc tccggatgac gaccgtagtg
atgaatctct cctggcggga acagcaaaat 720 atcacccggt cggcaaacaa
attctcgtcc ctgatttttc accaccccct gaccgcgaat 780 ggtgagattg
agaatataac ctttcattcc cagcggtcgg tcgataaaaa aatcgagata 840
accgttggcc tcaatcggcg ttaaacccgc caccagatgg gcattaaacg agtatcccgg
900 cagcagggga tcattttgcg cttcagccat acttttcata ctcccgccat
tcagagaaga 960 aaccaattgt ccatattgca tcagacattg ccgtcactgc
gtcttttact ggctcttctc 1020 gctaaccaaa ccggtaaccc cgcttattaa
aagcattctg taacaaagcg ggaccaaagc 1080 catgacaaaa acgcgtaaca
aaagtgtcta taatcacggc agaaaagtcc acattgatta 1140 tttgcacggc
gtcacacttt gctatgccat agcattttta tccataagat tagcggatcc 1200
tacctgacgc tttttatcgc aactctctac tgtttctcca tacccgtttt tttgggctag
1260 caggaggaat tcaccctgca gatgtttgaa ccaatggaac ttaccaatga
cgcggtgatt 1320 aaagtcatcg gcgtcggcgg cggcggcggt aatgctgttg
aacacatggt gcgcgagcgc 1380 attgaaggtg ttgaattctt cgcggtaaat
accgatgcac aagcgctgcg taaaacagcg 1440 gttggacaga cgattcaaat
cggtagcggt atcaccaaag gactgggcgc tggcgctaat 1500 ccagaagttg
gccgcaatgc ggctgatgag gatcgcgatg cattgcgtgc ggcgctggaa 1560
ggtgcagaca tggtctttat tgctgcgggt atgggtggtg gtaccggtac aggtgcagca
1620 ccagtcgtcg ctgaagtggc aaaagatttg ggtatcctga ccgttgctgt
cgtcactaag 1680 cctttcaact ttgaaggcaa gaagcgtatg gcattcgcgg
agcaggggat cactgaactg 1740 tccaagcatg tggactctct gatcactatc
ccgaacgaca aactgctgaa agttctgggc 1800 cgcggtatct ccctgctgga
tgcgtttggc gcagcgaacg atgtactgaa aggcgctgtg 1860 caaggtatcg
ctgaactgat tactcgtccg ggtttgatga acgtggactt tgcagacgta 1920
cgcaccgtaa tgtctgagat gggctacgca atgatgggtt ctggcgtggc gagcggtgaa
1980 gaccgtgcgg aagaagctgc tgaaatggct atctcttctc cgctgctgga
agatatcgac 2040 ctgtctggcg cgcgcggcgt gctggttaac atcacggcgg
gcttcgacct gcgtctggat 2100 gagttcgaaa cggtaggtaa caccatccgt
gcatttgctt ccgacaacgc gactgtggtt 2160 atcggtactt ctcttgaccc
ggatatgaat gacgagctgc gcgtaaccgt tgttgcgaca 2220 ggtatcggca
tggacaaacg tcctgaaatc actctggtga ccaataagca ggttcagcag 2280
ccagtgatgg atcgctacca gcagcatggg atggctccgc tgacccagga gcagaagccg
2340 gttgctaaag tcgtgaatga caatgcgccg caaactgcga aagagccgga
ttatctggat 2400 atcccagcat tcctgcgtaa gcaagctgat taataatcta
gaggcgttac caattatgac 2460 aacttgacgg gaagttccta tactttctag
agaataggaa cttcccaaag ccagtatcaa 2520 ctcagacaaa ggcaaagcat cttg
2544 <210> SEQ ID NO 4 <211> LENGTH: 3350 <212>
TYPE: DNA <213> ORGANISM: E. coli <400> SEQUENCE: 4
aagcctgcat tgcggcgctt cagtctccgc tgcatactgt ccttaatctt tctgcgaatt
60 gagatgacgc cactggctgg gcgtcatccc ggtttcccgg gtaaacacca
ccgaaaaata 120 gttactatct tcaaagccac attcggtcga aatatcactg
attaacaggc ggctatgctg 180 gagaagatat tgcgcatgac acactctgac
ctgtcgcaga tattgattga tggtcattcc 240 agtctgctgg cgaaattgct
gacgcaaaac gcgctcactg cacgatgcct catcacaaaa 300 tttatccagc
gcaaagggac ttttcaggct agccgccagc cgggtaatca gcttatccag 360
caacgtttcg ctggatgttg gcggcaacga atcactggtg taacgatggc gattcagcaa
420 catcaccaac tgcccgaaca gcaactcagc catttcgtta gcaaacggca
catgctgact 480 actttcatgc tcaagctgac cgataacctg ccgcgcctgc
gccatcccca tgctacctaa 540 gcgccagtgt ggttgccctg cgctggcgtt
aaatcccgga atcgccccct gccagtcaag 600 attcagcttc agacgctccg
ggcaataaat aatattctgc aaaaccagat cgttaacgga 660 agcgtaggag
tgtttatcgt cagcatgaat gtaaaagaga tcgccacggg taatgcgata 720
agggcgatcg ttgagtacat gcaggccatt accgcgccag acaatcacca gctcacaaaa
780 atcatgtgta tgttcagcaa agacatcttg cggataacgg tcagccacag
cgactgcctg 840 ctggtcgctg gcaaaaaaat catctttgag aagttttaac
tgatgcgcca ccgtggctac 900 ctcggccaga gaacgaagtt gattattcgc
aatatggcgt acaaatacgt tgagaagatt 960 cgcgttattg cagaaagcca
tcccgtccct ggcgaatatc acgcggtgac cagttaaact 1020 ctcggcgaaa
aagcgtcgaa aagtggttac tgtcgctgaa tccacagcga taggcgatgt 1080
cagtaacgct ggcctcgctg tggcgtagca gatgtcgggc tttcatcagt cgcaggcggt
1140 tcaggtatcg ctgaggcgtc agtcccgttt gctgcttaag ctgccgatgt
agcgtacgca 1200 gtgaaagaga aaattgatcc gccacggcat cccaattcac
ctcatcggca aaatggtcct 1260 ccagccaggc cagaagcaag ttgagacgtg
atgcgctgtt ttccaggttc tcctgcaaac 1320
tgcttttacg cagcaagagc agtaattgca taaacaagat ctcgcgactg gcggtcgagg
1380 gtaaatcatt ttccccttcc tgctgttcca tctgtgcaac cagctgtcgc
acctgctgca 1440 atacgctgtg gttaacgcgc cagtgagacg gatactgccc
atccagctct tgtggcagca 1500 actgattcag cccggcgaga aactgaaatc
gatccggcga gcgatacagc acattggtca 1560 gacacagatt atcggtatgt
tcatacagat gccgatcatg atcgcgtacg aaacagaccg 1620 tgccaccggt
gatggtatag ggctgcccat taaacacatg aatacccgtg ccatgttcga 1680
caatcacaat ttcatgaaaa tcatgatgat gttcaggaaa atccgcctgc gggagccggg
1740 gttctatcgc cacggacgcg ttaccagacg gaaaaaaatc cacactatgt
aatacggtca 1800 tactggcctc ctgatgtcgt caacacggcg aaatagtaat
cacgaggtca ggttcttacc 1860 ttaaattttc gacggaaaac cacgtaaaaa
acgtcgattt ttcaagatac agcgtgaatt 1920 ttcaggaaat gcggtgagca
tcacatcacc acaattcagc aaattgtgaa catcatcacg 1980 ttcatctttc
cctggttgcc aatggcccat tttcctgtca gtaacgagaa ggtcgcgaat 2040
tcaggcgctt tttagactgg tcgtaatgaa attcagcagg atcacatatg tttgaaccaa
2100 tggaacttac caatgacgcg gtgattaaag tcatcggcgt cggcggcggc
ggcggtaatg 2160 ctgttgaaca catggtgcgc gagcgcattg aaggtgttga
attcttcgcg gtaaataccg 2220 atgcacaagc gctgcgtaaa acagcggttg
gacagacgat tcaaatcggt agcggtatca 2280 ccaaaggact gggcgctggc
gctaatccag aagttggccg caatgcggct gatgaggatc 2340 gcgatgcatt
gcgtgcggcg ctggaaggtg cagacatggt ctttattgct gcgggtatgg 2400
gtggtggtac cggtacaggt gcagcaccag tcgtcgctga agtggcaaaa gatttgggta
2460 tcctgaccgt tgctgtcgtc actaagcctt tcaactttga aggcaagaag
cgtatggcat 2520 tcgcggagca ggggatcact gaactgtcca agcatgtgga
ctctctgatc actatcccga 2580 acgacaaact gctgaaagtt ctgggccgcg
gtatctccct gctggatgcg tttggcgcag 2640 cgaacgatgt actgaaaggc
gctgtgcaag gtatcgctga actgattact cgtccgggtt 2700 tgatgaacgt
ggactttgca gacgtacgca ccgtaatgtc tgagatgggc tacgcaatga 2760
tgggttctgg cgtggcgagc ggtgaagacc gtgcggaaga agctgctgaa atggctatct
2820 cttctccgct gctggaagat atcgacctgt ctggcgcgcg cggcgtgctg
gttaacatca 2880 cggcgggctt cgacctgcgt ctggatgagt tcgaaacggt
aggtaacacc atccgtgcat 2940 ttgcttccga caacgcgact gtggttatcg
gtacttctct tgacccggat atgaatgacg 3000 agctgcgcgt aaccgttgtt
gcgacaggta tcggcatgga caaacgtcct gaaatcactc 3060 tggtgaccaa
taagcaggtt cagcagccag tgatggatcg ctaccagcag catgggatgg 3120
ctccgctgac ccaggagcag aagccggttg ctaaagtcgt gaatgacaat gcgccgcaaa
3180 ctgcgaaaga gccggattat ctggatatcc cagcattcct gcgtaagcaa
gctgattaat 3240 aatctagagg cgttaccaat tatgacaact tgacgggaag
ttcctatact ttctagagaa 3300 taggaacttc ccaaagccag tatcaactca
gacaaaggca aagcatcttg 3350 <210> SEQ ID NO 5 <211>
LENGTH: 2280 <212> TYPE: DNA <213> ORGANISM: E. coli
<400> SEQUENCE: 5 aagcctgcat tgcggcgctt cagtctccgc tgcatactgt
ccttaataaa gtgagtcgat 60 attgtctttg ttgaccagta ataccttatg
gaaacggata attcgcttat ccatatctac 120 gtcggcctta cccagattct
gcatttctaa tccaggcttg atctcttcac ccttcagcaa 180 cgtgctggcg
acggctgcga gtgcgtaacc tgcagaggcc ggatcgtaag taatcccttc 240
ggtgatatca ccacttttaa tcagtgatgc cgcctgtgaa gggatcatca tgccatagac
300 tgcgacttta tttttcgccc gtttctcttt caccgcacgt cccgcgccaa
tcggaccgtt 360 tgaaccaaag gagacaaccg ctttcaagtc aggataggtt
ttcatcaggt ccagtgtagt 420 acgacgtgag acatccacac tctcggcaac
cggcatgcgg cgggtaactt catgcatatc 480 cgggtaatgc tctttctggt
atttcaccag caagtcagcc cataagttat gctgcggcac 540 ggtcaaacta
cccacgtaaa tcacatagcc gcccttgcca cccatgcgtt tcgccatatg 600
ctcaacatat tcagcggcaa atttttcgtt atcaatgatt tcgatatccc agttagcact
660 tggctgaccg ggggattcgt tggtcagaac cacaattccg gcatctcgcg
cttttttgaa 720 taccggttcc agcacgttgg catcgtttgg cacgatagta
attgcattaa ccttacgggc 780 gattaaatcc tcaataattt taacttgttg
cggagcatca gtacttgaag gccccacctg 840 tgaggcatta acaccaaagg
ctttacccgc ctcaaccaca ccttcgccca tgcgattaaa 900 ccacggcata
ccatcgactt tagaaatatt caccacgact ttttccgctg cctggagcgg 960
cgcagaaatt agcgcagcgc ctaataacag cgaagacacc atattgataa caaaacgttt
1020 attcatcata tggaacttac caatgacgcg gtgattaaag tcatcggcgt
cggcggcggc 1080 ggcggtaatg ctgttgaaca catggtgcgc gagcgcattg
aaggtgttga attcttcgcg 1140 gtaaataccg atgcacaagc gctgcgtaaa
acagcggttg gacagacgat tcaaatcggt 1200 agcggtatca ccaaaggact
gggcgctggc gctaatccag aagttggccg caatgcggct 1260 gatgaggatc
gcgatgcatt gcgtgcggcg ctggaaggtg cagacatggt ctttattgct 1320
gcgggtatgg gtggtggtac cggtacaggt gcagcaccag tcgtcgctga agtggcaaaa
1380 gatttgggta tcctgaccgt tgctgtcgtc actaagcctt tcaactttga
aggcaagaag 1440 cgtatggcat tcgcggagca ggggatcact gaactgtcca
agcatgtgga ctctctgatc 1500 actatcccga acgacaaact gctgaaagtt
ctgggccgcg gtatctccct gctggatgcg 1560 tttggcgcag cgaacgatgt
actgaaaggc gctgtgcaag gtatcgctga actgattact 1620 cgtccgggtt
tgatgaacgt ggactttgca gacgtacgca ccgtaatgtc tgagatgggc 1680
tacgcaatga tgggttctgg cgtggcgagc ggtgaagacc gtgcggaaga agctgctgaa
1740 atggctatct cttctccgct gctggaagat atcgacctgt ctggcgcgcg
cggcgtgctg 1800 gttaacatca cggcgggctt cgacctgcgt ctggatgagt
tcgaaacggt aggtaacacc 1860 atccgtgcat ttgcttccga caacgcgact
gtggttatcg gtacttctct tgacccggat 1920 atgaatgacg agctgcgcgt
aaccgttgtt gcgacaggta tcggcatgga caaacgtcct 1980 gaaatcactc
tggtgaccaa taagcaggtt cagcagccag tgatggatcg ctaccagcag 2040
catgggatgg ctccgctgac ccaggagcag aagccggttg ctaaagtcgt gaatgacaat
2100 gcgccgcaaa ctgcgaaaga gccggattat ctggatatcc cagcattcct
gcgtaagcaa 2160 gctgattaat aatctagagg cgttaccaat tatgacaact
tgacgggaag ttcctattct 2220 ctagaaagta taggaacttc ccaaagccag
tatcaactca gacaaaggca aagcatcttg 2280 <210> SEQ ID NO 6
<211> LENGTH: 4728 <212> TYPE: DNA <213>
ORGANISM: E. coli <400> SEQUENCE: 6 tcgcgcgttt cggtgatgac
ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct
gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc
ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg
caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt
aaaacgacgg ccagtgccaa gcttaattaa tctttctgcg 420 aattgagatg
acgccactgg ctgggcgtca tcccggtttc ccgggtaaac accaccgaaa 480
aatagttact atcttcaaag ccacattcgg tcgaaatatc actgattaac aggcggctat
540 gctggagaag atattgcgca tgacacactc tgacctgtcg cagatattga
ttgatggtca 600 ttccagtctg ctggcgaaat tgctgacgca aaacgcgctc
actgcacgat gcctcatcac 660 aaaatttatc cagcgcaaag ggacttttca
ggctagccgc cagccgggta atcagcttat 720 ccagcaacgt ttcgctggat
gttggcggca acgaatcact ggtgtaacga tggcgattca 780 gcaacatcac
caactgcccg aacagcaact cagccatttc gttagcaaac ggcacatgct 840
gactactttc atgctcaagc tgaccgataa cctgccgcgc ctgcgccatc cccatgctac
900 ctaagcgcca gtgtggttgc cctgcgctgg cgttaaatcc cggaatcgcc
ccctgccagt 960 caagattcag cttcagacgc tccgggcaat aaataatatt
ctgcaaaacc agatcgttaa 1020 cggaagcgta ggagtgttta tcgtcagcat
gaatgtaaaa gagatcgcca cgggtaatgc 1080 gataagggcg atcgttgagt
acatgcaggc cattaccgcg ccagacaatc accagctcac 1140 aaaaatcatg
tgtatgttca gcaaagacat cttgcggata acggtcagcc acagcgactg 1200
cctgctggtc gctggcaaaa aaatcatctt tgagaagttt taactgatgc gccaccgtgg
1260 ctacctcggc cagagaacga agttgattat tcgcaatatg gcgtacaaat
acgttgagaa 1320 gattcgcgtt attgcagaaa gccatcccgt ccctggcgaa
tatcacgcgg tgaccagtta 1380 aactctcggc gaaaaagcgt cgaaaagtgg
ttactgtcgc tgaatccaca gcgataggcg 1440 atgtcagtaa cgctggcctc
gctgtggcgt agcagatgtc gggctttcat cagtcgcagg 1500 cggttcaggt
atcgctgagg cgtcagtccc gtttgctgct taagctgccg atgtagcgta 1560
cgcagtgaaa gagaaaattg atccgccacg gcatcccaat tcacctcatc ggcaaaatgg
1620 tcctccagcc aggccagaag caagttgaga cgtgatgcgc tgttttccag
gttctcctgc 1680 aaactgcttt tacgcagcaa gagcagtaat tgcataaaca
agatctcgcg actggcggtc 1740 gagggtaaat cattttcccc ttcctgctgt
tccatctgtg caaccagctg tcgcacctgc 1800 tgcaatacgc tgtggttaac
gcgccagtga gacggatact gcccatccag ctcttgtggc 1860 agcaactgat
tcagcccggc gagaaactga aatcgatccg gcgagcgata cagcacattg 1920
gtcagacaca gattatcggt atgttcatac agatgccgat catgatcgcg tacgaaacag
1980 accgtgccac cggtgatggt atagggctgc ccattaaaca catgaatacc
cgtgccatgt 2040 tcgacaatca caatttcatg aaaatcatga tgatgttcag
gaaaatccgc ctgcgggagc 2100 cggggttcta tcgccacgga cgcgttacca
gacggaaaaa aatccacact atgtaatacg 2160 gtcatactgg cctcctgatg
tcgtcaacac ggcgaaatag taatcacgag gtcaggttct 2220 taccttaaat
tttcgacgga aaaccacgta aaaaacgtcg atttttcaag atacagcgtg 2280
aattttcagg aaatgcggtg agcatcacat caccacaatt cagcaaattg tgaacatcat
2340 cacgttcatc tttccctggt tgccaatggc ccattttcct gtcagtaacg
agaaggtcgc 2400 gaattcaggc gctttttaga ctggtcgtaa tgaaattcag
caggatcaca ttctgcaggt 2460 cgactctaga ggatccccgg gtaccgagct
cgaattcgta atcatggtca tagctgtttc 2520 ctgtgtgaaa ttgttatccg
ctcacaattc cacacaacat acgagccgga agcataaagt 2580 gtaaagcctg
gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 2640
ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
2700
ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
2760 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
cggttatcca 2820 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
aggccagcaa aaggccagga 2880 accgtaaaaa ggccgcgttg ctggcgtttt
tccataggct ccgcccccct gacgagcatc 2940 acaaaaatcg acgctcaagt
cagaggtggc gaaacccgac aggactataa agataccagg 3000 cgtttccccc
tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 3060
acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
3120 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
ccccccgttc 3180 agcccgaccg ctgcgcctta tccggtaact atcgtcttga
gtccaacccg gtaagacacg 3240 acttatcgcc actggcagca gccactggta
acaggattag cagagcgagg tatgtaggcg 3300 gtgctacaga gttcttgaag
tggtggccta actacggcta cactagaagg acagtatttg 3360 gtatctgcgc
tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 3420
gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
3480 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
gctcagtgga 3540 acgaaaactc acgttaaggg attttggtca tgagattatc
aaaaaggatc ttcacctaga 3600 tccttttaaa ttaaaaatga agttttaaat
caatctaaag tatatatgag taaacttggt 3660 ctgacagtta ccaatgctta
atcagtgagg cacctatctc agcgatctgt ctatttcgtt 3720 catccatagt
tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat 3780
ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
3840 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
ttatccgcct 3900 ccatccagtc tattaattgt tgccgggaag ctagagtaag
tagttcgcca gttaatagtt 3960 tgcgcaacgt tgttgccatt gctacaggca
tcgtggtgtc acgctcgtcg tttggtatgg 4020 cttcattcag ctccggttcc
caacgatcaa ggcgagttac atgatccccc atgttgtgca 4080 aaaaagcggt
tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt 4140
tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
4200 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
atgcggcgac 4260 cgagttgctc ttgcccggcg tcaatacggg ataataccgc
gccacatagc agaactttaa 4320 aagtgctcat cattggaaaa cgttcttcgg
ggcgaaaact ctcaaggatc ttaccgctgt 4380 tgagatccag ttcgatgtaa
cccactcgtg cacccaactg atcttcagca tcttttactt 4440 tcaccagcgt
ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa 4500
gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
4560 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
aataaacaaa 4620 taggggttcc gcgcacattt ccccgaaaag tgccacctga
cgtctaagaa accattatta 4680 tcatgacatt aacctataaa aataggcgta
tcacgaggcc ctttcgtc 4728 <210> SEQ ID NO 7 <211>
LENGTH: 1440 <212> TYPE: DNA <213> ORGANISM: E. coli
<400> SEQUENCE: 7 gaattcaggc gctttttaga ctggtcgtaa tgaaattcag
caggatcaca ttctgcagat 60 gcctgttctg gaaaaccggg ctgctcaggg
cgatattact gcacccggcg gtgctcgccg 120 tttaacgggt gatcagactg
ccgctctgcg tgattctctt agcgataaac ctgcaaaaaa 180 tattattttg
ctgattggcg atgggatggg ggactcggaa attactgccg cacgtaatta 240
tgccgaaggt gcgggcggct tttttaaagg tatagatgcc ttaccgctta ccgggcaata
300 cactcactat gcgctgaata aaaaaaccgg caaaccggac tacgtcaccg
actcggctgc 360 atcagcaacc gcctggtcaa ccggtgtcaa aacctataac
ggcgcgctgg gcgtcgatat 420 tcacgaaaaa gatcacccaa cgattctgga
aatggcaaaa gccgcaggtc tggcgaccgg 480 taacgtttct accgcagagt
tgcaggatgc cacgcccgct gcgctggtgg cacatgtgac 540 ctcgcgcaaa
tgctacggtc cgagcgcgac cagtgaaaaa tgtccgggta acgctctgga 600
aaaaggcgga aaaggatcga ttaccgaaca gctgcttaac gctcgtgccg acgttacgct
660 tggcggcggc gcaaaaacct ttgctgaaac ggcaaccgct ggtgaatggc
agggaaaaac 720 gctgcgtgaa caggcacagg cgcgtggtta tcagttggtg
agcgatgctg cctcactgaa 780 ttcggtgacg gaagcgaatc agcaaaaacc
cctgcttggc ctgtttgctg acggcaatat 840 gccagtgcgc tggctaggac
cgaaagcaac gtaccatggc aatatcgata agcccgcagt 900 cacctgtacg
ccaaatccgc aacgtaatga cagtgtacca accctggcgc agatgaccga 960
caaagccatt gaattgttga gtaaaaatga gaaaggcttt ttcctgcaag ttgaaggtgc
1020 gtcaatcgat aaacaggatc atgctgcgaa tccttgtggg caaattggcg
agacggtcga 1080 tctcgatgaa gccgtacaac gggcgctgga attcgctaaa
aaggagggta acacgctggt 1140 catagtcacc gctgatcacg cccacgccag
ccagattgtt gcgccggata ccaaagctcc 1200 gggcctcacc caggcgctaa
ataccaaaga tggcgcagtg atggtgatga gttacgggaa 1260 ctccgaagag
gattcacaag aacataccgg cagtcagttg cgtattgcgg cgtatggccc 1320
gcatgccgcc aatgttgttg gactgaccga ccagaccgat ctcttctaca ccatgaaagc
1380 cgctctgggg ctgaaataat aatctagagg atccccgggt accgagctcg
aattcgtaat 1440 <210> SEQ ID NO 8 <211> LENGTH: 1560
<212> TYPE: DNA <213> ORGANISM: E. coli <400>
SEQUENCE: 8 gaattcaggc gctttttaga ctggtcgtaa tgaaattcag caggatcaca
ttctgcagat 60 gtcacggccg agacttatag tcgctttgtt tttatttttt
aatgtatttg tacatggaga 120 aaataaagtg aaacaaagca ctattgcact
ggcactctta ccgttactgt ttacccctgt 180 gacaaaagcc cggacaccag
aaatgcctgt tctggaaaac cgggctgctc agggcgatat 240 tactgcaccc
ggcggtgctc gccgtttaac gggtgatcag actgccgctc tgcgtgattc 300
tcttagcgat aaacctgcaa aaaatattat tttgctgatt ggcgatggga tgggggactc
360 ggaaattact gccgcacgta attatgccga aggtgcgggc ggctttttta
aaggtataga 420 tgccttaccg cttaccgggc aatacactca ctatgcgctg
aataaaaaaa ccggcaaacc 480 ggactacgtc accgactcgg ctgcatcagc
aaccgcctgg tcaaccggtg tcaaaaccta 540 taacggcgcg ctgggcgtcg
atattcacga aaaagatcac ccaacgattc tggaaatggc 600 aaaagccgca
ggtctggcga ccggtaacgt ttctaccgca gagttgcagg atgccacgcc 660
cgctgcgctg gtggcacatg tgacctcgcg caaatgctac ggtccgagcg cgaccagtga
720 aaaatgtccg ggtaacgctc tggaaaaagg cggaaaagga tcgattaccg
aacagctgct 780 taacgctcgt gccgacgtta cgcttggcgg cggcgcaaaa
acctttgctg aaacggcaac 840 cgctggtgaa tggcagggaa aaacgctgcg
tgaacaggca caggcgcgtg gttatcagtt 900 ggtgagcgat gctgcctcac
tgaattcggt gacggaagcg aatcagcaaa aacccctgct 960 tggcctgttt
gctgacggca atatgccagt gcgctggcta ggaccgaaag caacgtacca 1020
tggcaatatc gataagcccg cagtcacctg tacgccaaat ccgcaacgta atgacagtgt
1080 accaaccctg gcgcagatga ccgacaaagc cattgaattg ttgagtaaaa
atgagaaagg 1140 ctttttcctg caagttgaag gtgcgtcaat cgataaacag
gatcatgctg cgaatccttg 1200 tgggcaaatt ggcgagacgg tcgatctcga
tgaagccgta caacgggcgc tggaattcgc 1260 taaaaaggag ggtaacacgc
tggtcatagt caccgctgat cacgcccacg ccagccagat 1320 tgttgcgccg
gataccaaag ctccgggcct cacccaggcg ctaaatacca aagatggcgc 1380
agtgatggtg atgagttacg ggaactccga agaggattca caagaacata ccggcagtca
1440 gttgcgtatt gcggcgtatg gcccgcatgc cgccaatgtt gttggactga
ccgaccagac 1500 cgatctcttc tacaccatga aagccgctct ggggctgaaa
taatctagag gatccccggg 1560 <210> SEQ ID NO 9 <211>
LENGTH: 1500 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Nucleic Acid Sequence <400> SEQUENCE: 9 gaattcaggc
gctttttaga ctggtcgtaa tgaaattcag caggatcaca ttctgcagat 60
gaacttgggg aatcgactgt ttattctgat agcggtctta cttcccctcg cagtattact
120 gctcatgcct gttctggaaa accgggctgc tcagggcgat attactgcac
ccggcggtgc 180 tcgccgttta acgggtgatc agactgccgc tctgcgtgat
tctcttagcg ataaacctgc 240 aaaaaatatt attttgctga ttggcgatgg
gatgggggac tcggaaatta ctgccgcacg 300 taattatgcc gaaggtgcgg
gcggcttttt taaaggtata gatgccttac cgcttaccgg 360 gcaatacact
cactatgcgc tgaataaaaa aaccggcaaa ccggactacg tcaccgactc 420
ggctgcatca gcaaccgcct ggtcaaccgg tgtcaaaacc tataacggcg cgctgggcgt
480 cgatattcac gaaaaagatc acccaacgat tctggaaatg gcaaaagccg
caggtctggc 540 gaccggtaac gtttctaccg cagagttgca ggatgccacg
cccgctgcgc tggtggcaca 600 tgtgacctcg cgcaaatgct acggtccgag
cgcgaccagt gaaaaatgtc cgggtaacgc 660 tctggaaaaa ggcggaaaag
gatcgattac cgaacagctg cttaacgctc gtgccgacgt 720 tacgcttggc
ggcggcgcaa aaacctttgc tgaaacggca accgctggtg aatggcaggg 780
aaaaacgctg cgtgaacagg cacaggcgcg tggttatcag ttggtgagcg atgctgcctc
840 actgaattcg gtgacggaag cgaatcagca aaaacccctg cttggcctgt
ttgctgacgg 900 caatatgcca gtgcgctggc taggaccgaa agcaacgtac
catggcaata tcgataagcc 960 cgcagtcacc tgtacgccaa atccgcaacg
taatgacagt gtaccaaccc tggcgcagat 1020 gaccgacaaa gccattgaat
tgttgagtaa aaatgagaaa ggctttttcc tgcaagttga 1080 aggtgcgtca
atcgataaac aggatcatgc tgcgaatcct tgtgggcaaa ttggcgagac 1140
ggtcgatctc gatgaagccg tacaacgggc gctggaattc gctaaaaagg agggtaacac
1200 gctggtcata gtcaccgctg atcacgccca cgccagccag attgttgcgc
cggataccaa 1260 agctccgggc ctcacccagg cgctaaatac caaagatggc
gcagtgatgg tgatgagtta 1320 cgggaactcc gaagaggatt cacaagaaca
taccggcagt cagttgcgta ttgcggcgta 1380 tggcccgcat gccgccaatg
ttgttggact gaccgaccag accgatctct tctacaccat 1440 gaaagccgct
ctggggctga aataataatc tagaggatcc ccgggtaccg agctcgaatt 1500
<210> SEQ ID NO 10 <211> LENGTH: 3968 <212> TYPE:
DNA <213> ORGANISM: E. coli <400> SEQUENCE: 10
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga
gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat
gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc
aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct
ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gcttcgcagc gctgttcctt
420 tgctcgcctg ctgcgagctg ggtaagcgga caaattctca ccgtctccgg
tggtggggta 480 caggagctca attaatacac taacggaccg gtaaacaacc
gtgcgtgttg tttaccggga 540 taaactcatc aacgtctctg ctaaataact
ggcagccaaa tcacggctat tggttaacca 600 atttcagagt gaaaagtata
cgaatagagt gtgccttcgc actattcaac agcaatgata 660 ggcgctcacc
tgacaacgcg gtaaactagt tattcacgct aactataatg gtttaatgat 720
ggataacatg cagactgaag cacaaccgac acggacccgg atcctcaatg ctgccagaga
780 gattttttca gaaaatggat ttcacagtgc ctcgatgaaa gccatctgta
aatcttgcgc 840 cattagtccc gggacgctct atcaccattt catctccaaa
gaagccttga ttcaggcgat 900 tatcttacag gaccaggaga gggcgctggc
ccgtttccgg gaaccgattg aagggattca 960 tttcgttgac tatatggtcg
agtccattgt ctctctcacc catgaagcct ttggacaacg 1020 ggcgctggtg
gttgaaatta tggcggaagg gatgcgtaac ccacaggtcg ccgccatgct 1080
taaaaataag catatgacga tcacggaatt tgttgcccag cggatgcgtg atgcccagca
1140 aaaaggcgag ataagcccag acatcaacac ggcaatgact tcacgtttac
tgctggatct 1200 gacctacggt gtactggccg atatcgaagc ggaagacctg
gcgcgtgaag cgtcgtttgc 1260 tcagggatta cgcgcgatga ttggcggtat
cttaaccgca tcctgattct ctctcttttt 1320 cggcgggctg gtgataactg
tgcccgcgtt tcatatcgta atttctctgt gcaaaaatta 1380 tccttcccgg
cttcggagaa ttccccccaa aatattcact gtagccatat gtcatgagag 1440
tttatcgttc ccaatacgct cgaacgaacg ttcggttgct tattttatgg cttctgtcaa
1500 cgctgtttta aagattaatg cgatctatat cacgctgtgg gtattgcagt
ttttggtttt 1560 ttgatcgcgg tgtcagttct ttttatttcc atttctcttc
catgggtttc tcacagataa 1620 ctgtgtgcaa cacagaattg gttaactaat
cagattaaag gttgaccagt attattatct 1680 taatgaggag tcctgcaggt
cgactctaga ggatccccgg gtaccgagct cgaattcgta 1740 atcatggtca
tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat 1800
acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
1860 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
agctgcatta 1920 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
gggcgctctt ccgcttcctc 1980 gctcactgac tcgctgcgct cggtcgttcg
gctgcggcga gcggtatcag ctcactcaaa 2040 ggcggtaata cggttatcca
cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 2100 aggccagcaa
aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 2160
ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
2220 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
ctcctgttcc 2280 gaccctgccg cttaccggat acctgtccgc ctttctccct
tcgggaagcg tggcgctttc 2340 tcatagctca cgctgtaggt atctcagttc
ggtgtaggtc gttcgctcca agctgggctg 2400 tgtgcacgaa ccccccgttc
agcccgaccg ctgcgcctta tccggtaact atcgtcttga 2460 gtccaacccg
gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 2520
cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
2580 cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct
tcggaaaaag 2640 agttggtagc tcttgatccg gcaaacaaac caccgctggt
agcggtggtt tttttgtttg 2700 caagcagcag attacgcgca gaaaaaaagg
atctcaagaa gatcctttga tcttttctac 2760 ggggtctgac gctcagtgga
acgaaaactc acgttaaggg attttggtca tgagattatc 2820 aaaaaggatc
ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag 2880
tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
2940 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
agataactac 3000 gatacgggag ggcttaccat ctggccccag tgctgcaatg
ataccgcgag acccacgctc 3060 accggctcca gatttatcag caataaacca
gccagccgga agggccgagc gcagaagtgg 3120 tcctgcaact ttatccgcct
ccatccagtc tattaattgt tgccgggaag ctagagtaag 3180 tagttcgcca
gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc 3240
acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
3300 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
tcgttgtcag 3360 aagtaagttg gccgcagtgt tatcactcat ggttatggca
gcactgcata attctcttac 3420 tgtcatgcca tccgtaagat gcttttctgt
gactggtgag tactcaacca agtcattctg 3480 agaatagtgt atgcggcgac
cgagttgctc ttgcccggcg tcaatacggg ataataccgc 3540 gccacatagc
agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact 3600
ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
3660 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
gaaggcaaaa 3720 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
atactcatac tcttcctttt 3780 tcaatattat tgaagcattt atcagggtta
ttgtctcatg agcggataca tatttgaatg 3840 tatttagaaa aataaacaaa
taggggttcc gcgcacattt ccccgaaaag tgccacctga 3900 cgtctaagaa
accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc 3960
ctttcgtc 3968 <210> SEQ ID NO 11 <211> LENGTH: 3872
<212> TYPE: DNA <213> ORGANISM: E. coli <400>
SEQUENCE: 11 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
gagacggtca 60 cagcttgtct gtaagcggat gccgggagca gacaagcccg
tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg
cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata
ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt
caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
360 tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gcttttagcc
gggaaacgtc 420 tggcggcgct gttggctaag tttgcggtat tgttgcggcg
acatgccgac atatttgccg 480 aacgtgctgt aaaaacgact acttgaacga
aagcctgccg tcagggcaat atcgagaata 540 cttttatcgg tatcgctcag
taacgcgcga acgtggttga tgcgcatcgc ggtaatgtac 600 tgtttcatcg
tcaattgcat gacccgctgg aatatcccca ttgcatagtt ggcgttaagt 660
ttgacgtgct cagccacatc gttgatggtc agcgcctgat catagttttc ggcaataaag
720 cccagcatct ggctaacata aaattgcgca tggcgcgaga cgctgttttt
gtgtgtgcgc 780 gaggttttat tgaccagaat cggttcccag ccagagaggc
taaatcgctt gagcatcagg 840 ccaatttcat caatggcgag ctggcgaatt
tgctcgttcg gactgtttaa ttcctgctgc 900 cagcggcgca cttcaaacgg
gctaagttgc tgtgtggcca gtgatttgat caccatgccg 960 tgagtgacgt
ggttaatcag gtctttatcc agcggccagg agagaaacag atgcatcggc 1020
agattaaaaa tcgccatgct ctgacaggtt ccggtatctg ttagttggtg cggtgtacag
1080 gcccagaaca gcgtgatatg accctgattg atattcactt tttcattgtt
gatcaggtat 1140 tccacatcgc catcgaaagg cacattcact tcgacctgac
catgccagtg gctggtgggc 1200 atgatatgcg gtgcgcgaaa ctcaatctcc
atccgctggt attccgaata cagcgacagc 1260 gggctgcggg tctgtttttc
gtcgctgctg cacataaacg tatctgtatt catggatggc 1320 tctctttcct
ggaatatcag aattatggca ggagtgaggg aggatgactg cgagtgggag 1380
cacggttttc accctcttcc cagaggggcg aggggactct ccgagtatca tgaggccgaa
1440 aactctgctt ttcaggtaat ttattcccat aaactcagat ttactgctgc
ttcacgcagg 1500 atctgagttt atgggaatgc tcaacctgga agccggaggt
tttctgcaga ttcgcctgcc 1560 atgatgaagt tattcaagca agccaggaga
tctggtaccc gggtcgactc tagaggatcc 1620 ccgggtaccg agctcgaatt
cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta 1680 tccgctcaca
attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc 1740
ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
1800 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag
gcggtttgcg 1860 tattgggcgc tcttccgctt cctcgctcac tgactcgctg
cgctcggtcg ttcggctgcg 1920 gcgagcggta tcagctcact caaaggcggt
aatacggtta tccacagaat caggggataa 1980 cgcaggaaag aacatgtgag
caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 2040 gttgctggcg
tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 2100
aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
2160 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
ccgcctttct 2220 cccttcggga agcgtggcgc tttctcatag ctcacgctgt
aggtatctca gttcggtgta 2280 ggtcgttcgc tccaagctgg gctgtgtgca
cgaacccccc gttcagcccg accgctgcgc 2340 cttatccggt aactatcgtc
ttgagtccaa cccggtaaga cacgacttat cgccactggc 2400 agcagccact
ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 2460
gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct
2520 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
aaaccaccgc 2580 tggtagcggt ggtttttttg tttgcaagca gcagattacg
cgcagaaaaa aaggatctca 2640 agaagatcct ttgatctttt ctacggggtc
tgacgctcag tggaacgaaa actcacgtta 2700 agggattttg gtcatgagat
tatcaaaaag gatcttcacc tagatccttt taaattaaaa 2760 atgaagtttt
aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 2820
cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
2880 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
ccagtgctgc 2940 aatgataccg cgagacccac gctcaccggc tccagattta
tcagcaataa accagccagc 3000 cggaagggcc gagcgcagaa gtggtcctgc
aactttatcc gcctccatcc agtctattaa 3060 ttgttgccgg gaagctagag
taagtagttc gccagttaat agtttgcgca acgttgttgc 3120 cattgctaca
ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 3180
ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
3240
cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
3300 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
ctgtgactgg 3360 tgagtactca accaagtcat tctgagaata gtgtatgcgg
cgaccgagtt gctcttgccc 3420 ggcgtcaata cgggataata ccgcgccaca
tagcagaact ttaaaagtgc tcatcattgg 3480 aaaacgttct tcggggcgaa
aactctcaag gatcttaccg ctgttgagat ccagttcgat 3540 gtaacccact
cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 3600
gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
3660 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
gttattgtct 3720 catgagcgga tacatatttg aatgtattta gaaaaataaa
caaatagggg ttccgcgcac 3780 atttccccga aaagtgccac ctgacgtcta
agaaaccatt attatcatga cattaaccta 3840 taaaaatagg cgtatcacga
ggccctttcg tc 3872 <210> SEQ ID NO 12 <211> LENGTH:
3934 <212> TYPE: DNA <213> ORGANISM: E. coli
<400> SEQUENCE: 12 tcgcgcgttt cggtgatgac ggtgaaaacc
tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat
gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg
tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg
ccagtgccaa gcttcaagcc gtcaattgtc 420 tgattcgtta ccaattatga
caacttgacg gctacatcat tcactttttc ttcacaaccg 480 gcacggaact
cgctcgggct ggccccggtg cattttttaa atacccgcga gaaatagagt 540
tgatcgtcaa aaccaacatt gcgaccgacg gtggcgatag gcatccgggt ggtgctcaaa
600 agcagcttcg cctggctgat acgttggtcc tcgcgccagc ttaagacgct
aatccctaac 660 tgctggcgga aaagatgtga cagacgcgac ggcgacaagc
aaacatgctg tgcgacgctg 720 gcgatatcaa aattgctgtc tgccaggtga
tcgctgatgt actgacaagc ctcgcgtacc 780 cgattatcca tcggtggatg
gagcgactcg ttaatcgctt ccatgcgccg cagtaacaat 840 tgctcaagca
gatttatcgc cagcagctcc gaatagcgcc cttccccttg cccggcgtta 900
atgatttgcc caaacaggtc gctgaaatgc ggctggtgcg cttcatccgg gcgaaagaac
960 cccgtattgg caaatattga cggccagtta agccattcat gccagtaggc
gcgcggacga 1020 aagtaaaccc actggtgata ccattcgcga gcctccggat
gacgaccgta gtgatgaatc 1080 tctcctggcg ggaacagcaa aatatcaccc
ggtcggcaaa caaattctcg tccctgattt 1140 ttcaccaccc cctgaccgcg
aatggtgaga ttgagaatat aacctttcat tcccagcggt 1200 cggtcgataa
aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacc cgccaccaga 1260
tgggcattaa acgagtatcc cggcagcagg ggatcatttt gcgcttcagc catacttttc
1320 atactcccgc cattcagaga agaaaccaat tgtccatatt gcatcagaca
ttgccgtcac 1380 tgcgtctttt actggctctt ctcgctaacc aaaccggtaa
ccccgcttat taaaagcatt 1440 ctgtaacaaa gcgggaccaa agccatgaca
aaaacgcgta acaaaagtgt ctataatcac 1500 ggcagaaaag tccacattga
ttatttgcac ggcgtcacac tttgctatgc catagcattt 1560 ttatccataa
gattagcgga tcctacctga cgctttttat cgcaactctc tactgtttct 1620
ccatacccgt ttttttgggc tagcaggagg aattcaccct gcaggtcgac tctagaggat
1680 ccccgggtac cgagctcgaa ttcgtaatca tggtcatagc tgtttcctgt
gtgaaattgt 1740 tatccgctca caattccaca caacatacga gccggaagca
taaagtgtaa agcctggggt 1800 gcctaatgag tgagctaact cacattaatt
gcgttgcgct cactgcccgc tttccagtcg 1860 ggaaacctgt cgtgccagct
gcattaatga atcggccaac gcgcggggag aggcggtttg 1920 cgtattgggc
gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 1980
cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
2040 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
taaaaaggcc 2100 gcgttgctgg cgtttttcca taggctccgc ccccctgacg
agcatcacaa aaatcgacgc 2160 tcaagtcaga ggtggcgaaa cccgacagga
ctataaagat accaggcgtt tccccctgga 2220 agctccctcg tgcgctctcc
tgttccgacc ctgccgctta ccggatacct gtccgccttt 2280 ctcccttcgg
gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 2340
taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
2400 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
atcgccactg 2460 gcagcagcca ctggtaacag gattagcaga gcgaggtatg
taggcggtgc tacagagttc 2520 ttgaagtggt ggcctaacta cggctacact
agaaggacag tatttggtat ctgcgctctg 2580 ctgaagccag ttaccttcgg
aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 2640 gctggtagcg
gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 2700
caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
2760 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
tttaaattaa 2820 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa
cttggtctga cagttaccaa 2880 tgcttaatca gtgaggcacc tatctcagcg
atctgtctat ttcgttcatc catagttgcc 2940 tgactccccg tcgtgtagat
aactacgata cgggagggct taccatctgg ccccagtgct 3000 gcaatgatac
cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 3060
gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
3120 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
caacgttgtt 3180 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg
gtatggcttc attcagctcc 3240 ggttcccaac gatcaaggcg agttacatga
tcccccatgt tgtgcaaaaa agcggttagc 3300 tccttcggtc ctccgatcgt
tgtcagaagt aagttggccg cagtgttatc actcatggtt 3360 atggcagcac
tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact 3420
ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
3480 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
gctcatcatt 3540 ggaaaacgtt cttcggggcg aaaactctca aggatcttac
cgctgttgag atccagttcg 3600 atgtaaccca ctcgtgcacc caactgatct
tcagcatctt ttactttcac cagcgtttct 3660 gggtgagcaa aaacaggaag
gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 3720 tgttgaatac
tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 3780
ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
3840 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat
gacattaacc 3900 tataaaaata ggcgtatcac gaggcccttt cgtc 3934
<210> SEQ ID NO 13 <211> LENGTH: 6013 <212> TYPE:
DNA <213> ORGANISM: E. coli <400> SEQUENCE: 13
tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
60 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
cccgcccatt 120 gacgtcaata atgacgtatg ttcccatagt aacgccaata
gggactttcc attgacgtca 180 atgggtggag tatttacggt aaactgccca
cttggcagta catcaagtgt atcatatgcc 240 aagtacgccc cctattgacg
tcaatgacgg taaatggccc gcctggcatt atgcccagta 300 catgacctta
tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360
catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
420 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
aaaatcaacg 480 ggactttcca aaatgtcgta acaactccgc cccattgacg
caaatgggcg gtaggcgtgt 540 acggtgggag gtctatataa gcagagctgg
tttagtgaac cgtcagatcc gctagcgcta 600 ccggtcgcca ccatggtgag
caagggcgag gagctgttca ccggggtggt gcccatcctg 660 gtcgagctgg
acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc 720
gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg
780 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag
ccgctacccc 840 gaccacatga agcagcacga cttcttcaag tccgccatgc
ccgaaggcta cgtccaggag 900 cgcaccatct tcttcaagga cgacggcaac
tacaagaccc gcgccgaggt gaagttcgag 960 ggcgacaccc tggtgaaccg
catcgagctg aagggcatcg acttcaagga ggacggcaac 1020 atcctggggc
acaagctgga gtacaactac aacagccaca acgtctatat catggccgac 1080
aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc
1140 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
cgtgctgctg 1200 cccgacaacc actacctgag cacccagtcc gccctgagca
aagaccccaa cgagaagcgc 1260 gatcacatgg tcctgctgga gttcgtgacc
gccgccggga tcactctcgg catggacgag 1320 ctgtacaagt ccggactcag
atctcgagct taataacaag ccgtcaattg tctgattcgt 1380 taccaattat
gacaacttga cggctacatc attcactttt tcttcacaac cggcacggaa 1440
ctcgctcggg ctggccccgg tgcatttttt aaatacccgc gagaaataga gttgatcgtc
1500 aaaaccaaca ttgcgaccga cggtggcgat aggcatccgg gtggtgctca
aaagcagctt 1560 cgcctggctg atacgttggt cctcgcgcca gcttaagacg
ctaatcccta actgctggcg 1620 gaaaagatgt gacagacgcg acggcgacaa
gcaaacatgc tgtgcgacgc tggcgatatc 1680 aaaattgctg tctgccaggt
gatcgctgat gtactgacaa gcctcgcgta cccgattatc 1740 catcggtgga
tggagcgact cgttaatcgc ttccatgcgc cgcagtaaca attgctcaag 1800
cagatttatc gccagcagct ccgaatagcg cccttcccct tgcccggcgt taatgatttg
1860 cccaaacagg tcgctgaaat gcggctggtg cgcttcatcc gggcgaaaga
accccgtatt 1920 ggcaaatatt gacggccagt taagccattc atgccagtag
gcgcgcggac gaaagtaaac 1980 ccactggtga taccattcgc gagcctccgg
atgacgaccg tagtgatgaa tctctcctgg 2040 cgggaacagc aaaatatcac
ccggtcggca aacaaattct cgtccctgat ttttcaccac 2100 cccctgaccg
cgaatggtga gattgagaat ataacctttc attcccagcg gtcggtcgat 2160
aaaaaaatcg agataaccgt tggcctcaat cggcgttaaa cccgccacca gatgggcatt
2220 aaacgagtat cccggcagca ggggatcatt ttgcgcttca gccatacttt
tcatactccc 2280 gccattcaga gaagaaacca attgtccata ttgcatcaga
cattgccgtc actgcgtctt 2340 ttactggctc ttctcgctaa ccaaaccggt
aaccccgctt attaaaagca ttctgtaaca 2400 aagcgggacc aaagccatga
caaaaacgcg taacaaaagt gtctataatc acggcagaaa 2460
agtccacatt gattatttgc acggcgtcac actttgctat gccatagcat ttttatccat
2520 aagattagcg gatcctacct gacgcttttt atcgcaactc tctactgttt
ctccataccc 2580 gtttttttgg gctagcagga ggaattcacc atggtacccg
gggatcctct agagtcgacc 2640 tgcaggcatg caagcttggc ccgcgggccc
gggatccacc ggatctagat aactgatcat 2700 aatcagccat accacatttg
tagaggtttt acttgcttta aaaaacctcc cacacctccc 2760 cctgaacctg
aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta 2820
taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
2880 gcattctagt tgtggtttgt ccaaactcat caatgtatct taacgcgtaa
attgtaagcg 2940 ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
cagctcattt tttaaccaat 3000 aggccgaaat cggcaaaatc ccttataaat
caaaagaata gaccgagata gggttgagtg 3060 ttgttccagt ttggaacaag
agtccactat taaagaacgt ggactccaac gtcaaagggc 3120 gaaaaaccgt
ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt 3180
tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag
3240 cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
aaaggagcgg 3300 gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
aaccaccaca cccgccgcgc 3360 ttaatgcgcc gctacagggc gcgtcaggtg
gcacttttcg gggaaatgtg cgcggaaccc 3420 ctatttgttt atttttctaa
atacattcaa atatgtatcc gctcatgaga caataaccct 3480 gataaatgct
tcaataatat tgaaaaagga agagtcctga ggcggaaaga accagctgtg 3540
gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
3600 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
ccccagcagg 3660 cagaagtatg caaagcatgc atctcaatta gtcagcaacc
atagtcccgc ccctaactcc 3720 gcccatcccg cccctaactc cgcccagttc
cgcccattct ccgccccatg gctgactaat 3780 tttttttatt tatgcagagg
ccgaggccgc ctcggcctct gagctattcc agaagtagtg 3840 aggaggcttt
tttggaggcc taggcttttg caaagatcga tcaagagaca ggatgaggat 3900
cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
3960 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
gccgtgttcc 4020 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac
cgacctgtcc ggtgccctga 4080 atgaactgca agacgaggca gcgcggctat
cgtggctggc cacgacgggc gttccttgcg 4140 cagctgtgct cgacgttgtc
actgaagcgg gaagggactg gctgctattg ggcgaagtgc 4200 cggggcagga
tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg 4260
atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
4320 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
caggatgatc 4380 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt
cgccaggctc aaggcgagca 4440 tgcccgacgg cgaggatctc gtcgtgaccc
atggcgatgc ctgcttgccg aatatcatgg 4500 tggaaaatgg ccgcttttct
ggattcatcg actgtggccg gctgggtgtg gcggaccgct 4560 atcaggacat
agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg 4620
accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
4680 gccttcttga cgagttcttc tgagcgggac tctggggttc gaaatgaccg
accaagcgac 4740 gcccaacctg ccatcacgag atttcgattc caccgccgcc
ttctatgaaa ggttgggctt 4800 cggaatcgtt ttccgggacg ccggctggat
gatcctccag cgcggggatc tcatgctgga 4860 gttcttcgcc caccctaggg
ggaggctaac tgaaacacgg aaggagacaa taccggaagg 4920 aacccgcgct
atgacggcaa taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt 4980
tcataaacgc ggggttcggt cccagggctg gcactctgtc gataccccac cgagacccca
5040 ttggggccaa tacgcccgcg tttcttcctt ttccccaccc caccccccaa
gttcgggtga 5100 aggcccaggg ctcgcagcca acgtcggggc ggcaggccct
gccatagcct caggttactc 5160 atatatactt tagattgatt taaaacttca
tttttaattt aaaaggatct aggtgaagat 5220 cctttttgat aatctcatga
ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc 5280 agaccccgta
gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg 5340
ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
5400 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
atactgtcct 5460 tctagtgtag ccgtagttag gccaccactt caagaactct
gtagcaccgc ctacatacct 5520 cgctctgcta atcctgttac cagtggctgc
tgccagtggc gataagtcgt gtcttaccgg 5580 gttggactca agacgatagt
taccggataa ggcgcagcgg tcgggctgaa cggggggttc 5640 gtgcacacag
cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga 5700
gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
5760 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
ggtatcttta 5820 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
tttttgtgat gctcgtcagg 5880 ggggcggagc ctatggaaaa acgccagcaa
cgcggccttt ttacggttcc tggccttttg 5940 ctggcctttt gctcacatgt
tctttcctgc gttatcccct gattctgtgg ataaccgtat 6000 taccgccatg cat
6013 <210> SEQ ID NO 14 <211> LENGTH: 1380 <212>
TYPE: DNA <213> ORGANISM: Rattus norvegicus <400>
SEQUENCE: 14 gaattcaggc gctttttaga ctggtcgtaa tgaaattcag caggatcaca
ttctgcaggt 60 cgacatggca accacgcacg cgcagggcca cccgccagtc
ttggggaatg atactctccg 120 ggaacattat gattacgtgg ggaagctggc
aggcaggctg cgggatcccc ctgagggtag 180 caccctcatc accaccatcc
tcttcttggt cacctgtagc ttcatcgtct tggagaacct 240 gatggttttg
attgccatct ggaaaaacaa taaatttcat aaccgcatgt actttttcat 300
cggcaacttg gctctctgcg acctgctggc cggcatagcc tacaaggtca acattctgat
360 gtccggtagg aagacgttca gcctgtctcc aacagtgtgg ttcctcaggg
agggcagtat 420 gttcgtagcc ctgggcgcat ccacatgcag cttattggcc
attgccattg agcggcacct 480 gaccatgatc aagatgaggc cgtacgacgc
caacaagaag caccgcgtgt tccttctgat 540 tgggatgtgc tggctaattg
ccttctcgct gggtgccctg cccatcctgg gctggaactg 600 cctggaaaac
tttcccgact gctctaccat cttgcccctc tactccaaga aatacattgc 660
ctttctcatc agcatcttca tagccattct ggtgaccatc gtcatcttgt acgcgcgcat
720 ctacttcctg gtcaagtcca gcagccgcag ggtggccaac cacaactccg
agagatccat 780 ggcccttctg cggaccgtag tgatcgtggt gagcgtgttc
atcgcctgtt ggtcccccct 840 tttcatcctc ttcctcatcg atgtggcctg
cagggcgaag gagtgctcca tcctcttcaa 900 gagtcagtgg ttcatcatgc
tggctgtcct caactcggcc atgaaccctg tcatctacac 960 gctggccagc
aaagagatgc ggcgtgcttt cttccggttg gtgtgcggct gtctggtcaa 1020
gggcaagggg acccaggcct ccccgatgca gcctgctctt gacccgagca gaagtaaatc
1080 aagctccagt aacaacagca gcagccactc tccaaaggtc aaggaagacc
tgccccatgt 1140 ggctacctct tcctgcgtta ctgacaaaac gaggtcgctt
cagaatgggg tcctctgcaa 1200 gaagggcaat tctgcagata tccagcacag
tggcggccgc tcgagtctag agggcccgcg 1260 gttcgaaggt aagcctatcc
ctaaccctct cctcggtctc gattctacgc gtaccggtca 1320 tcatcaccat
caccattgat aaggtaccga gctcgaattc gtaatcatgg tcatagctgt 1380
<210> SEQ ID NO 15 <211> LENGTH: 1320 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 15
gaattcaggc gctttttaga ctggtcgtaa tgaaattcag caggatcaca ttctgcaggt
60 cgacatgggg caacccggga acggcagcgc cttcttgctg gcacccaatg
gaagccatgc 120 gccggaccac gacgtcacgc agcaaaggga cgaggtgtgg
gtggtgggca tgggcatcgt 180 catgtctctc atcgtcctgg ccatcgtgtt
tggcaatgtg ctggtcatca cagccattgc 240 caagttcgag cgtctgcaga
cggtcaccaa ctacttcatc acttcactgg cctgtgctga 300 tctggtcatg
ggcctagcag tggtgccctt tggggccgcc catattctta tgaaaatgtg 360
gacttttggc aacttctggt gcgagttttg gacttccatt gatgtgctgt gcgtcacggc
420 cagcattgag accctgtgcg tgatcgcagt ggatcgctac tttgccatta
cttcaccttt 480 caagtaccag agcctgctga ccaagaataa ggcccgggtg
atcattctga tggtgtggat 540 tgtgtcaggc cttayctcct tcttgcccat
tcagatgcac tggtacaggg ccacccacca 600 ggaagccatc aactgctatg
ccaatgagac ctgctgtgac ttcttcacga accaagccta 660 tgccattgcc
tcttccatcg tgtccttcta cgttcccctg gtgatcatgg tcttcgtcta 720
ctccagggtc tttcaggagg ccaaaaggca gctccagaag attgacaaat ctgagggccg
780 cttccatgtc cagaacctta gccaggtgga gcaggatggg cggacggggc
atggactccg 840 cagatcttcc aagttctgct tgaaggagca caaagccctc
aagacgttag gcatcatcat 900 gggcactttc accctctgct ggctgccctt
cttcatcgtt aacattgtgc atgtgatcca 960 ggataacctc atccgtaagg
aagtttacat cctcctaaat tggataggct atgtcaattc 1020 tggtttcaat
ccccttatct actgccggag cccagatttc aggattgcct tccaggagct 1080
tctgtgcctg cgcaggtctt ctttgaaggc ctatggcaat ggctactcca gcaacggcaa
1140 cacaggggag cagagtggat atcacgtgga acaggagaaa gaaaataaac
tgctgtgtga 1200 agacctccca ggcacggaag actttgtggg ccatcaaggt
actgtgccta gcgataacat 1260 tgattcacaa gggaggaatt gtagtacaaa
tgactcactg ctataataag gatccccggg 1320 <210> SEQ ID NO 16
<211> LENGTH: 52 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide sequence <400>
SEQUENCE: 16 aattggtacc tcaatgatga tgatgatgat gcttgcagag gaccccattc
tg 52 <210> SEQ ID NO 17 <211> LENGTH: 1320 <212>
TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:
17 tcgcaactct ctactgtttc tccatacccg tttttttggg ctagcaggag
gaattcacca 60 tggatagtgt gtgtccccaa ggaaaatata tccaccctca
aaataattcg atttgctgta 120
ccaagtgcca caaaggaacc tacttgtaca atgactgtcc aggcccgggg caggatacgg
180 actgcaggga gtgtgagagc ggctccttca ccgcttcaga aaaccacctc
agacactgcc 240 tcagctgctc caaatgccga aaggaaatgg gtcaggtgga
gatctcttct tgcacagtgg 300 accgggacac cgtgtgtggc tgcaggaaga
accagtaccg gcattattgg agtgaaaacc 360 ttttccagtg cttcaattgc
agcctctgcc tcaatgggac cgtgcacctc tcctgccagg 420 agaaacagaa
caccgtgtgc acctgccatg caggtttctt tctaagagaa aacgagtgtg 480
tctcctgtag taactgtaag aaaagcctgg agtgcacgaa gttgtgccta ccccagattg
540 agaatgttaa gggcactgag gactcaggca ccacagtgct gttgcccctg
gtcattttct 600 ttggtctttg ccttttatcc ctcctcttca ttggtttaat
gtatcgctac caacggtgga 660 agtccaagct ctactccatt gtttgtggga
aatcgacacc tgaaaaagag ggggagcttg 720 aaggaactac tactaagccc
ctggccccaa acccaagctt cagtcccact ccaggcttca 780 cccccaccct
gggcttcagt cccgtgccca gttccacctt cacctccagc tccacctata 840
cccccggtga ctgtcccaac tttgcggctc cccgcagaga ggtggcacca ccctatcagg
900 gggctgaccc catccttgcg acagccctcg cctccgaccc catccccaac
ccccttcaga 960 agtgggagga cagcgcccac aagccacaga gcctagacac
tgatgacccc gcgacgctgt 1020 acgccgtggt ggagaacgtg cccccgttgc
gctggaagga attcgtgcgg cgcctagggc 1080 tgagcgacca cgagatcgat
cggctggagc tgcagaacgg gcgctgcctg cgcgaggcgc 1140 aatacagcat
gctggcgacc tggaggcggc gcacgccgcg gcgcgaggcc acgctggagc 1200
tgctgggacg cgtgctccgc gacatggacc tgctgggctg cctggaggac atcgaggagg
1260 cgctttgcgg ccccgccgcc ctcccgcccg cgcccagtct tctcagatga
tctagagtcg 1320 <210> SEQ ID NO 18 <211> LENGTH: 1380
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 18 ccatacccgt ttttttgggc tagcaggagg
aattcaccct gcaggtcgac atgggactgg 60 tccctcacct aggggacagg
gagaagagag atagtgtgtg tccccaagga aaatatatcc 120 accctcaaaa
taattcgatt tgctgtacca agtgccacaa aggaacctac ttgtacaatg 180
actgtccagg cccggggcag gatacggact gcagggagtg tgagagcggc tccttcaccg
240 cttcagaaaa ccacctcaga cactgcctca gctgctccaa atgccgaaag
gaaatgggtc 300 aggtggagat ctcttcttgc acagtggacc gggacaccgt
gtgtggctgc aggaagaacc 360 agtaccggca ttattggagt gaaaaccttt
tccagtgctt caattgcagc ctctgcctca 420 atgggaccgt gcacctctcc
tgccaggaga aacagaacac cgtgtgcacc tgccatgcag 480 gtttctttct
aagagaaaac gagtgtgtct cctgtagtaa ctgtaagaaa agcctggagt 540
gcacgaagtt gtgcctaccc cagattgaga atgttaaggg cactgaggac tcaggcacca
600 cagtgctgtt gcccctggtc attttctttg gtctttgcct tttatccctc
ctcttcattg 660 gtttaatgta tcgctaccaa cggtggaagt ccaagctcta
ctccattgtt tgtgggaaat 720 cgacacctga aaaagagggg gagcttgaag
gaactactac taagcccctg gccccaaacc 780 caagcttcag tcccactcca
ggcttcaccc ccaccctggg cttcagtccc gtgcccagtt 840 ccaccttcac
ctccagctcc acctataccc ccggtgactg tcccaacttt gcggctcccc 900
gcagagaggt ggcaccaccc tatcaggggg ctgaccccat ccttgcgaca gccctcgcct
960 ccgaccccat ccccaacccc cttcagaagt gggaggacag cgcccacaag
ccacagagcc 1020 tagacactga tgaccccgcg acgctgtacg ccgtggtgga
gaacgtgccc ccgttgcgct 1080 ggaaggaatt cgtgcggcgc ctagggctga
gcgaccacga gatcgatcgg ctggagctgc 1140 agaacgggcg ctgcctgcgc
gaggcgcaat acagcatgct ggcgacctgg aggcggcgca 1200 cgccgcggcg
cgaggccacg ctggagctgc tgggacgcgt gctccgcgac atggacctgc 1260
tgggctgcct ggaggacatc gaggaggcgc tttgcggccc cgccgccctc ccgcccgcgc
1320 ccagtcttct cagataataa ggtaccgagc tcgaattcgt aatcatggtc
atagctgttt 1380 <210> SEQ ID NO 19 <211> LENGTH: 780
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 19 gtttttttgg gctagcagga ggaattcatg
agcactgaaa gcatgatccg ggacgtggag 60 ctggccgagg aggcgctccc
caagaagaca ggggggcccc agggctccag gcggtgcttg 120 ttcctcagcc
tcttctcctt cctgatcgtg gcaggcgcca ccacgctctt ctgcctgctg 180
cactttggag tgatcggccc ccagagggaa gagttcccca gggacctctc tctaatcagc
240 cctctggccc aggcagtcag atcatcttct cgaaccccga gtgacaagcc
tgtagcccat 300 gttgtagcaa accctcaagc tgaggggcag ctccagtggc
tgaaccgccg ggccaatgcc 360 ctcctggcca atggcgtgga gctgagagat
aaccagctgg tggtgccatc agagggcctg 420 tacctcatct actcccaggt
cctcttcaag ggccaaggct gcccctccac ccatgtgctc 480 ctcacccaca
ccatcagccg catcgccgtc tcctaccaga ccaaggtcaa cctcctctct 540
gccatcaaga gcccctgcca gagggagacc ccagaggggg ctgaggccaa gccctggtat
600 gagcccatct atctgggagg ggtcttccag ctggagaagg gtgaccgact
cagcgctgag 660 atcaatcggc ccgactatct cgactttgcc gagtctgggc
aggtctactt tgggatcatt 720 gccctgtgat aagcttggcc cgcgggcccg
ggatccaccg gatctagata actgatcata 780 <210> SEQ ID NO 20
<211> LENGTH: 300 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic nucleic acid sequence <400> SEQUENCE:
20 gtttttttgg gctagcagga ggaattcacc atggtaccat gaacttgggg
aatcgactgt 60 ttattctgat agcggtctta cttcccctcg cagtattact
gctcaatagt gactctgaat 120 gtcccctgtc ccacgatggg tactgcctcc
atgatggtgt gtgcatgtat attgaagcat 180 tggacaagta tgcatgcaac
tgtgttgttg gctacatcgg ggagcgatgt cagtaccgag 240 acctgaagtg
gtgggaactg cgctaataag cttggcccgc gggcccggga tccaccggat 300
<210> SEQ ID NO 21 <211> LENGTH: 1620 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleic acid sequence
<400> SEQUENCE: 21 gtttttttgg gctagcagga ggaattcacc
atgaacttgg ggaatcgact gtttattctg 60 atagcggtct tacttcccct
cgcagtatta ctgctctcat tcacattgag cgtcaccgtt 120 cagcagcctc
agttgacatt aacggcggcc gtcattggtg atggcgcacc ggctaatggg 180
aaaactgcaa tcaccgttga gttcaccgtt gctgattttg aggggaaacc cttagccggg
240 caggaggtgg tgataaccac caataatggt gcgctaccga ataaaatcac
ggaaaagaca 300 gatgcaaatg gcgtcgcgcg cattgcatta accaatacga
cagatggcgt gacggtagtc 360 acagcagaag tggaggggca acggcaaagt
gttgataccc actttgttaa gggtactatc 420 gcggcggata aatccactct
ggctgcggta ccgacatcta tcatcgctga tggtctaatg 480 gcttcaacca
tcacgttgga gttgaaggat acctatgggg acccgcaggc tggcgcgaat 540
gtggcttttg acacaacctt aggcaatatg ggcgttatca cggatcacaa tgacggcact
600 tatagcgcac cattgaccag taccacgttg ggggtagcaa cagtaacggt
gaaagtggat 660 ggggctgcgt tcagtgtgcc gagtgtgacg gttaatttca
cggcagatcc tattccagat 720 gctggccgct ccagtttcac cgtctccaca
ccggatatct tggctgatgg cacgatgagt 780 tccacattat cctttgtccc
tgtcgataag aatggccatt ttatcagtgg gatgcagggc 840 ttgagtttta
ctcaaaacgg tgtgccggtg agtattagcc ccattaccga gcagccagat 900
agctataccg cgacggtggt tgggaatagt gtcggtgatg tcacaatcac gccgcaggtt
960 gataccctga tactgagtac attgcagaaa aaaatatccc tattcccggt
acctacgctg 1020 accggtattc tggttaacgg gcaaaatttc gctacggata
aagggttccc gaaaacgatc 1080 tttaaaaacg ccacattcca gttacagatg
gataacgatg ttgctaataa tactcagtat 1140 gagtggtcgt cgtcattcac
acccaatgta tcggttaacg atcagggtca ggtgacgatt 1200 acctaccaaa
cctatagcga agtggctgtg acggcgaaaa gtaaaaaatt cccaagttat 1260
tcggtgagtt atcggttcta cccaaatcgg tggatatacg atggcggcag atcgctggta
1320 tccagtctcg aggccagcag acaatgccaa ggttcagata tgtctgcggt
tcttgaatcc 1380 tcacgtgcaa ccaacggaac gcgtgcgcct gacgggacat
tgtggggcga gtgggggagc 1440 ttgaccgcgt atagttctga ttggcaatct
ggtgaatatt gggtcaaaaa gaccagcacg 1500 gattttgaaa ccatgaatat
ggacacaggc gcactgcaac cagggcctgc atacttggcg 1560 ttcccgctct
gtgcgctgtc aatataactg caggcatgca agcttggccc gcgggcccgg 1620
<210> SEQ ID NO 22 <211> LENGTH: 208 <212> TYPE:
DNA <213> ORGANISM: E. coli <400> SEQUENCE: 22
gaattcaggc gctttttaga ctggtcgtaa tgaaattcag caggatcaca ttctgcagat
60 gtcacggccg agacttatag tcgctttgtt tttatttttt aatgtatttg
tacatggaga 120 aaataaagtg aaacaaagca ctattgcact ggcactctta
ccgttactgt ttacccctgt 180 gacaaaagcc cggacaccag aatctaga 208
<210> SEQ ID NO 23 <211> LENGTH: 1546 <212> TYPE:
DNA <213> ORGANISM: E. coli <400> SEQUENCE: 23
gaattcaggc gctttttaga ctggtcgtaa tgaaattcag caggatcaca ttctgcagat
60 gtcacggccg agacttatag tcgctttgtt tttatttttt aatgtatttg
tacatggaga 120 aaataaagtg aaacaaagca ctattgcact ggcactctta
ccgttactgt ttacccctgt 180 gacaaaagcc cggacaccag aaatgcctgt
tctggaaaac cgggctgctc agggcgatat 240 tactgcaccc ggcggtgctc
gccgtttaac gggtgatcag actgccgctc tgcgtgattc 300 tcttagcgat
aaacctgcaa aaaatattat tttgctgatt ggcgatggga tgggggactc 360
ggaaattact gccgcacgta attatgccga aggtgcgggc ggctttttta aaggtataga
420 tgccttaccg cttaccgggc aatacactca ctatgcgctg aataaaaaaa
ccggcaaacc 480
ggactacgtc accgactcgg ctgcatcagc aaccgcctgg tcaaccggtg tcaaaaccta
540 taacggcgcg ctgggcgtcg atattcacga aaaagatcac ccaacgattc
tggaaatggc 600 aaaagccgca ggtctggcga ccggtaacgt ttctaccgca
gagttgcagg atgccacgcc 660 cgctgcgctg gtggcacatg tgacctcgcg
caaatgctac ggtccgagcg cgaccagtga 720 aaaatgtccg ggtaacgctc
tggaaaaagg cggaaaagga tcgattaccg aacagctgct 780 taacgctcgt
gccgacgtta cgcttggcgg cggcgcaaaa acctttgctg aaacggcaac 840
cgctggtgaa tggcagggaa aaacgctgcg tgaacaggca caggcgcgtg gttatcagtt
900 ggtgagcgat gctgcctcac tgaattcggt gacggaagcg aatcagcaaa
aacccctgct 960 tggcctgttt gctgacggca atatgccagt gcgctggcta
ggaccgaaag caacgtacca 1020 tggcaatatc gataagcccg cagtcacctg
tacgccaaat ccgcaacgta atgacagtgt 1080 accaaccctg gcgcagatga
ccgacaaagc cattgaattg ttgagtaaaa atgagaaagg 1140 ctttttcctg
caagttgaag gtgcgtcaat cgataaacag gatcatgctg cgaatccttg 1200
tgggcaaatt ggcgagacgg tcgatctcga tgaagccgta caacgggcgc tggaattcgc
1260 taaaaaggag ggtaacacgc tggtcatagt caccgctgat cacgcccacg
ccagccagat 1320 tgttgcgccg gataccaaag ctccgggcct cacccaggcg
ctaaatacca aagatggcgc 1380 agtgatggtg atgagttacg ggaactccga
agaggattca caagaacata ccggcagtca 1440 gttgcgtatt gcggcgtatg
gcccgcatgc cgccaatgtt gttggactga ccgaccagac 1500 cgatctcttc
tacaccatga aagccgctct ggggctgaaa tctaga 1546 <210> SEQ ID NO
24 <211> LENGTH: 148 <212> TYPE: DNA <213>
ORGANISM: E. coli <400> SEQUENCE: 24 gaattcaggc gctttttaga
ctggtcgtaa tgaaattcag caggatcaca ttctgcagat 60 gaaaataaaa
acaggtgcac gcatcctcgc attatccgca ttaacgacga tgatgttttc 120
cgcctcggct ctcgccaaaa tctctaga 148 <210> SEQ ID NO 25
<211> LENGTH: 1174 <212> TYPE: DNA <213>
ORGANISM: E. coli <400> SEQUENCE: 25 gaattcaggc gctttttaga
ctggtcgtaa tgaaattcag caggatcaca ttctgcagat 60 gaaaataaaa
acaggtgcac gcatcctcgc attatccgca ttaacgacga tgatgttttc 120
cgcctcggct ctcgccaaaa tcgaagaagg taaactggta atctggatta acggcgataa
180 aggctataac ggtctcgctg aagtcggtaa gaaattcgag aaagataccg
gaattaaagt 240 caccgttgag catccggata aactggaaga gaaattccca
caggttgcgg caactggcga 300 tggccctgac attatcttct gggcacacga
ccgctttggt ggctacgctc aatctggcct 360 gttggctgaa atcaccccgg
acaaagcgtt ccaggacaag ctgtatccgt ttacctggga 420 tgccgtacgt
tacaacggca agctgattgc ttacccgatc gctgttgaag cgttatcgct 480
gatttataac aaagatctgc tgccgaaccc gccaaaaacc tgggaagaga tcccggcgct
540 ggataaagaa ctgaaagcga aaggtaagag cgcgctgatg ttcaacctgc
aagaaccgta 600 cttcacctgg ccgctgattg ctgctgacgg gggttatgcg
ttcaagtatg aaaacggcaa 660 gtacgacatt aaagacgtgg gcgtggataa
cgctggcgcg aaagcgggtc tgaccttcct 720 ggttgacctg attaaaaaca
aacacatgaa tgcagacacc gattactcca tcgcagaagc 780 tgcctttaat
aaaggcgaaa cagcgatgac catcaacggc ccgtgggcat ggtccaacat 840
cgacaccagc aaagtgaatt atggtgtaac ggtactgccg accttcaagg gtcaaccatc
900 caaaccgttc gttggcgtgc tgagcgcagg tattaacgcc gccagtccga
acaaagagct 960 ggcgaaagag ttcctcgaaa actatctgct gactgatgaa
ggtctggaag cggttaataa 1020 agacaaaccg ctgggtgccg tagcgctgaa
gtcttacgag gaagagttgg cgaaagatcc 1080 acgtattgcc gccaccatgg
aaaacgccca gaaaggtgaa atcatgccga acatcccgca 1140 gatgtccgct
ttctggtatg ccgtgcgttc taga 1174 <210> SEQ ID NO 26
<211> LENGTH: 3840 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic nucleic acid sequence <400>
SEQUENCE: 26 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
atcaggcgcc 60 atacgcgaca gcgcgcaata accgttctcg actcataaaa
gtgatgccgc tataatgccg 120 cgtcctattt gaatgctttc gggatgattc
tggtaacagg gaatgtgatt gattataaga 180 acatcccggt tccgcgaagc
caacaacctg tgcttgcggg gtaagagttg accgagcact 240 gtgatttttt
gaggtaacaa gatgcaagtt tcagttgaaa ccactcaagg ccttggccgc 300
cgtgtaacga ttactatcgc tgctgacagc atcgagaccg ctgttaaaag cgagctggtc
360 aacgttgcga aaaaagtacg tattgacggc ttccgcaaag gcaaagtgcc
aatgaatatc 420 gttgctcagc gttatggcgc gtctgtacgc caggacgttc
tgggtgacct gatgagccgt 480 aacttcattg acgccatcat taaagaaaaa
atcaatccgg ctggcgcacc gacttatgtt 540 ccgggcgaat acaagctggg
tgaagacttc acttactctg tagagtttga agtttatccg 600 gaagttgaac
tgcagggtct ggaagcgatc gaagttgaaa aaccgatcgt tgaagtgacc 660
gacgctgacg ttgacggcat gctggatact ctgcgtaaac agcaggcgac ctggaaagaa
720 aaagacggcg ctgttgaagc agaagaccgc gtaaccatcg acttcaccgg
ttctgtagac 780 ggcgaagagt tcgaaggcgg taaagcgtct gatttcgtac
tggcgatggg ccagggtcgt 840 atgatcccgg gctttgaaga cggtatcaaa
ggccacaaag ctggcgaaga gttcaccatc 900 gacgtgacct tcccggaaga
ataccacgca gaaaacctga aaggtaaagc agcgaaattc 960 gctatcaacc
tgaagaaagt tgaagagcgt gaactgccgg aactgactgc agaattcatc 1020
aaacgtttcg gcgttgaaga tggttccgta gaaggtctgc gcgctgaagt gcgtaaaaac
1080 atggagcgcg agctgaagag cgccatccgt aaccgcgtta agtctcaggc
gatcgaaggt 1140 ctggtaaaag ctaacgacat cgacgtaccg gctgcgctga
tcgacagcga aatcgacgtt 1200 ctgcgtcgcc aggctgcaca gcgtttcggt
ggcaacgaaa aacaagctct ggaactgccg 1260 cgcgaactgt tcgaagaaca
ggctaaacgc cgcgtagttg ttggcctgct gctgggcgaa 1320 gttatccgca
ccaacgagct gaaagctgac gaagagcgcg tgaaaggcct gatcgaagag 1380
atggcttctg cgtacgaaga tccgaaagaa gttatcgagt tctacagcaa aaacaaagaa
1440 ctgatggaca acatgcgcaa tgttgctctg gaagaacagg ctgttgaagc
tgtactggcg 1500 aaagcgaaag tgactgaaaa agaaaccact ttcaacgagc
tgatgaacca gcaggcgtaa 1560 taataatcta gaggtagcac aatcagattc
gcttatgacg gcgatgaaga aattgcgatg 1620 aaatgtgagg tgaatcaggg
ttttcacccg attttgtgct gatcagaatt ttttttcttt 1680 ttcccccttg
aaggggcgaa gcctcatccc catttctctg gtcaccagcc gggaaaccac 1740
gtaagctccg gcgtcaccca taacagatac ggactttctc aaaggagagt tatcaatgaa
1800 tattcgtcca ttgcatgatc gcgtgatcgt caagcgtaaa gaagttgaaa
ctaaatctgc 1860 tggcggcatc gttctgaccg gctctgcagc ggctaaatcc
acccgcggcg aagtgctggc 1920 tgtcggcaat ggccgtatcc ttgaaaatgg
cgaagtgaag ccgctggatg tgaaagttgg 1980 cgacatcgtt attttcaacg
atggctacgg tgtgaaatct gagaagatcg acaatgaaga 2040 agtgttgatc
atgtccgaaa gcgacattct ggcaattgtt gaagcgtaat ccgcgcacga 2100
cactgaacat acgaatttaa ggaataaaga taatggcagc taaagacgta aaattcggta
2160 acgacgctcg tgtgaaaatg ctgcgcggcg taaacgtact ggcagatgca
gtgaaagtta 2220 ccctcggtcc aaaaggccgt aacgtagttc tggataaatc
tttcggtgca ccgaccatca 2280 ccaaagatgg tgtttccgtt gctcgtgaaa
tcgaactgga agacaagttc gaaaatatgg 2340 gtgcgcagat ggtgaaagaa
gttgcctcta aagcaaacga cgctgcaggc gacggtacca 2400 ccactgcaac
cgtactggct caggctatca tcactgaagg tctgaaagct gttgctgcgg 2460
gcatgaaccc gatggacctg aaacgtggta tcgacaaagc ggttaccgct gcagttgaag
2520 aactgaaagc gctgtccgta ccatgctctg actctaaagc gattgctcag
gttggtacca 2580 tctccgctaa ctccgacgaa accgtaggta aactgatcgc
tgaagcgatg gacaaagtcg 2640 gtaaagaagg cgttatcacc gttgaagacg
gtaccggtct gcaggacgaa ctggacgtgg 2700 ttgaaggtat gcagttcgac
cgtggctacc tgtctcctta cttcatcaac aagccggaaa 2760 ctggcgcagt
agaactggaa agcccgttca tcctgctggc tgacaagaaa atctccaaca 2820
tccgcgaaat gctgccggtt ctggaagctg ttgccaaagc aggcaaaccg ctgctgatca
2880 tcgctgaaga tgtagaaggc gaagcgctgg caactctggt tgttaacacc
atgcgtggca 2940 tcgtgaaagt cgctgcggtt aaagcaccgg gcttcggcga
tcgtcgtaaa gctatgctgc 3000 aggatatcgc aaccctgact ggcggtaccg
tgatctctga agagatcggt atggagctgg 3060 aaaaagcaac cctggaagac
ctgggtcagg ctaaacgtgt tgtgatcaac aaagacacca 3120 ccactatcat
cgatggcgtg ggtgaagaag ctgcaatcca gggccgtgtt gctcagatcc 3180
gtcagcagat tgaagaagca acttctgact acgaccgtga aaaactgcag gaacgcgtag
3240 cgaaactggc aggcggcgtt gcagttatca aagtgggtgc tgctaccgaa
gttgaaatga 3300 aagagaaaaa agcacgcgtt gaagatgccc tgcacgcgac
ccgtgctgcg gtagaagaag 3360 gcgtggttgc tggtggtggt gttgcgctga
tccgcgtagc gtctaaactg gctgacctgc 3420 gtggtcagaa cgaagaccag
aacgtgggta tcaaagttgc actgcgtgca atggaagctc 3480 cgctgcgtca
gatcgtattg aactgcggcg aagaaccgtc tgttgttgct aacaccgtta 3540
aaggcggcga cggcaactac ggttacaacg cagcaaccga agaatacggc aacatgatcg
3600 acatgggtat cctggatcca accaaagtaa ctcgttctgc tctgcagtac
gcagcttctg 3660 tggctggcct gatgatcacc accgaatgca tggttaccga
cctgccgaaa aacgatgcag 3720 ctgacttagg cgctgctggc ggtatgggcg
gcatgggtgg catgggcggc atgatgtaat 3780 aataagcttg catgcctgca
ggtcgactct agaggatccc cgggtaccga gctcgaattc 3840 <210> SEQ ID
NO 27 <211> LENGTH: 480 <212> TYPE: DNA <213>
ORGANISM: E. coli <400> SEQUENCE: 27 gaattcaggc gctttttaga
ctggtcgtaa tgaaattcag caggatcaca ttctgcagat 60 gatcgaagcc
cgctctagac tcgagagcga taaaattatt cacctgactg acgacagttt 120
tgacacggat gtactcaaag cggacggggc gatcctcgtc gatttctggg cagagtggtg
180
cggtccgtgc aaaatgatcg ccccgattct ggatgaaatc gctgacgaat atcagggcaa
240 actgaccgtt gcaaaactga acatcgatca aaaccctggc actgcgccga
aatatggcat 300 ccgtggtatc ccgactctgc tgctgttcaa aaacggtgaa
gtggcggcaa ccaaagtggg 360 tgcactgtct aaaggtcagt tgaaagagtt
cctcgacgct aacctggcgc tcgaggatta 420 taaagatcat gatggcgatt
ataaagatca tgatgattaa taaggatccc cgggtaccga 480 <210> SEQ ID
NO 28 <211> LENGTH: 1140 <212> TYPE: DNA <213>
ORGANISM: Rattus norvegicus <400> SEQUENCE: 28 atggcaacca
cgcacgcgca ggggcacccg ccagtcttgg ggaatgatac tctccgggaa 60
cattatgatt acgtggggaa gctggcaggc aggctgcggg atccccctga gggtagcacc
120 ctcatcacca ccatcctctt cttggtcacc tgtagcttca tcgtcttgga
gaacctgatg 180 gttttgattg ccatctggaa aaacaataaa tttcataacc
gcatgtactt tttcatcggc 240 aacttggctc tctgcgacct gctggccggc
atagcctaca aggtcaacat tctgatgtcc 300 ggtaggaaga cgttcagcct
gtctccaaca gtgtggttcc tcagggaggg cagtatgttc 360 gtagccctgg
gcgcatccac atgcagctta ttggccattg ccattgagcg gcacctgacc 420
atgatcaaga tgaggccgta cgacgccaac aagaagcacc gcgtgttcct tctgattggg
480 atgtgctggc taattgcctt ctcgctgggt gccctgccca tcctgggctg
gaactgcctg 540 gagaactttc ccgactgctc taccatcttg cccctctact
ccaagaaata cattgccttt 600 ctcatcagca tcttcacagc cattctggtg
accatcgtca tcttgtacgc gcgcatctac 660 ttcctggtca agtccagcag
ccgcagggtg gccaaccaca actccgagag atccatggcc 720 cttctgcgga
ccgtagtgat cgtggtgagc gtgttcatcg cctgttggtc cccccttttc 780
atcctcttcc tcatcgatgt ggcctgcagg gcgaaggagt gctccatcct cttcaagagt
840 cagtggttca tcatgctggc tgtcctcaac tcggccatga accctgtcat
ctacacgctg 900 gccagcaaag agatgcggcg tgctttcttc cggttggtgt
gcggctgtct ggtcaagggc 960 aaggggaccc aggcctcccc gatgcagcct
gctcttgacc cgagcagaag taaatcaagc 1020 tccagtaaca acagcagcag
ccactctcca aaggtcaagg aagacctgcc ccatgtggct 1080 acctcttcct
gcgtcactga caaaacgagg tcgcttcaga atggggtcct ctgcaagtga 1140
<210> SEQ ID NO 29 <211> LENGTH: 379 <212> TYPE:
PRT <213> ORGANISM: Rattus norvegicus <400> SEQUENCE:
29 Met Ala Thr Thr His Ala Gln Gly His Pro Pro Val Leu Gly Asn Asp
1 5 10 15 Thr Leu Arg Glu His Tyr Asp Tyr Val Gly Lys Leu Ala Gly
Arg Leu 20 25 30 Arg Asp Pro Pro Glu Gly Ser Thr Leu Ile Thr Thr
Ile Leu Phe Leu 35 40 45 Val Thr Cys Ser Phe Ile Val Leu Glu Asn
Leu Met Val Leu Ile Ala 50 55 60 Ile Trp Lys Asn Asn Lys Phe His
Asn Arg Met Tyr Phe Phe Ile Gly 65 70 75 80 Asn Leu Ala Leu Cys Asp
Leu Leu Ala Gly Ile Ala Tyr Lys Val Asn 85 90 95 Ile Leu Met Ser
Gly Arg Lys Thr Phe Ser Leu Ser Pro Thr Val Trp 100 105 110 Phe Leu
Arg Glu Gly Ser Met Phe Val Ala Leu Gly Ala Ser Thr Cys 115 120 125
Ser Leu Leu Ala Ile Ala Ile Glu Arg His Leu Thr Met Ile Lys Met 130
135 140 Arg Pro Tyr Asp Ala Asn Lys Lys His Arg Val Phe Leu Leu Ile
Gly 145 150 155 160 Met Cys Trp Leu Ile Ala Phe Ser Leu Gly Ala Leu
Pro Ile Leu Gly 165 170 175 Trp Asn Cys Leu Glu Asn Phe Pro Asp Cys
Ser Thr Ile Leu Pro Leu 180 185 190 Tyr Ser Lys Lys Tyr Ile Ala Phe
Leu Ile Ser Ile Phe Thr Ala Ile 195 200 205 Leu Val Thr Ile Val Ile
Leu Tyr Ala Arg Ile Tyr Phe Leu Val Lys 210 215 220 Ser Ser Ser Arg
Arg Val Ala Asn His Asn Ser Glu Arg Ser Met Ala 225 230 235 240 Leu
Leu Arg Thr Val Val Ile Val Val Ser Val Phe Ile Ala Cys Trp 245 250
255 Ser Pro Leu Phe Ile Leu Phe Leu Ile Asp Val Ala Cys Arg Ala Lys
260 265 270 Glu Cys Ser Ile Leu Phe Lys Ser Gln Trp Phe Ile Met Leu
Ala Val 275 280 285 Leu Asn Ser Ala Met Asn Pro Val Ile Tyr Thr Leu
Ala Ser Lys Glu 290 295 300 Met Arg Arg Ala Phe Phe Arg Leu Val Cys
Gly Cys Leu Val Lys Gly 305 310 315 320 Lys Gly Thr Gln Ala Ser Pro
Met Gln Pro Ala Leu Asp Pro Ser Arg 325 330 335 Ser Lys Ser Ser Ser
Ser Asn Asn Ser Ser Ser His Ser Pro Lys Val 340 345 350 Lys Glu Asp
Leu Pro His Val Ala Thr Ser Ser Cys Val Thr Asp Lys 355 360 365 Thr
Arg Ser Leu Gln Asn Gly Val Leu Cys Lys 370 375 <210> SEQ ID
NO 30 <400> SEQUENCE: 30 000 <210> SEQ ID NO 31
<211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic nucleic acid primer sequence <400>
SEQUENCE: 31 aattgctagc tccaccagca tcccagtggt ta 32 <210> SEQ
ID NO 32 <211> LENGTH: 32 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic nucleic acid primer sequence
<400> SEQUENCE: 32 aattggatcc ttaagaagaa gaattgacgt tt 32
<210> SEQ ID NO 33 <211> LENGTH: 32 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleic acid primer
sequence <400> SEQUENCE: 33 aattggatcc agaagaagaa ttgacgtttc
ca 32 <210> SEQ ID NO 34 <211> LENGTH: 52 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic nucleic acid
primer sequence <400> SEQUENCE: 34 aattggatcc ttaatgatga
tgatgatgat gagaagaaga attgacgttt cc 52 <210> SEQ ID NO 35
<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic nucleic acid primer sequence <400>
SEQUENCE: 35 ttatggcaac cacgcacgcg cagg 24 <210> SEQ ID NO 36
<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic nucleic acid primer sequence <400>
SEQUENCE: 36 agaccgtcac ttgcagagga c 21 <210> SEQ ID NO 37
<211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic nucleic acid primer sequence <400>
SEQUENCE: 37 aattgctagc acgcacgcgc aggggcaccc gc 32 <210> SEQ
ID NO 38 <211> LENGTH: 34 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic nucleic acid primer sequence
<400> SEQUENCE: 38 aattggtacc tcacttgcag aggaccccat tctg 34
<210> SEQ ID NO 39 <211> LENGTH: 32 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleic acid primer
sequence <400> SEQUENCE: 39 aattgctagc acgcacgcgc aggggcaccc
gc 32
<210> SEQ ID NO 40 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleic acid primer
sequence <400> SEQUENCE: 40 ggtcgccacc atggtgagca a 21
<210> SEQ ID NO 41 <211> LENGTH: 31 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleic acid primer
sequence <400> SEQUENCE: 41 ttaaggatcc ttacttgtac agctcgtcca
t 31 <210> SEQ ID NO 42 <211> LENGTH: 21 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic nucleic acid
primer sequence <400> SEQUENCE: 42 ggtcgccacc atggtgagca a 21
<210> SEQ ID NO 43 <211> LENGTH: 31 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleic acid primer
sequence <400> SEQUENCE: 43 ttaaggatcc cttgtacagc tcgtccatgc
c 31 <210> SEQ ID NO 44 <211> LENGTH: 25 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 44 ccaatggaac ttaccaatga
cgcgg 25 <210> SEQ ID NO 45 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 45 gcttgcttac
gcaggaatgc tggg 24 <210> SEQ ID NO 46 <211> LENGTH: 45
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 46 cgcggctgca
gatgtttgaa ccaatggaac ttaccaatga cgcgg 45 <210> SEQ ID NO 47
<211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 47 gcgcctctag attattaatc agcttgctta cgcaggaatg ctggg 45
<210> SEQ ID NO 48 <211> LENGTH: 31 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 48 gctagactgg gcggttttat ggacagcaag
c 31 <210> SEQ ID NO 49 <211> LENGTH: 35 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 49 gcgttaataa ttcagaagaa
ctcgtcaaga aggcg 35 <210> SEQ ID NO 50 <211> LENGTH: 99
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 50 gcgcctactg
acgtagttcg accgtcggac tagcgaagtt cctatacttt ctagagaata 60
ggaacttcgc tagactgggc ggttttatgg acagcaagc 99 <210> SEQ ID NO
51 <211> LENGTH: 109 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 51 caagatgctt tgcctttgtc tgagttgata
ctggctttgg gaagttccta ttctctagaa 60 agtataggaa cttcgcgtta
ataattcaga agaactcgtc aagaaggcg 109 <210> SEQ ID NO 52
<211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 52 cgttaccaat tatgacaact tgacgg 26 <210> SEQ ID NO
53 <211> LENGTH: 29 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 53 ttaatctttc tgcgaattga gatgacgcc 29
<210> SEQ ID NO 54 <211> LENGTH: 28 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 54 gtgagtcgat attgtctttg ttgaccag 28
<210> SEQ ID NO 55 <211> LENGTH: 66 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 55 gcctgcattg cggcgcttca gtctccgctg
catactgtcc cgttaccaat tatgacaact 60 tgacgg 66 <210> SEQ ID NO
56 <211> LENGTH: 69 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 56 gcctgcattg cggcgcttca gtctccgctg
catactgtcc ttaatctttc tgcgaattga 60 gatgacgcc 69 <210> SEQ ID
NO 57 <211> LENGTH: 76 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 57 gcctgcattg cggcgcttca gtctccgctg
catactgtcc ttaataaagt gagtcgatat 60 tgtctttgtt gaccag 76
<210> SEQ ID NO 58 <211> LENGTH: 66 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 58 gcctgcattg cggcgcttca gtctccgctg
catactgtcc cgttaccaat tatgacaact 60 tgacgg 66 <210> SEQ ID NO
59 <211> LENGTH: 30 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 59 gcctgttctg gaaaaccggg ctgctcaggg 30
<210> SEQ ID NO 60 <211> LENGTH: 28 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 60 gcggctttca tggtgtagaa gagatcgg 28
<210> SEQ ID NO 61 <211> LENGTH: 43 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 61 ccgcgctgca gatgcctgtt ctggaaaacc
gggctgctca ggg 43 <210> SEQ ID NO 62 <211> LENGTH: 58
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 62 gcgcctctag
attattattt cagccccaga gcggctttca tggtgtagaa gagatcgg 58 <210>
SEQ ID NO 63 <211> LENGTH: 24 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 63 gtcacggccg agacttatag tcgc 24
<210> SEQ ID NO 64 <211> LENGTH: 28 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 64 gcggctttca tggtgtagaa gagatcgg 28
<210> SEQ ID NO 65 <211> LENGTH: 37 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 65 ccgcgctgca gatgtcacgg ccgagactta
tagtcgc 37 <210> SEQ ID NO 66 <211> LENGTH: 58
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 66 gcgcctctag
attattattt cagccccaga gcggctttca tggtgtagaa gagatcgg 58 <210>
SEQ ID NO 67 <211> LENGTH: 109 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 67 ccgcgctgca gatgaacttg gggaatcgac
tgtttattct gatagcggtc ttacttcccc 60 tcgcagtatt actgctcatg
cctgttctgg aaaaccgggc tgctcaggg 109 <210> SEQ ID NO 68
<211> LENGTH: 58 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 68 gcgcctctag attattattt cagccccaga gcggctttca tggtgtagaa
gagatcgg 58 <210> SEQ ID NO 69 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 69 gcgaattgag
atgacgccac tggc 24 <210> SEQ ID NO 70 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 70 cctgctgaat
ttcattaacg accag 25 <210> SEQ ID NO 71 <211> LENGTH: 48
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 71 cggcgaagct
taattaatct ttctgcgaat tgagatgacg ccactggc 48 <210> SEQ ID NO
72 <211> LENGTH: 53 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 72 cgccgtaatc gccgctgcag aatgtgatcc
tgctgaattt cattaacgac cag 53 <210> SEQ ID NO 73 <211>
LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 73
cgcagcgctg ttcctttgct cg 22 <210> SEQ ID NO 74 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 74
cctcattaag ataataatac tgg 23 <210> SEQ ID NO 75 <211>
LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 75
gccgcaagct tcgcagcgct gttcctttgc tcg 33 <210> SEQ ID NO 76
<211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 76 ccaatgcatt ggttctgcag gactcctcat taagataata atactgg 47
<210> SEQ ID NO 77 <211> LENGTH: 19 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 77 cgtctttagc cgggaaacg 19
<210> SEQ ID NO 78 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 78 gcagatctcc tggcttgc 18
<210> SEQ ID NO 79 <211> LENGTH: 30 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 79 gccgcaagct tcgtctttag ccgggaaacg
30 <210> SEQ ID NO 80 <211> LENGTH: 26 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence
<400> SEQUENCE: 80 cggtcgacgc agatctcctg gcttgc 26
<210> SEQ ID NO 81 <211> LENGTH: 23 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 81 caagccgtca attgtctgat tcg 23
<210> SEQ ID NO 82 <211> LENGTH: 22 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 82 ggtgaattcc tcctgctagc cc 22
<210> SEQ ID NO 83 <211> LENGTH: 34 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 83 gcgccaagct tcaagccgtc aattgtctga
ttcg 34 <210> SEQ ID NO 84 <211> LENGTH: 28 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 84 ctgcagggtg aattcctcct
gctagccc 28 <210> SEQ ID NO 85 <211> LENGTH: 42
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 85 gcttaactcg
agcttaataa caagccgtca attgtctgat tc 42 <210> SEQ ID NO 86
<211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 86 gcttaaccgc gggccaagct tgcatgcctg ctcc 34 <210>
SEQ ID NO 87 <211> LENGTH: 26 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 87 ggcaaccacg cacgcgcagg gccacc 26
<210> SEQ ID NO 88 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 88 caatggtgat ggtgatgatg accgg 25
<210> SEQ ID NO 89 <211> LENGTH: 38 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 89 cgcggtcgac atggcaacca cgcacgcgca
gggccacc 38 <210> SEQ ID NO 90 <211> LENGTH: 40
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 90 gcgccggtac
cttatcaatg gtgatggtga tgatgaccgg 40 <210> SEQ ID NO 91
<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 91 ggggcaaccc gggaacggca gcgcc 25 <210> SEQ ID NO
92 <211> LENGTH: 30 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 92 gcagtgagtc atttgtacta caattcctcc 30
<210> SEQ ID NO 93 <211> LENGTH: 37 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 93 cgcggtcgac atggggcaac ccgggaacgg
cagcgcc 37 <210> SEQ ID NO 94 <211> LENGTH: 49
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 94 gcgccggatc
cttattatag cagtgagtca tttgtactac aattcctcc 49 <210> SEQ ID NO
95 <211> LENGTH: 28 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 95 ggactggtcc ctcacctagg ggacaggg 28
<210> SEQ ID NO 96 <211> LENGTH: 27 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 96 ctgagaagac tgggcgcggg cgggagg 27
<210> SEQ ID NO 97 <211> LENGTH: 42 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 97 cgcgggtcga catgggactg gtccctcacc
taggggacag gg 42 <210> SEQ ID NO 98 <211> LENGTH: 44
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 98 gcgccggtac
cttattactg agaagactgg gcgcgggcgg gagg 44 <210> SEQ ID NO 99
<211> LENGTH: 16 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 99 gatagtgtgt gtcccc 16 <210> SEQ ID NO 100
<211> LENGTH: 17 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 100 ctgagaagac tgggcgc 17 <210> SEQ ID NO 101
<211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 101 gggagaccat ggatagtgtg tgtcccc 27
<210> SEQ ID NO 102 <211> LENGTH: 32 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 102 gcctcatcta gattactgag aagactgggc
gc 32 <210> SEQ ID NO 103 <211> LENGTH: 26 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 103 gagcactgaa agcatgatcc
gggacg 26 <210> SEQ ID NO 104 <211> LENGTH: 28
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 104
cagggcaatg atcccaaagt agacctgc 28 <210> SEQ ID NO 105
<211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 105 ccgcggaatt catgagcact gaaagcatga tccgggacg 39
<210> SEQ ID NO 106 <211> LENGTH: 43 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 106 ggcgcaagct tatcacaggg caatgatccc
aaagtagacc tgc 43 <210> SEQ ID NO 107 <211> LENGTH: 110
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 107
tctgatagcg gtcttacttc ccctcgcagt attactgctc aatagtgact ctgaatgtcc
60 cctgtcccac gatgggtact gcctccatga tggtgtgtgc atgtatattg 110
<210> SEQ ID NO 108 <211> LENGTH: 101 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 108 aggtctcggt actgacatcg ctccccgatg
tagccaacaa cacagttgca tgcatacttg 60 tccaatgctt caatatacat
gcacacacca tcatggaggc a 101 <210> SEQ ID NO 109 <211>
LENGTH: 63 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 109
ccgcgggtac catgaacttg gggaatcgac tgtttattct gatagcggtc ttacttcccc
60 tcg 63 <210> SEQ ID NO 110 <211> LENGTH: 61
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 110
gcgccaagct tattagcgca gttcccacca cttcaggtct cggtactgac atcgctcccc
60 g 61 <210> SEQ ID NO 111 <211> LENGTH: 22
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 111
tcattcacat tgagcgtcac cg 22 <210> SEQ ID NO 112 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 112
ttatattgac agcgcacaga gcgg 24 <210> SEQ ID NO 113 <211>
LENGTH: 101 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 113
gcaagaattc accatgaact tggggaatcg actgtttatt ctgatagcgg tcttacttcc
60 cctcgcagta ttactgctct cattcacatt gagcgtcacc g 101 <210>
SEQ ID NO 114 <211> LENGTH: 47 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 114 cgcggttacg taagcaactg cagttatatt
gacagcgcac agagcgg 47 <210> SEQ ID NO 115 <211> LENGTH:
24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 115
gtcacggccg agacttatag tcgc 24 <210> SEQ ID NO 116 <400>
SEQUENCE: 116 000 <210> SEQ ID NO 117 <211> LENGTH: 37
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 117
cgcggctgca gatgtcacgg ccgagactta tagtcgc 37 <210> SEQ ID NO
118 <211> LENGTH: 38 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 118 cgcggtctag attctggtgt ccgggctttt gtcacagg
38 <210> SEQ ID NO 119 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 119 cagccccaga gcggctttca tgg
23 <210> SEQ ID NO 120 <211> LENGTH: 37 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 120 cgcggtctag atttcagccc
cagagcggct ttcatgg 37 <210> SEQ ID NO 121 <211> LENGTH:
106 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 121
cgcggctgca gatgaaaata aaaacaggtg cacgcatcct cgcattatcc gcattaacga
60 cgatgatgtt ttccgcctcg gctctcgcca aaatctctag acgcgg 106
<210> SEQ ID NO 122 <211> LENGTH: 106 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 122 ccgcgtctag agattttggc gagagccgag
gcggaaaaca tcatcgtcgt taatgcggat 60 aatgcgagga tgcgtgcacc
tgtttttatt ttcatctgca gccgcg 106 <210> SEQ ID NO 123
<211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 123 ggtgcacgca tcctcgcatt atccgc 26 <210> SEQ ID NO
124 <211> LENGTH: 26 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 124 cggcatacca gaaagcggac atctgc 26
<210> SEQ ID NO 125 <211> LENGTH: 52 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 125 cgcggctgca gatgaaaata aaaacaggtg
cacgcatcct cgcattatcc gc 52 <210> SEQ ID NO 126 <211>
LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 126
cgcggtctag aacgcacggc ataccagaaa gcggacatct gc 42 <210> SEQ
ID NO 127 <211> LENGTH: 27 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 127 cgcgacagcg cgcaataacc gttctcg 27
<210> SEQ ID NO 128 <211> LENGTH: 27 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 128 gctggttcat cagctcgttg aaagtgg 27
<210> SEQ ID NO 129 <211> LENGTH: 41 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 129 gcgccggcgc catacgcgac agcgcgcaat
aaccgttctc g 41 <210> SEQ ID NO 130 <211> LENGTH: 52
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 130
ggcgctctag attattatta cgcctgctgg ttcatcagct cgttgaaagt gg 52
<210> SEQ ID NO 131 <211> LENGTH: 29 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 131 ggtagcacaa tcagattcgc ttatgacgg
29 <210> SEQ ID NO 132 <211> LENGTH: 25 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 132 gccgcccatg ccacccatgc
cgccc 25 <210> SEQ ID NO 133 <211> LENGTH: 38
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 133
gcgtctagag gtagcacaat cagattcgct tatgacgg 38 <210> SEQ ID NO
134 <211> LENGTH: 49 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 134 ggcgcaagct tattattaca tcatgccgcc
catgccaccc atgccgccc 49 <210> SEQ ID NO 135 <211>
LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 135
gcgataaaat tattcacctg actgacg 27 <210> SEQ ID NO 136
<211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 136 gcgtcgagga actctttcaa ctgacc 26 <210> SEQ ID NO
137 <211> LENGTH: 66 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 137 cgcggctgca gatgatcgaa gcccgctcta
gactcgagag cgataaaatt attcacctga 60 ctgacg 66 <210> SEQ ID NO
138 <211> LENGTH: 101 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 138 ccgcgggatc cttattaatc atcatgatct
ttataatcgc catcatgatc tttataatcc 60 tcgagcgcca ggttagcgtc
gaggaactct ttcaactgac c 101 <210> SEQ ID NO 139 <400>
SEQUENCE: 139 000 <210> SEQ ID NO 140 <400> SEQUENCE:
140 000 <210> SEQ ID NO 141 <400> SEQUENCE: 141 000
<210> SEQ ID NO 142 <400> SEQUENCE: 142 000 <210>
SEQ ID NO 143 <211> LENGTH: 65 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 143 tatgtaagga ggttgtcgac cggctcagtc
tagaggtacc cgccctcatc cgaaagggcg 60
tattg 65 <210> SEQ ID NO 144 <211> LENGTH: 67
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 144
gatccaatac gccctttcgg atgagggcgg gtacctctag actgagccgg tcgacaacct
60 ccttaca 67 <210> SEQ ID NO 145 <211> LENGTH: 65
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 145
tatgtaagga ggttctgcag cggctcagtc tagaggtacc cgccctcatc cgaaagggcg
60 tattg 65 <210> SEQ ID NO 146 <211> LENGTH: 67
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 146
gatccaatac gccctttcgg atgagggcgg gtacctctag actgagccgc tgcagaacct
60 ccttaca 67 <210> SEQ ID NO 147 <211> LENGTH: 66
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 147
gatcctaagg aggttgtcga ccggctcagt ctagaggtac ccgccctcat ccgaaagggc
60 gtattc 66 <210> SEQ ID NO 148 <211> LENGTH: 66
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 148
tcgagaatac gccctttcgg atgagggcgg gtacctctag actgagccgg tcgacaacct
60 ccttag 66 <210> SEQ ID NO 149 <211> LENGTH: 66
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 149
gatcctaagg aggttctgca gcggctcagt ctagaggtac ccgccctcat ccgaaagggc
60 gtattc 66 <210> SEQ ID NO 150 <211> LENGTH: 66
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 150
tcgagaatac gccctttcgg atgagggcgg gtacctctag actgagccgc tgcagaacct
60 ccttag 66 <210> SEQ ID NO 151 <211> LENGTH: 4740
<212> TYPE: DNA <213> ORGANISM: E. coli <400>
SEQUENCE: 151 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca
gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg
cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg
gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa
gcttaattaa tctttctgcg 420 aattgagatg acgccactgg ctgggcgtca
tcccggtttc ccgggtaaac accaccgaaa 480 aatagttact atcttcaaag
ccacattcgg tcgaaatatc actgattaac aggcggctat 540 gctggagaag
atattgcgca tgacacactc tgacctgtcg cagatattga ttgatggtca 600
ttccagtctg ctggcgaaat tgctgacgca aaacgcgctc actgcacgat gcctcatcac
660 aaaatttatc cagcgcaaag ggacttttca ggctagccgc cagccgggta
atcagcttat 720 ccagcaacgt ttcgctggat gttggcggca acgaatcact
ggtgtaacga tggcgattca 780 gcaacatcac caactgcccg aacagcaact
cagccatttc gttagcaaac ggcacatgct 840 gactactttc atgctcaagc
tgaccgataa cctgccgcgc ctgcgccatc cccatgctac 900 ctaagcgcca
gtgtggttgc cctgcgctgg cgttaaatcc cggaatcgcc ccctgccagt 960
caagattcag cttcagacgc tccgggcaat aaataatatt ctgcaaaacc agatcgttaa
1020 cggaagcgta ggagtgttta tcgtcagcat gaatgtaaaa gagatcgcca
cgggtaatgc 1080 gataagggcg atcgttgagt acatgcaggc cattaccgcg
ccagacaatc accagctcac 1140 aaaaatcatg tgtatgttca gcaaagacat
cttgcggata acggtcagcc acagcgactg 1200 cctgctggtc gctggcaaaa
aaatcatctt tgagaagttt taactgatgc gccaccgtgg 1260 ctacctcggc
cagagaacga agttgattat tcgcaatatg gcgtacaaat acgttgagaa 1320
gattcgcgtt attgcagaaa gccatcccgt ccctggcgaa tatcacgcgg tgaccagtta
1380 aactctcggc gaaaaagcgt cgaaaagtgg ttactgtcgc tgaatccaca
gcgataggcg 1440 atgtcagtaa cgctggcctc gctgtggcgt agcagatgtc
gggctttcat cagtcgcagg 1500 cggttcaggt atcgctgagg cgtcagtccc
gtttgctgct taagctgccg atgtagcgta 1560 cgcagtgaaa gagaaaattg
atccgccacg gcatcccaat tcacctcatc ggcaaaatgg 1620 tcctccagcc
aggccagaag caagttgaga cgtgatgcgc tgttttccag gttctcctgc 1680
aaactgcttt tacgcagcaa gagcagtaat tgcataaaca agatctcgcg actggcggtc
1740 gagggtaaat cattttcccc ttcctgctgt tccatctgtg caaccagctg
tcgcacctgc 1800 tgcaatacgc tgtggttaac gcgccagtga gacggatact
gcccatccag ctcttgtggc 1860 agcaactgat tcagcccggc gagaaactga
aatcgatccg gcgagcgata cagcacattg 1920 gtcagacaca gattatcggt
atgttcatac agatgccgat catgatcgcg tacgaaacag 1980 accgtgccac
cggtgatggt atagggctgc ccattaaaca catgaatacc cgtgccatgt 2040
tcgacaatca caatttcatg aaaatcatga tgatgttcag gaaaatccgc ctgcgggagc
2100 cggggttcta tcgccacgga cgcgttacca gacggaaaaa aatccacact
atgtaatacg 2160 gtcatactgg cctcctgatg tcgtcaacac ggcgaaatag
taatcacgag gtcaggttct 2220 taccttaaat tttcgacgga aaaccacgta
aaaaacgtcg atttttcaag atacagcgtg 2280 aattttcagg aaatgcggtg
agcatcacat caccacaatt cagcaaattg tgaacatcat 2340 cacgttcatc
tttccctggt tgccaatggc ccattttcct gtcagtaacg agaaggtcgc 2400
gaattcaggc gctttttaga ctggtcgtaa tgaaattcag gaggttgtcg actctagagg
2460 atccccgcgc cctcatccga aagggcgtat tggtaccgag ctcgaattcg
taatcatggt 2520 catagctgtt tcctgtgtga aattgttatc cgctcacaat
tccacacaac atacgagccg 2580 gaagcataaa gtgtaaagcc tggggtgcct
aatgagtgag ctaactcaca ttaattgcgt 2640 tgcgctcact gcccgctttc
cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg 2700 gccaacgcgc
ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg 2760
actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
2820 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
aaaggccagc 2880 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
tttccatagg ctccgccccc 2940 ctgacgagca tcacaaaaat cgacgctcaa
gtcagaggtg gcgaaacccg acaggactat 3000 aaagatacca ggcgtttccc
cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc 3060 cgcttaccgg
atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct 3120
cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
3180 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
gagtccaacc 3240 cggtaagaca cgacttatcg ccactggcag cagccactgg
taacaggatt agcagagcga 3300 ggtatgtagg cggtgctaca gagttcttga
agtggtggcc taactacggc tacactagaa 3360 ggacagtatt tggtatctgc
gctctgctga agccagttac cttcggaaaa agagttggta 3420 gctcttgatc
cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc 3480
agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
3540 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
tcaaaaagga 3600 tcttcaccta gatcctttta aattaaaaat gaagttttaa
atcaatctaa agtatatatg 3660 agtaaacttg gtctgacagt taccaatgct
taatcagtga ggcacctatc tcagcgatct 3720 gtctatttcg ttcatccata
gttgcctgac tccccgtcgt gtagataact acgatacggg 3780 agggcttacc
atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc 3840
cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
3900 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
agtagttcgc 3960 cagttaatag tttgcgcaac gttgttgcca ttgctacagg
catcgtggtg tcacgctcgt 4020 cgtttggtat ggcttcattc agctccggtt
cccaacgatc aaggcgagtt acatgatccc 4080 ccatgttgtg caaaaaagcg
gttagctcct tcggtcctcc gatcgttgtc agaagtaagt 4140 tggccgcagt
gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc 4200
catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
4260 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
gcgccacata 4320
gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
4380 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
tgatcttcag 4440 catcttttac tttcaccagc gtttctgggt gagcaaaaac
aggaaggcaa aatgccgcaa 4500 aaaagggaat aagggcgaca cggaaatgtt
gaatactcat actcttcctt tttcaatatt 4560 attgaagcat ttatcagggt
tattgtctca tgagcggata catatttgaa tgtatttaga 4620 aaaataaaca
aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag 4680
aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
4740 <210> SEQ ID NO 152 <211> LENGTH: 4746 <212>
TYPE: DNA <213> ORGANISM: E. coli <400> SEQUENCE: 152
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
60 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
tcagcgggtg 120 ttggcgggtg tcggggctgg cttaactatg cggcatcaga
gcagattgta ctgagagtgc 180 accatatgcg gtgtgaaata ccgcacagat
gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc
aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct
ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gcttaattaa tctttctgcg
420 aattgagatg acgccactgg ctgggcgtca tcccggtttc ccgggtaaac
accaccgaaa 480 aatagttact atcttcaaag ccacattcgg tcgaaatatc
actgattaac aggcggctat 540 gctggagaag atattgcgca tgacacactc
tgacctgtcg cagatattga ttgatggtca 600 ttccagtctg ctggcgaaat
tgctgacgca aaacgcgctc actgcacgat gcctcatcac 660 aaaatttatc
cagcgcaaag ggacttttca ggctagccgc cagccgggta atcagcttat 720
ccagcaacgt ttcgctggat gttggcggca acgaatcact ggtgtaacga tggcgattca
780 gcaacatcac caactgcccg aacagcaact cagccatttc gttagcaaac
ggcacatgct 840 gactactttc atgctcaagc tgaccgataa cctgccgcgc
ctgcgccatc cccatgctac 900 ctaagcgcca gtgtggttgc cctgcgctgg
cgttaaatcc cggaatcgcc ccctgccagt 960 caagattcag cttcagacgc
tccgggcaat aaataatatt ctgcaaaacc agatcgttaa 1020 cggaagcgta
ggagtgttta tcgtcagcat gaatgtaaaa gagatcgcca cgggtaatgc 1080
gataagggcg atcgttgagt acatgcaggc cattaccgcg ccagacaatc accagctcac
1140 aaaaatcatg tgtatgttca gcaaagacat cttgcggata acggtcagcc
acagcgactg 1200 cctgctggtc gctggcaaaa aaatcatctt tgagaagttt
taactgatgc gccaccgtgg 1260 ctacctcggc cagagaacga agttgattat
tcgcaatatg gcgtacaaat acgttgagaa 1320 gattcgcgtt attgcagaaa
gccatcccgt ccctggcgaa tatcacgcgg tgaccagtta 1380 aactctcggc
gaaaaagcgt cgaaaagtgg ttactgtcgc tgaatccaca gcgataggcg 1440
atgtcagtaa cgctggcctc gctgtggcgt agcagatgtc gggctttcat cagtcgcagg
1500 cggttcaggt atcgctgagg cgtcagtccc gtttgctgct taagctgccg
atgtagcgta 1560 cgcagtgaaa gagaaaattg atccgccacg gcatcccaat
tcacctcatc ggcaaaatgg 1620 tcctccagcc aggccagaag caagttgaga
cgtgatgcgc tgttttccag gttctcctgc 1680 aaactgcttt tacgcagcaa
gagcagtaat tgcataaaca agatctcgcg actggcggtc 1740 gagggtaaat
cattttcccc ttcctgctgt tccatctgtg caaccagctg tcgcacctgc 1800
tgcaatacgc tgtggttaac gcgccagtga gacggatact gcccatccag ctcttgtggc
1860 agcaactgat tcagcccggc gagaaactga aatcgatccg gcgagcgata
cagcacattg 1920 gtcagacaca gattatcggt atgttcatac agatgccgat
catgatcgcg tacgaaacag 1980 accgtgccac cggtgatggt atagggctgc
ccattaaaca catgaatacc cgtgccatgt 2040 tcgacaatca caatttcatg
aaaatcatga tgatgttcag gaaaatccgc ctgcgggagc 2100 cggggttcta
tcgccacgga cgcgttacca gacggaaaaa aatccacact atgtaatacg 2160
gtcatactgg cctcctgatg tcgtcaacac ggcgaaatag taatcacgag gtcaggttct
2220 taccttaaat tttcgacgga aaaccacgta aaaaacgtcg atttttcaag
atacagcgtg 2280 aattttcagg aaatgcggtg agcatcacat caccacaatt
cagcaaattg tgaacatcat 2340 cacgttcatc tttccctggt tgccaatggc
ccattttcct gtcagtaacg agaaggtcgc 2400 gaattcaggc gctttttaga
ctggtcgtaa tgaaattcag gaggttctgc aggtcgactc 2460 tagaggatcc
ccgcgccctc atccgaaagg gcgtattggt accgagctcg aattcgtaat 2520
catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
2580 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
ctcacattaa 2640 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
gtcgtgccag ctgcattaat 2700 gaatcggcca acgcgcgggg agaggcggtt
tgcgtattgg gcgctcttcc gcttcctcgc 2760 tcactgactc gctgcgctcg
gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg 2820 cggtaatacg
gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag 2880
gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
2940 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
aacccgacag 3000 gactataaag ataccaggcg tttccccctg gaagctccct
cgtgcgctct cctgttccga 3060 ccctgccgct taccggatac ctgtccgcct
ttctcccttc gggaagcgtg gcgctttctc 3120 atagctcacg ctgtaggtat
ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg 3180 tgcacgaacc
ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt 3240
ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
3300 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
tacggctaca 3360 ctagaaggac agtatttggt atctgcgctc tgctgaagcc
agttaccttc ggaaaaagag 3420 ttggtagctc ttgatccggc aaacaaacca
ccgctggtag cggtggtttt tttgtttgca 3480 agcagcagat tacgcgcaga
aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg 3540 ggtctgacgc
tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa 3600
aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
3660 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
cctatctcag 3720 cgatctgtct atttcgttca tccatagttg cctgactccc
cgtcgtgtag ataactacga 3780 tacgggaggg cttaccatct ggccccagtg
ctgcaatgat accgcgagac ccacgctcac 3840 cggctccaga tttatcagca
ataaaccagc cagccggaag ggccgagcgc agaagtggtc 3900 ctgcaacttt
atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta 3960
gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
4020 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
cgagttacat 4080 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
tcctccgatc gttgtcagaa 4140 gtaagttggc cgcagtgtta tcactcatgg
ttatggcagc actgcataat tctcttactg 4200 tcatgccatc cgtaagatgc
ttttctgtga ctggtgagta ctcaaccaag tcattctgag 4260 aatagtgtat
gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc 4320
cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
4380 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
cccaactgat 4440 cttcagcatc ttttactttc accagcgttt ctgggtgagc
aaaaacagga aggcaaaatg 4500 ccgcaaaaaa gggaataagg gcgacacgga
aatgttgaat actcatactc ttcctttttc 4560 aatattattg aagcatttat
cagggttatt gtctcatgag cggatacata tttgaatgta 4620 tttagaaaaa
taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 4680
tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
4740 ttcgtc 4746 <210> SEQ ID NO 153 <211> LENGTH: 3946
<212> TYPE: DNA <213> ORGANISM: E. coli <400>
SEQUENCE: 153 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat gccgggagca
gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg tcggggctgg
cttaactatg cggcatcaga gcagattgta ctgagagtgc 180 accatatgcg
gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa
gcttcaagcc gtcaattgtc 420 tgattcgtta ccaattatga caacttgacg
gctacatcat tcactttttc ttcacaaccg 480 gcacggaact cgctcgggct
ggccccggtg cattttttaa atacccgcga gaaatagagt 540 tgatcgtcaa
aaccaacatt gcgaccgacg gtggcgatag gcatccgggt ggtgctcaaa 600
agcagcttcg cctggctgat acgttggtcc tcgcgccagc ttaagacgct aatccctaac
660 tgctggcgga aaagatgtga cagacgcgac ggcgacaagc aaacatgctg
tgcgacgctg 720 gcgatatcaa aattgctgtc tgccaggtga tcgctgatgt
actgacaagc ctcgcgtacc 780 cgattatcca tcggtggatg gagcgactcg
ttaatcgctt ccatgcgccg cagtaacaat 840 tgctcaagca gatttatcgc
cagcagctcc gaatagcgcc cttccccttg cccggcgtta 900 atgatttgcc
caaacaggtc gctgaaatgc ggctggtgcg cttcatccgg gcgaaagaac 960
cccgtattgg caaatattga cggccagtta agccattcat gccagtaggc gcgcggacga
1020 aagtaaaccc actggtgata ccattcgcga gcctccggat gacgaccgta
gtgatgaatc 1080 tctcctggcg ggaacagcaa aatatcaccc ggtcggcaaa
caaattctcg tccctgattt 1140 ttcaccaccc cctgaccgcg aatggtgaga
ttgagaatat aacctttcat tcccagcggt 1200 cggtcgataa aaaaatcgag
ataaccgttg gcctcaatcg gcgttaaacc cgccaccaga 1260 tgggcattaa
acgagtatcc cggcagcagg ggatcatttt gcgcttcagc catacttttc 1320
atactcccgc cattcagaga agaaaccaat tgtccatatt gcatcagaca ttgccgtcac
1380 tgcgtctttt actggctctt ctcgctaacc aaaccggtaa ccccgcttat
taaaagcatt 1440 ctgtaacaaa gcgggaccaa agccatgaca aaaacgcgta
acaaaagtgt ctataatcac 1500 ggcagaaaag tccacattga ttatttgcac
ggcgtcacac tttgctatgc catagcattt 1560 ttatccataa gattagcgga
tcctacctga cgctttttat cgcaactctc tactgtttct 1620 ccatacccgt
ttttttgggc tagcaggagg ccgtcgactc tagaggatcc ccgcgccctc 1680
atccgaaagg gcgtattggt accgagctcg aattcgtaat catggtcata gctgtttcct
1740 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
cataaagtgt 1800
aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
1860 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
acgcgcgggg 1920 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
tcactgactc gctgcgctcg 1980 gtcgttcggc tgcggcgagc ggtatcagct
cactcaaagg cggtaatacg gttatccaca 2040 gaatcagggg ataacgcagg
aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 2100 cgtaaaaagg
ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 2160
aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
2220 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
taccggatac 2280 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
atagctcacg ctgtaggtat 2340 ctcagttcgg tgtaggtcgt tcgctccaag
ctgggctgtg tgcacgaacc ccccgttcag 2400 cccgaccgct gcgccttatc
cggtaactat cgtcttgagt ccaacccggt aagacacgac 2460 ttatcgccac
tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 2520
gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt
2580 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
ttgatccggc 2640 aaacaaacca ccgctggtag cggtggtttt tttgtttgca
agcagcagat tacgcgcaga 2700 aaaaaaggat ctcaagaaga tcctttgatc
ttttctacgg ggtctgacgc tcagtggaac 2760 gaaaactcac gttaagggat
tttggtcatg agattatcaa aaaggatctt cacctagatc 2820 cttttaaatt
aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 2880
gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
2940 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
cttaccatct 3000 ggccccagtg ctgcaatgat accgcgagac ccacgctcac
cggctccaga tttatcagca 3060 ataaaccagc cagccggaag ggccgagcgc
agaagtggtc ctgcaacttt atccgcctcc 3120 atccagtcta ttaattgttg
ccgggaagct agagtaagta gttcgccagt taatagtttg 3180 cgcaacgttg
ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct 3240
tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
3300 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
cgcagtgtta 3360 tcactcatgg ttatggcagc actgcataat tctcttactg
tcatgccatc cgtaagatgc 3420 ttttctgtga ctggtgagta ctcaaccaag
tcattctgag aatagtgtat gcggcgaccg 3480 agttgctctt gcccggcgtc
aatacgggat aataccgcgc cacatagcag aactttaaaa 3540 gtgctcatca
ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg 3600
agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
3660 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
gggaataagg 3720 gcgacacgga aatgttgaat actcatactc ttcctttttc
aatattattg aagcatttat 3780 cagggttatt gtctcatgag cggatacata
tttgaatgta tttagaaaaa taaacaaata 3840 ggggttccgc gcacatttcc
ccgaaaagtg ccacctgacg tctaagaaac cattattatc 3900 atgacattaa
cctataaaaa taggcgtatc acgaggccct ttcgtc 3946 <210> SEQ ID NO
154 <211> LENGTH: 3952 <212> TYPE: DNA <213>
ORGANISM: E. coli <400> SEQUENCE: 154 tcgcgcgttt cggtgatgac
ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60 cagcttgtct
gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc
ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg
caaggcgatt aagttgggta acgccagggt 360 tttcccagtc acgacgttgt
aaaacgacgg ccagtgccaa gcttcaagcc gtcaattgtc 420 tgattcgtta
ccaattatga caacttgacg gctacatcat tcactttttc ttcacaaccg 480
gcacggaact cgctcgggct ggccccggtg cattttttaa atacccgcga gaaatagagt
540 tgatcgtcaa aaccaacatt gcgaccgacg gtggcgatag gcatccgggt
ggtgctcaaa 600 agcagcttcg cctggctgat acgttggtcc tcgcgccagc
ttaagacgct aatccctaac 660 tgctggcgga aaagatgtga cagacgcgac
ggcgacaagc aaacatgctg tgcgacgctg 720 gcgatatcaa aattgctgtc
tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc 780 cgattatcca
tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg cagtaacaat 840
tgctcaagca gatttatcgc cagcagctcc gaatagcgcc cttccccttg cccggcgtta
900 atgatttgcc caaacaggtc gctgaaatgc ggctggtgcg cttcatccgg
gcgaaagaac 960 cccgtattgg caaatattga cggccagtta agccattcat
gccagtaggc gcgcggacga 1020 aagtaaaccc actggtgata ccattcgcga
gcctccggat gacgaccgta gtgatgaatc 1080 tctcctggcg ggaacagcaa
aatatcaccc ggtcggcaaa caaattctcg tccctgattt 1140 ttcaccaccc
cctgaccgcg aatggtgaga ttgagaatat aacctttcat tcccagcggt 1200
cggtcgataa aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacc cgccaccaga
1260 tgggcattaa acgagtatcc cggcagcagg ggatcatttt gcgcttcagc
catacttttc 1320 atactcccgc cattcagaga agaaaccaat tgtccatatt
gcatcagaca ttgccgtcac 1380 tgcgtctttt actggctctt ctcgctaacc
aaaccggtaa ccccgcttat taaaagcatt 1440 ctgtaacaaa gcgggaccaa
agccatgaca aaaacgcgta acaaaagtgt ctataatcac 1500 ggcagaaaag
tccacattga ttatttgcac ggcgtcacac tttgctatgc catagcattt 1560
ttatccataa gattagcgga tcctacctga cgctttttat cgcaactctc tactgtttct
1620 ccatacccgt ttttttgggc tagcaggagg ccctgcaggt cgactctaga
ggatccccgc 1680 gccctcatcc gaaagggcgt attggtaccg agctcgaatt
cgtaatcatg gtcatagctg 1740 tttcctgtgt gaaattgtta tccgctcaca
attccacaca acatacgagc cggaagcata 1800 aagtgtaaag cctggggtgc
ctaatgagtg agctaactca cattaattgc gttgcgctca 1860 ctgcccgctt
tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc 1920
gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
1980 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
aatacggtta 2040 tccacagaat caggggataa cgcaggaaag aacatgtgag
caaaaggcca gcaaaaggcc 2100 aggaaccgta aaaaggccgc gttgctggcg
tttttccata ggctccgccc ccctgacgag 2160 catcacaaaa atcgacgctc
aagtcagagg tggcgaaacc cgacaggact ataaagatac 2220 caggcgtttc
cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 2280
ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
2340 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
cgaacccccc 2400 gttcagcccg accgctgcgc cttatccggt aactatcgtc
ttgagtccaa cccggtaaga 2460 cacgacttat cgccactggc agcagccact
ggtaacagga ttagcagagc gaggtatgta 2520 ggcggtgcta cagagttctt
gaagtggtgg cctaactacg gctacactag aaggacagta 2580 tttggtatct
gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 2640
tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
2700 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
tgacgctcag 2760 tggaacgaaa actcacgtta agggattttg gtcatgagat
tatcaaaaag gatcttcacc 2820 tagatccttt taaattaaaa atgaagtttt
aaatcaatct aaagtatata tgagtaaact 2880 tggtctgaca gttaccaatg
cttaatcagt gaggcaccta tctcagcgat ctgtctattt 2940 cgttcatcca
tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 3000
ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
3060 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
aactttatcc 3120 gcctccatcc agtctattaa ttgttgccgg gaagctagag
taagtagttc gccagttaat 3180 agtttgcgca acgttgttgc cattgctaca
ggcatcgtgg tgtcacgctc gtcgtttggt 3240 atggcttcat tcagctccgg
ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 3300 tgcaaaaaag
cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 3360
gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
3420 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
gtgtatgcgg 3480 cgaccgagtt gctcttgccc ggcgtcaata cgggataata
ccgcgccaca tagcagaact 3540 ttaaaagtgc tcatcattgg aaaacgttct
tcggggcgaa aactctcaag gatcttaccg 3600 ctgttgagat ccagttcgat
gtaacccact cgtgcaccca actgatcttc agcatctttt 3660 actttcacca
gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 3720
ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
3780 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
gaaaaataaa 3840 caaatagggg ttccgcgcac atttccccga aaagtgccac
ctgacgtcta agaaaccatt 3900 attatcatga cattaaccta taaaaatagg
cgtatcacga ggccctttcg tc 3952 <210> SEQ ID NO 155 <211>
LENGTH: 3886 <212> TYPE: DNA <213> ORGANISM: E. coli
<400> SEQUENCE: 155 tcgcgcgttt cggtgatgac ggtgaaaacc
tctgacacat gcagctcccg gagacggtca 60 cagcttgtct gtaagcggat
gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120 ttggcgggtg
tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc
tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
aagttgggta acgccagggt 360 tttcccagtc acgacgttgt aaaacgacgg
ccagtgccaa gcttttagcc gggaaacgtc 420 tggcggcgct gttggctaag
tttgcggtat tgttgcggcg acatgccgac atatttgccg 480 aacgtgctgt
aaaaacgact acttgaacga aagcctgccg tcagggcaat atcgagaata 540
cttttatcgg tatcgctcag taacgcgcga acgtggttga tgcgcatcgc ggtaatgtac
600 tgtttcatcg tcaattgcat gacccgctgg aatatcccca ttgcatagtt
ggcgttaagt 660 ttgacgtgct cagccacatc gttgatggtc agcgcctgat
catagttttc ggcaataaag 720 cccagcatct ggctaacata aaattgcgca
tggcgcgaga cgctgttttt gtgtgtgcgc 780 gaggttttat tgaccagaat
cggttcccag ccagagaggc taaatcgctt gagcatcagg 840 ccaatttcat
caatggcgag ctggcgaatt tgctcgttcg gactgtttaa ttcctgctgc 900
cagcggcgca cttcaaacgg gctaagttgc tgtgtggcca gtgatttgat caccatgccg
960
tgagtgacgt ggttaatcag gtctttatcc agcggccagg agagaaacag atgcatcggc
1020 agattaaaaa tcgccatgct ctgacaggtt ccggtatctg ttagttggtg
cggtgtacag 1080 gcccagaaca gcgtgatatg accctgattg atattcactt
tttcattgtt gatcaggtat 1140 tccacatcgc catcgaaagg cacattcact
tcgacctgac catgccagtg gctggtgggc 1200 atgatatgcg gtgcgcgaaa
ctcaatctcc atccgctggt attccgaata cagcgacagc 1260 gggctgcggg
tctgtttttc gtcgctgctg cacataaacg tatctgtatt catggatggc 1320
tctctttcct ggaatatcag aattatggca ggagtgaggg aggatgactg cgagtgggag
1380 cacggttttc accctcttcc cagaggggcg aggggactct ccgagtatca
tgaggccgaa 1440 aactctgctt ttcaggtaat ttattcccat aaactcagat
ttactgctgc ttcacgcagg 1500 atctgagttt atgggaatgc tcaacctgga
agccggaggt tttctgcaga ttcgcctgcc 1560 atgatgaagt tattcaagca
agccaggagg tcgtcgactc tagaggatcc ccgcgccctc 1620 atccgaaagg
gcgtattggt accgagctcg aattcgtaat catggtcata gctgtttcct 1680
gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
1740 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
ctcactgccc 1800 gctttccagt cgggaaacct gtcgtgccag ctgcattaat
gaatcggcca acgcgcgggg 1860 agaggcggtt tgcgtattgg gcgctcttcc
gcttcctcgc tcactgactc gctgcgctcg 1920 gtcgttcggc tgcggcgagc
ggtatcagct cactcaaagg cggtaatacg gttatccaca 1980 gaatcagggg
ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 2040
cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
2100 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
ataccaggcg 2160 tttccccctg gaagctccct cgtgcgctct cctgttccga
ccctgccgct taccggatac 2220 ctgtccgcct ttctcccttc gggaagcgtg
gcgctttctc atagctcacg ctgtaggtat 2280 ctcagttcgg tgtaggtcgt
tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 2340 cccgaccgct
gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 2400
ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
2460 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac
agtatttggt 2520 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
ttggtagctc ttgatccggc 2580 aaacaaacca ccgctggtag cggtggtttt
tttgtttgca agcagcagat tacgcgcaga 2640 aaaaaaggat ctcaagaaga
tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 2700 gaaaactcac
gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 2760
cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
2820 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
atttcgttca 2880 tccatagttg cctgactccc cgtcgtgtag ataactacga
tacgggaggg cttaccatct 2940 ggccccagtg ctgcaatgat accgcgagac
ccacgctcac cggctccaga tttatcagca 3000 ataaaccagc cagccggaag
ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc 3060 atccagtcta
ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg 3120
cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
3180 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
gttgtgcaaa 3240 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
gtaagttggc cgcagtgtta 3300 tcactcatgg ttatggcagc actgcataat
tctcttactg tcatgccatc cgtaagatgc 3360 ttttctgtga ctggtgagta
ctcaaccaag tcattctgag aatagtgtat gcggcgaccg 3420 agttgctctt
gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa 3480
gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
3540 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
ttttactttc 3600 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
ccgcaaaaaa gggaataagg 3660 gcgacacgga aatgttgaat actcatactc
ttcctttttc aatattattg aagcatttat 3720 cagggttatt gtctcatgag
cggatacata tttgaatgta tttagaaaaa taaacaaata 3780 ggggttccgc
gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc 3840
atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc 3886 <210>
SEQ ID NO 156 <211> LENGTH: 28 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 156 gcagaacctc ctgaatttca ttacgacc
28 <210> SEQ ID NO 157 <211> LENGTH: 86 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 157 ccgcgggtac caatacgccc
tttcggatga gggcgcgggg atcctctaga gtcgacgtcg 60 acaacctcct
gaatttcatt acgacc 86 <210> SEQ ID NO 158 <211> LENGTH:
86 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 158
ccgcgggtac caatacgccc tttcggatga gggcgcgggg atcctctaga gtcgacctgc
60 agaacctcct gaatttcatt acgacc 86 <210> SEQ ID NO 159
<211> LENGTH: 38 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 159 ctgcagggcc tcctgctagc ccaaaaaaac gggtatgg 38
<210> SEQ ID NO 160 <211> LENGTH: 94 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 160 ccgcgggtac caatacgccc tttcggatga
gggcgcgggg atcctctaga gtcgacgtcg 60 acggcctcct gctagcccaa
aaaaacgggt atgg 94 <210> SEQ ID NO 161 <211> LENGTH: 94
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 161
ccgcgggtac caatacgccc tttcggatga gggcgcgggg atcctctaga gtcgacctgc
60 agggcctcct gctagcccaa aaaaacgggt atgg 94 <210> SEQ ID NO
162 <211> LENGTH: 32 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 162 cctcctggct tgcttgaata acttcatcat gg 32
<210> SEQ ID NO 163 <211> LENGTH: 91 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 163 ccgcgggtac caatacgccc tttcggatga
gggcgcgggg atcctctaga gtcgaccccc 60 tcctggcttg cttgaataac
ttcatcatgg c 91 <210> SEQ ID NO 164 <400> SEQUENCE: 164
000 <210> SEQ ID NO 165 <400> SEQUENCE: 165 000
<210> SEQ ID NO 166 <211> LENGTH: 2319 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic nucleic acid
sequence <400> SEQUENCE: 166 gtcgacatga aaataaaaac aggtgcacgc
atcctcgcat tatccgcatt aacgacgatg 60 atgttttccg cctcggctct
cgccaaaatc gaagaaggta aactggtaat ctggattaac 120 ggcgataaag
gctataacgg tctcgctgaa gtcggtaaga aattcgagaa agataccgga 180
attaaagtca ccgttgagca tccggataaa ctggaagaga aattcccaca ggttgcggca
240 actggcgatg gccctgacat tatcttctgg gcacacgacc gctttggtgg
ctacgctcaa 300 tctggcctgt tggctgaaat caccccggac aaagcgttcc
aggacaagct gtatccgttt 360 acctgggatg ccgtacgtta caacggcaag
ctgattgctt acccgatcgc tgttgaagcg 420 ttatcgctga tttataacaa
agatctgctg ccgaacccgc caaaaacctg ggaagagatc 480 ccggcgctgg
ataaagaact gaaagcgaaa ggtaagagcg cgctgatgtt caacctgcaa 540
gaaccgtact tcacctggcc gctgattgct gctgacgggg gttatgcgtt caagtatgaa
600 aacggcaagt acgacattaa agacgtgggc gtggataacg ctggcgcgaa
agcgggtctg 660
accttcctgg ttgacctgat taaaaacaaa cacatgaatg cagacaccga ttactccatc
720 gcagaagctg cctttaataa aggcgaaaca gcgatgacca tcaacggccc
gtgggcatgg 780 tccaacatcg acaccagcaa agtgaattat ggtgtaacgg
tactgccgac cttcaagggt 840 caaccatcca aaccgttcgt tggcgtgctg
agcgcaggta ttaacgccgc cagtccgaac 900 aaagagctgg cgaaagagtt
cctcgaaaac tatctgctga ctgatgaagg tctggaagcg 960 gttaataaag
acaaaccgct gggtgccgta gcgctgaagt cttacgagga agagttggcg 1020
aaagatccac gtattgccgc caccatggaa aacgcccaga aaggtgaaat catgccgaac
1080 atcccgcaga tgtccgcttt ctggtatgcc gtgctgatcg aagcccgcac
ctcggaatcc 1140 gacacggcag ggcccaacag cgacctggac gtgaacactg
acatttattc caaggtgctg 1200 gtgactgcta tatacctggc actcttcgtg
gtgggcactg tgggcaactc cgtgacagcc 1260 ttcactctag cgcggaagaa
gtcactgcag agcctgcaga gcactgtgca ttaccacctg 1320 ggcagcctgg
cactgtcgga cctgcttatc cttctgctgg ccatgcccgt ggagctatac 1380
aacttcatct gggtacacca tccctgggcc tttggggacg ctggctgccg tggctactat
1440 ttcctgcgtg atgcctgcac ctatgccaca gccctcaatg tagccagcct
gagtgtggag 1500 cgctacttgg ccatctgcca tcccttcaag gccaagaccc
tcatgtcccg cagccgcacc 1560 aagaaattca tcagtgccat atggctagct
tcggcgctgc tggctatacc catgcttttc 1620 accatgggcc tgcagaaccg
cagtggtgac ggcacgcacc ctggcggcct ggtgtgcaca 1680 cccattgtgg
acacagccac tgtcaaggtc gtcatccagg ttaacacctt catgtccttc 1740
ctgtttccca tgttggtcat ctccatccta aacaccgtga ttgccaacaa actgacagtc
1800 atggtgcacc aggccgccga gcagggccga gtgtgcaccg tgggcacaca
caacggttta 1860 gagcacagca cgttcaacat gaccatcgag ccgggtcgtg
tccaggccct gcgccacgga 1920 gtcctcgtct tacgtgctgt ggtcattgcc
tttgtggtct gctggctgcc ctaccacgtg 1980 cgacgcctga tgttctgcta
tatctcggat gaacagtgga ctacgttcct cttcgatttc 2040 taccactatt
tctacatgct aaccaacgct ctcttctacg tcagctccgc catcaatccc 2100
atcctctaca acctggtctc cgccaacttc cgccaggtct ttctgtccac gctggcctgc
2160 ctttgtcctg ggtggcgcca ccgccgaaag aagaggccaa cgttctccag
gaagcccaac 2220 agcatgtcca gcaaccatgc cttttccacc agcgccaccc
gggagaccct gtacgcggcc 2280 gcagattata aagatgacga tgacaaataa
taaggtacc 2319 <210> SEQ ID NO 167 <211> LENGTH: 1293
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleic acid sequence <400> SEQUENCE: 167 gtcgacatga
aaataaaaac aggtgcacgc atcctcgcat tatccgcatt aacgacgatg 60
atgttttccg cctcggctct cgccaaaatc atcgaagccc gcacctcgga atccgacacg
120 gcagggccca acagcgacct ggacgtgaac actgacattt attccaaggt
gctggtgact 180 gctatatacc tggcactctt cgtggtgggc actgtgggca
actccgtgac agccttcact 240 ctagcgcgga agaagtcact gcagagcctg
cagagcactg tgcattacca cctgggcagc 300 ctggcactgt cggacctgct
tatccttctg ctggccatgc ccgtggagct atacaacttc 360 atctgggtac
accatccctg ggcctttggg gacgctggct gccgtggcta ctatttcctg 420
cgtgatgcct gcacctatgc cacagccctc aatgtagcca gcctgagtgt ggagcgctac
480 ttggccatct gccatccctt caaggccaag accctcatgt cccgcagccg
caccaagaaa 540 ttcatcagtg ccatatggct agcttcggcg ctgctggcta
tacccatgct tttcaccatg 600 ggcctgcaga accgcagtgg tgacggcacg
caccctggcg gcctggtgtg cacacccatt 660 gtggacacag ccactgtcaa
ggtcgtcatc caggttaaca ccttcatgtc cttcctgttt 720 cccatgttgg
tcatctccat cctaaacacc gtgattgcca acaaactgac agtcatggtg 780
caccaggccg ccgagcaggg ccgagtgtgc accgtgggca cacacaacgg tttagagcac
840 agcacgttca acatgaccat cgagccgggt cgtgtccagg ccctgcgcca
cggagtcctc 900 gtcttacgtg ctgtggtcat tgcctttgtg gtctgctggc
tgccctacca cgtgcgacgc 960 ctgatgttct gctatatctc ggatgaacag
tggactacgt tcctcttcga tttctaccac 1020 tatttctaca tgctaaccaa
cgctctcttc tacgtcagct ccgccatcaa tcccatcctc 1080 tacaacctgg
tctccgccaa cttccgccag gtctttctgt ccacgctggc ctgcctttgt 1140
cctgggtggc gccaccgccg aaagaagagg ccaacgttct ccaggaagcc caacagcatg
1200 tccagcaacc atgccttttc caccagcgcc acccgggaga ccctgtacgc
ggccgcagat 1260 tataaagatg acgatgacaa ataataaggt acc 1293
<210> SEQ ID NO 168 <400> SEQUENCE: 168 000 <210>
SEQ ID NO 169 <211> LENGTH: 2652 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleic acid sequence
<400> SEQUENCE: 169 gtcgacatga aaataaaaac aggtgcacgc
atcctcgcat tatccgcatt aacgacgatg 60 atgttttccg cctcggctct
cgccaaaatc gaagaaggta aactggtaat ctggattaac 120 ggcgataaag
gctataacgg tctcgctgaa gtcggtaaga aattcgagaa agataccgga 180
attaaagtca ccgttgagca tccggataaa ctggaagaga aattcccaca ggttgcggca
240 actggcgatg gccctgacat tatcttctgg gcacacgacc gctttggtgg
ctacgctcaa 300 tctggcctgt tggctgaaat caccccggac aaagcgttcc
aggacaagct gtatccgttt 360 acctgggatg ccgtacgtta caacggcaag
ctgattgctt acccgatcgc tgttgaagcg 420 ttatcgctga tttataacaa
agatctgctg ccgaacccgc caaaaacctg ggaagagatc 480 ccggcgctgg
ataaagaact gaaagcgaaa ggtaagagcg cgctgatgtt caacctgcaa 540
gaaccgtact tcacctggcc gctgattgct gctgacgggg gttatgcgtt caagtatgaa
600 aacggcaagt acgacattaa agacgtgggc gtggataacg ctggcgcgaa
agcgggtctg 660 accttcctgg ttgacctgat taaaaacaaa cacatgaatg
cagacaccga ttactccatc 720 gcagaagctg cctttaataa aggcgaaaca
gcgatgacca tcaacggccc gtgggcatgg 780 tccaacatcg acaccagcaa
agtgaattat ggtgtaacgg tactgccgac cttcaagggt 840 caaccatcca
aaccgttcgt tggcgtgctg agcgcaggta ttaacgccgc cagtccgaac 900
aaagagctgg cgaaagagtt cctcgaaaac tatctgctga ctgatgaagg tctggaagcg
960 gttaataaag acaaaccgct gggtgccgta gcgctgaagt cttacgagga
agagttggcg 1020 aaagatccac gtattgccgc caccatggaa aacgcccaga
aaggtgaaat catgccgaac 1080 atcccgcaga tgtccgcttt ctggtatgcc
gtgctgatcg aagcccgcac ctcggaatcc 1140 gacacggcag ggcccaacag
cgacctggac gtgaacactg acatttattc caaggtgctg 1200 gtgactgcta
tatacctggc actcttcgtg gtgggcactg tgggcaactc cgtgacagcc 1260
ttcactctag cgcggaagaa gtcactgcag agcctgcaga gcactgtgca ttaccacctg
1320 ggcagcctgg cactgtcgga cctgcttatc cttctgctgg ccatgcccgt
ggagctatac 1380 aacttcatct gggtacacca tccctgggcc tttggggacg
ctggctgccg tggctactat 1440 ttcctgcgtg atgcctgcac ctatgccaca
gccctcaatg tagccagcct gagtgtggag 1500 cgctacttgg ccatctgcca
tcccttcaag gccaagaccc tcatgtcccg cagccgcacc 1560 aagaaattca
tcagtgccat atggctagct tcggcgctgc tggctatacc catgcttttc 1620
accatgggcc tgcagaaccg cagtggtgac ggcacgcacc ctggcggcct ggtgtgcaca
1680 cccattgtgg acacagccac tgtcaaggtc gtcatccagg ttaacacctt
catgtccttc 1740 ctgtttccca tgttggtcat ctccatccta aacaccgtga
ttgccaacaa actgacagtc 1800 atggtgcacc aggccgccga gcagggccga
gtgtgcaccg tgggcacaca caacggttta 1860 gagcacagca cgttcaacat
gaccatcgag ccgggtcgtg tccaggccct gcgccacgga 1920 gtcctcgtct
tacgtgctgt ggtcattgcc tttgtggtct gctggctgcc ctaccacgtg 1980
cgacgcctga tgttctgcta tatctcggat gaacagtgga ctacgttcct cttcgatttc
2040 taccactatt tctacatgct aaccaacgct ctcttctacg tcagctccgc
catcaatccc 2100 atcctctaca acctggtctc cgccaacttc cgccaggtct
ttctgtccac gctggcctgc 2160 ctttgtcctg ggtggcgcca ccgccgaaag
aagaggccaa cgttctccag gaagcccaac 2220 agcatgtcca gcaaccatgc
cttttccacc agcgccaccc gggagaccct gtacgcggcc 2280 gcaagcgata
aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg 2340
gacggggcga tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc
2400 ccgattctgg atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc
aaaactgaac 2460 atcgatcaaa accctggcac tgcgccgaaa tatggcatcc
gtggtatccc gactctgctg 2520 ctgttcaaaa acggtgaagt ggcggcaacc
aaagtgggtg cactgtctaa aggtcagttg 2580 aaagagttcc tcgacgctaa
cctggcggcg gccgcagatt ataaagatga cgatgacaaa 2640 taataaggta cc 2652
<210> SEQ ID NO 170 <211> LENGTH: 1626 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic nucleic acid
sequence <400> SEQUENCE: 170 gtcgacatga aaataaaaac aggtgcacgc
atcctcgcat tatccgcatt aacgacgatg 60 atgttttccg cctcggctct
cgccaaaatc atcgaagccc gcacctcgga atccgacacg 120 gcagggccca
acagcgacct ggacgtgaac actgacattt attccaaggt gctggtgact 180
gctatatacc tggcactctt cgtggtgggc actgtgggca actccgtgac agccttcact
240 ctagcgcgga agaagtcact gcagagcctg cagagcactg tgcattacca
cctgggcagc 300 ctggcactgt cggacctgct tatccttctg ctggccatgc
ccgtggagct atacaacttc 360 atctgggtac accatccctg ggcctttggg
gacgctggct gccgtggcta ctatttcctg 420 cgtgatgcct gcacctatgc
cacagccctc aatgtagcca gcctgagtgt ggagcgctac 480 ttggccatct
gccatccctt caaggccaag accctcatgt cccgcagccg caccaagaaa 540
ttcatcagtg ccatatggct agcttcggcg ctgctggcta tacccatgct tttcaccatg
600 ggcctgcaga accgcagtgg tgacggcacg caccctggcg gcctggtgtg
cacacccatt 660 gtggacacag ccactgtcaa ggtcgtcatc caggttaaca
ccttcatgtc cttcctgttt 720
cccatgttgg tcatctccat cctaaacacc gtgattgcca acaaactgac agtcatggtg
780 caccaggccg ccgagcaggg ccgagtgtgc accgtgggca cacacaacgg
tttagagcac 840 agcacgttca acatgaccat cgagccgggt cgtgtccagg
ccctgcgcca cggagtcctc 900 gtcttacgtg ctgtggtcat tgcctttgtg
gtctgctggc tgccctacca cgtgcgacgc 960 ctgatgttct gctatatctc
ggatgaacag tggactacgt tcctcttcga tttctaccac 1020 tatttctaca
tgctaaccaa cgctctcttc tacgtcagct ccgccatcaa tcccatcctc 1080
tacaacctgg tctccgccaa cttccgccag gtctttctgt ccacgctggc ctgcctttgt
1140 cctgggtggc gccaccgccg aaagaagagg ccaacgttct ccaggaagcc
caacagcatg 1200 tccagcaacc atgccttttc caccagcgcc acccgggaga
ccctgtacgc ggccgcaagc 1260 gataaaatta ttcacctgac tgacgacagt
tttgacacgg atgtactcaa agcggacggg 1320 gcgatcctcg tcgatttctg
ggcagagtgg tgcggtccgt gcaaaatgat cgccccgatt 1380 ctggatgaaa
tcgctgacga atatcagggc aaactgaccg ttgcaaaact gaacatcgat 1440
caaaaccctg gcactgcgcc gaaatatggc atccgtggta tcccgactct gctgctgttc
1500 aaaaacggtg aagtggcggc aaccaaagtg ggtgcactgt ctaaaggtca
gttgaaagag 1560 ttcctcgacg ctaacctggc agcggccgca gattataaag
atgacgatga caaataataa 1620 ggtacc 1626 <210> SEQ ID NO 171
<211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 171 ggtgcacgca tcctcgcatt atccgc 26 <210> SEQ ID NO
172 <211> LENGTH: 31 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 172 cgcacggcat accagaaagc ggacatctgc g 31
<210> SEQ ID NO 173 <211> LENGTH: 43 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 173 ccgcggtcga catgaaaata aaaacaggtg
cacgcatcct cgc 43 <210> SEQ ID NO 174 <211> LENGTH: 66
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 174
gccgtgtcgg attccgaggt gcggccttcg atacgcacgg cataccaaga aagcgggatg
60 ttcggc 66 <210> SEQ ID NO 175 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 175
cctcggaatc cgacacggca gggc 24 <210> SEQ ID NO 176 <211>
LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 176
gtacagggtc tcccgggtgg cgctgg 26 <210> SEQ ID NO 177
<211> LENGTH: 43 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 177 ccgcgatcga aggccgcacc tcggaatccg acacggcagg gcc 43
<210> SEQ ID NO 178 <211> LENGTH: 73 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 178 ggcgcggtac ctttgtcatc gtcatcttta
taatctgcgg ccgcgtacag ggtctcccgg 60 gtggcgctgg tgg 73 <210>
SEQ ID NO 179 <211> LENGTH: 51 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 179 gcggcggtac cttattattt gtcatcgtca
tctttataat ctgcggccgc g 51 <210> SEQ ID NO 180 <211>
LENGTH: 79 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 180
ccgcattaac gacgatgatg ttttccgcct cggctctcgc caaaatcatc gaaggccgca
60 cctcggaatc cgacacggc 79 <210> SEQ ID NO 181 <211>
LENGTH: 82 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 181
ccgcggtcga catgaaaata aaaacaggtg cacgcatcct cgcattatcc gcattaacga
60 cgatgatgtt ttccgcctcg gc 82 <210> SEQ ID NO 182
<211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 182 ccgcgagcga taaaattatt cacctgactg acg 33 <210>
SEQ ID NO 183 <211> LENGTH: 39 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 183 gcccgccagg ttagcgtcga ggaactcttt
caactgacc 39 <210> SEQ ID NO 184 <211> LENGTH: 37
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 184
gcggccgcaa gcgataaaat tattcacctg actgacg 37 <210> SEQ ID NO
185 <211> LENGTH: 38 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 185 ggcgctgcgg ccgcatcatc atgatcttta taatcgcc
38 <210> SEQ ID NO 186 <400> SEQUENCE: 186 000
<210> SEQ ID NO 187 <400> SEQUENCE: 187 000 <210>
SEQ ID NO 188 <211> LENGTH: 2465 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleic acid sequence
<400> SEQUENCE: 188 gtcgacatgg ggcaacccgg gaacggcagc
gccttcttgc tggcacccaa tggaagccat 60 gcgccggacc acgacgtcac
gcagcaaagg gacgaggtgt gggtggtggg catgggcatc 120 gtcatgtctc
tcatcgtcct ggccatcgtg tttggcaatg tgctggtcat cacagccatt 180
gccaagttcg agcgtctgca gacggtcacc aactacttca tcacttcact ggcctgtgct
240
gatctggtca tgggcctagc agtggtgccc tttggggccg cccatattct tatgaaaatg
300 tggacttttg gcaacttctg gtgcgagttt tggacttcca ttgatgtgct
gtgcgtcacg 360 gccagcattg agaccctgtg cgtgatcgca gtggatcgct
actttgccat tacttcacct 420 ttcaagtacc agagcctgct gaccaagaat
aaggcccggg tgatcattct gatggtgtgg 480 attgtgtcag gccttayctc
cttcttgccc attcagatgc actggtacag ggccacccac 540 caggaagcca
tcaactgcta tgccaatgag acctgctgtg acttcttcac gaaccaagcc 600
tatgccattg cctcttccat cgtgtccttc tacgttcccc tggtgatcat ggtcttcgtc
660 tactccaggg tctttcagga ggccaaaagg cagctccaga agattgacaa
atctgagggc 720 cgcttccatg tccagaacct tagccaggtg gagcaggatg
ggcggacggg gcatggactc 780 cgcagatctt ccaagttctg cttgaaggag
cacaaagccc tcaagacgtt aggcatcatc 840 atgggcactt tcaccctctg
ctggctgccc ttcttcatcg ttaacattgt gcatgtgatc 900 caggataacc
tcatccgtaa ggaagtttac atcctcctaa attggatagg ctatgtcaat 960
tctggtttca atccccttat ctactgccgg agcccagatt tcaggattgc cttccaggag
1020 cttctgtgcc tgcgcaggtc ttctttgaag gcctatggca atggctactc
cagcaacggc 1080 aacacagggg agcagagtgg atatcacgtg gaacaggaga
aagaaaataa actgctgtgt 1140 gaagacctcc caggcacgga agactttgtg
ggccatcaag gtactgtgcc tagcgataac 1200 attgattcac aagggaggaa
ttgtagtaca aatgactcac tgctagagcg tggccagacg 1260 gtcaccaacc
tgcagctcga gggctgcctc gggaacagta agaccgagga ccagcgcaac 1320
gaggagaagg cgcagcgtga ggccaacaaa aagatcgaga agcagctgca gaaggacaag
1380 caggtctacc gggccacgca ccgcctgctg ctgctgggtg ctggagaatc
tggtaaaagc 1440 accattgtga agcagatgag gatcctgcat gttaatgggt
ttaatggaga cagtgagaag 1500 gcaaccaaag tgcaggacat caaaaacaac
ctgaaagagg cgattgaaac cattgtggcc 1560 gccatgagca acctggtgcc
ccccgtggag ctggccaacc ccgagaacca gttcagagtg 1620 gactacatcc
tgagtgtgat gaacgtgcct gactttgact tccctcccga attctatgag 1680
catgccaagg ctctgtggga ggatgaagga gtgcgtgcct gctacgaacg ctccaacgag
1740 taccagctga ttgactgtgc ccagtacttc ctggacaaga tcgacgtgat
caagcaggct 1800 gactatgtgc cgagcgatca ggacctgctt cgctgccgtg
tcctgacttc tggaatcttt 1860 gagaccaagt tccaggtgga caaagtcaac
ttccacatgt ttgacgtggg tggccagcgc 1920 gatgaacgcc gcaagtggat
ccagtgcttc aacgatgtga ctgccatcat cttcgtggtg 1980 gccagcagca
gctacaacat ggtcatccgg gaggacaacc agaccaaccg cctgcaggag 2040
gctctgaacc tcttcaagag catctggaac aacagatggc tgcgcaccat ctctgtgatc
2100 ctgttcctca acaagcaaga tctgctcgct gagaaagtcc ttgctgggaa
atcgaagatt 2160 gaggactact ttccagaatt tgctcgctac actactcctg
aggatgctac tcccgagccc 2220 ggagaggacc cacgcgtgac ccgggccaag
tacttcattc gagatgagtt tctgaggatc 2280 agcactgcca gtggagatgg
gcgtcactac tgctaccctc atttcacctg cgctgtggac 2340 actgagaaca
tccgccgtgt gttcaacgac tgccgtgaca tcattcagcg catgcacctt 2400
cgtcagtacg agctgctcat cgattaataa tctagaggat ccccgcgccc tcatccgaaa
2460 gggcg 2465 <210> SEQ ID NO 189 <400> SEQUENCE: 189
000 <210> SEQ ID NO 190 <211> LENGTH: 2485 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic nucleic acid
sequence <400> SEQUENCE: 190 gtcgacatgg ggcaacccgg gaacggcagc
gccttcttgc tggcacccaa tggaagccat 60 gcgccggacc acgacgtcac
gcagcaaagg gacgaggtgt gggtggtggg catgggcatc 120 gtcatgtctc
tcatcgtcct ggccatcgtg tttggcaatg tgctggtcat cacagccatt 180
gccaagttcg agcgtctgca gacggtcacc aactacttca tcacttcact ggcctgtgct
240 gatctggtca tgggcctagc agtggtgccc tttggggccg cccatattct
tatgaaaatg 300 tggacttttg gcaacttctg gtgcgagttt tggacttcca
ttgatgtgct gtgcgtcacg 360 gccagcattg agaccctgtg cgtgatcgca
gtggatcgct actttgccat tacttcacct 420 ttcaagtacc agagcctgct
gaccaagaat aaggcccggg tgatcattct gatggtgtgg 480 attgtgtcag
gccttayctc cttcttgccc attcagatgc actggtacag ggccacccac 540
caggaagcca tcaactgcta tgccaatgag acctgctgtg acttcttcac gaaccaagcc
600 tatgccattg cctcttccat cgtgtccttc tacgttcccc tggtgatcat
ggtcttcgtc 660 tactccaggg tctttcagga ggccaaaagg cagctccaga
agattgacaa atctgagggc 720 cgcttccatg tccagaacct tagccaggtg
gagcaggatg ggcggacggg gcatggactc 780 cgcagatctt ccaagttctg
cttgaaggag cacaaagccc tcaagacgtt aggcatcatc 840 atgggcactt
tcaccctctg ctggctgccc ttcttcatcg ttaacattgt gcatgtgatc 900
caggataacc tcatccgtaa ggaagtttac atcctcctaa attggatagg ctatgtcaat
960 tctggtttca atccccttat ctactgccgg agcccagatt tcaggattgc
cttccaggag 1020 cttctgtgcc tgcgcaggtc ttctttgaag gcctatggca
atggctactc cagcaacggc 1080 aacacagggg agcagagtgg atatcacgtg
gaacaggaga aagaaaataa actgctgtgt 1140 gaagacctcc caggcacgga
agactttgtg ggccatcaag gtactgtgcc tagcgataac 1200 attgattcac
aagggaggaa ttgtagtaca aatgactcac tgctagagcg tggccagacg 1260
gtcaccaacc tgcagtaata atcaaggagg ccctcgagat gggctgcctc gggaacagta
1320 agaccgagga ccagcgcaac gaggagaagg cgcagcgtga ggccaacaaa
aagatcgaga 1380 agcagctgca gaaggacaag caggtctacc gggccacgca
ccgcctgctg ctgctgggtg 1440 ctggagaatc tggtaaaagc accattgtga
agcagatgag gatcctgcat gttaatgggt 1500 ttaatggaga cagtgagaag
gcaaccaaag tgcaggacat caaaaacaac ctgaaagagg 1560 cgattgaaac
cattgtggcc gccatgagca acctggtgcc ccccgtggag ctggccaacc 1620
ccgagaacca gttcagagtg gactacatcc tgagtgtgat gaacgtgcct gactttgact
1680 tccctcccga attctatgag catgccaagg ctctgtggga ggatgaagga
gtgcgtgcct 1740 gctacgaacg ctccaacgag taccagctga ttgactgtgc
ccagtacttc ctggacaaga 1800 tcgacgtgat caagcaggct gactatgtgc
cgagcgatca ggacctgctt cgctgccgtg 1860 tcctgacttc tggaatcttt
gagaccaagt tccaggtgga caaagtcaac ttccacatgt 1920 ttgacgtggg
tggccagcgc gatgaacgcc gcaagtggat ccagtgcttc aacgatgtga 1980
ctgccatcat cttcgtggtg gccagcagca gctacaacat ggtcatccgg gaggacaacc
2040 agaccaaccg cctgcaggag gctctgaacc tcttcaagag catctggaac
aacagatggc 2100 tgcgcaccat ctctgtgatc ctgttcctca acaagcaaga
tctgctcgct gagaaagtcc 2160 ttgctgggaa atcgaagatt gaggactact
ttccagaatt tgctcgctac actactcctg 2220 aggatgctac tcccgagccc
ggagaggacc cacgcgtgac ccgggccaag tacttcattc 2280 gagatgagtt
tctgaggatc agcactgcca gtggagatgg gcgtcactac tgctaccctc 2340
atttcacctg cgctgtggac actgagaaca tccgccgtgt gttcaacgac tgccgtgaca
2400 tcattcagcg catgcacctt cgtcagtacg agctgctcat cgattaataa
tctagaggat 2460 ccccgcgccc tcatccgaaa gggcg 2485 <210> SEQ ID
NO 191 <400> SEQUENCE: 191 000 <210> SEQ ID NO 192
<211> LENGTH: 1152 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 192 ctcgagatgg
gctgcctcgg gaacagtaag accgaggacc agcgcaacga ggagaaggcg 60
cagcgtgagg ccaacaaaaa gatcgagaag cagctgcaga aggacaagca ggtctaccgg
120 gccacgcacc gcctgctgct gctgggtgct ggagaatctg gtaaaagcac
cattgtgaag 180 cagatgagga tcctgcatgt taatgggttt aatggagaca
gtgagaaggc aaccaaagtg 240 caggacatca aaaacaacct gaaagaggcg
attgaaacca ttgtggccgc catgagcaac 300 ctggtgcccc ccgtggagct
ggccaacccc gagaaccagt tcagagtgga ctacatcctg 360 agtgtgatga
acgtgcctga ctttgacttc cctcccgaat tctatgagca tgccaaggct 420
ctgtgggagg atgaaggagt gcgtgcctgc tacgaacgct ccaacgagta ccagctgatt
480 gactgtgccc agtacttcct ggacaagatc gacgtgatca agcaggctga
ctatgtgccg 540 agcgatcagg acctgcttcg ctgccgtgtc ctgacttctg
gaatctttga gaccaagttc 600 caggtggaca aagtcaactt ccacatgttt
gacgtgggtg gccagcgcga tgaacgccgc 660 aagtggatcc agtgcttcaa
cgatgtgact gccatcatct tcgtggtggc cagcagcagc 720 tacaacatgg
tcatccggga ggacaaccag accaaccgcc tgcaggaggc tctgaacctc 780
ttcaagagca tctggaacaa cagatggctg cgcaccatct ctgtgatcct gttcctcaac
840 aagcaagatc tgctcgctga gaaagtcctt gctgggaaat cgaagattga
ggactacttt 900 ccagaatttg ctcgctacac tactcctgag gatgctactc
ccgagcccgg agaggaccca 960 cgcgtgaccc gggccaagta cttcattcga
gatgagtttc tgaggatcag cactgccagt 1020 ggagatgggc gtcactactg
ctaccctcat ttcacctgcg ctgtggacac tgagaacatc 1080 cgccgtgtgt
tcaacgactg ccgtgacatc attcagcgca tgcaccttcg tcagtacgag 1140
ctgctcatcg at 1152 <210> SEQ ID NO 193 <211> LENGTH:
1194 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 193 ctcgagatgg gctgcctcgg gaacagtaag
accgaggacc agcgcaacga ggagaaggcg 60 cagcgtgagg ccaacaaaaa
gatcgagaag cagctgcaga aggacaagca ggtctaccgg 120 gccacgcacc
gcctgctgct gctgggtgct ggagaatctg gtaaaagcac cattgtgaag 180
cagatgagga tcctgcatgt taatgggttt aatggagagg gcggcgaaga ggacccgcag
240 gctgcaagga gcaacagcga tggtgagaag gcaaccaaag tgcaggacat
caaaaacaac 300
ctgaaagagg cgattgaaac cattgtggcc gccatgagca acctggtgcc ccccgtggag
360 ctggccaacc ccgagaacca gttcagagtg gactacatcc tgagtgtgat
gaacgtgcct 420 gactttgact tccctcccga attctatgag catgccaagg
ctctgtggga ggatgaagga 480 gtgcgtgcct gctacgaacg ctccaacgag
taccagctga ttgactgtgc ccagtacttc 540 ctggacaaga tcgacgtgat
caagcaggct gactatgtgc cgagcgatca ggacctgctt 600 cgctgccgtg
tcctgacttc tggaatcttt gagaccaagt tccaggtgga caaagtcaac 660
ttccacatgt ttgacgtggg tggccagcgc gatgaacgcc gcaagtggat ccagtgcttc
720 aacgatgtga ctgccatcat cttcgtggtg gccagcagca gctacaacat
ggtcatccgg 780 gaggacaacc agaccaaccg cctgcaggag gctctgaacc
tcttcaagag catctggaac 840 aacagatggc tgcgcaccat ctctgtgatc
ctgttcctca acaagcaaga tctgctcgct 900 gagaaagtcc ttgctgggaa
atcgaagatt gaggactact ttccagaatt tgctcgctac 960 actactcctg
aggatgctac tcccgagccc ggagaggacc cacgcgtgac ccgggccaag 1020
tacttcattc gagatgagtt tctgaggatc agcactgcca gtggagatgg gcgtcactac
1080 tgctaccctc atttcacctg cgctgtggac actgagaaca tccgccgtgt
gttcaacgac 1140 tgccgtgaca tcattcagcg catgcacctt cgtcagtacg
agctgctcat cgat 1194 <210> SEQ ID NO 194 <211> LENGTH:
1089 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 194 ctcgagatga ctctggagtc catcatggcg
tgctgcctga gcgaggaggc caaggaagcc 60 cggcggatca acgacgagat
cgagcggcag ctccgcaggg acaagcggga cgcccgccgg 120 gagctcaagc
tgctgctgct cgggacagga gagagtggca agagtacgtt tatcaagcag 180
atgagaatca tccatgggtc aggatactct gatgaagata aaaggggctt caccaagctg
240 gtgtatcaga acatcttcac ggccatgcag gccatgatca gagccatgga
cacactcaag 300 atcccataca agtatgagca caataaggct catgcacaat
tagttcgaga agttgatgtg 360 gagaaggtgt ctgcttttga gaatccatat
gtagatgcaa taaagagttt atggaatgat 420 cctggaatcc aggaatgcta
tgatagacga cgagaatatc aattatctga ctctaccaaa 480 tactatctta
atgacttgga ccgcgtagct gaccctgcct acctgcctac gcaacaagat 540
gtgcttagag ttcgagtccc caccacaggg atcatcgaat acccctttga cttacaaagt
600 gtcattttca gaatggtcga tgtagggggc caaaggtcag agagaagaaa
atggatacac 660 tgctttgaaa atgtcacctc tatcatgttt ctagtagcgc
ttagtgaata tgatcaagtt 720 ctcgtggagt cagacaatga gaaccgaatg
gaggaaagca aggctctctt tagaacaatt 780 atcacatacc cctggttcca
gaactcctcg gttattctgt tcttaaacaa gaaagatctt 840 ctagaggaga
aaatcatgta ttcccatcta gtcgactact tcccagaata tgatggaccc 900
cagagagatg cccaggcagc ccgagaattc attctgaaga tgttcgtgga cctgaaccca
960 gacagtgaca aaattatcta ctcccacttc acgtgcgcca cagacaccga
gaatatccgc 1020 tttgtctttg ctgccgtcaa ggacaccatc ctccagttga
acctgaagga gtacaatctg 1080 gtcatcgat 1089 <210> SEQ ID NO 195
<211> LENGTH: 1077 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 195 ctcgagatgg
gctgcaccgt gagcgccgag gacaaggcgg cggccgagcg ctctaagatg 60
atcgacaaga acctgcggga ggacggagag aaggcggcgc gggaggtgaa gttgctgctg
120 ttgggtgctg gggagtcagg gaagagcacc atcgtcaagc agatgaagat
catccacgag 180 gatggctact ccgaggagga atgccggcag taccgggcgg
ttgtctacag caacaccatc 240 cagtccatca tggccattgt caaagccatg
ggaaacctgc agatcgactt tgccgacccc 300 tccagagcgg acgacgccag
gcagctattt gcactgtcct gcaccgccga ggagcaaggc 360 gtgctccctg
atgacctgtc cggcgtcatc cggaggctct gggctgacca tggtgtgcag 420
gcctgctttg gccgctcaag ggaataccag ctcaacgact cagctgccta ctacctgaac
480 gacctggagc gtattgcaca gagtgactac atccccacac agcaagatgt
gctacggacc 540 cgcgtaaaga ccacggggat cgtggagaca cacttcacct
tcaaggacct acacttcaag 600 atgtttgatg tgggtggtca gcggtctgag
cggaagaagt ggatccactg ctttgagggc 660 gtcacagcca tcatcttctg
cgtagccttg agcgcctatg acttggtgct agctgaggac 720 gaggagatga
accgcatgca tgagagcatg aagctattcg atagcatctg caacaacaag 780
tggttcacag acacgtccat catcctcttc ctcaacaaga aggacctgtt tgaggagaag
840 atcacacaca gtcccctgac catctgcttc cctgagtaca caggggccaa
caaatatgat 900 gaggcagcca gctacatcca gagtaagttt gaggacctga
ataagcgcaa agacaccaag 960 gagatctaca cgcacttcac gtgcgccacc
gacaccaaga acgtgcagtt cgtgtttgac 1020 gccgtcaccg atgtcatcat
caagaacaac ctgaaggact gcggcctctt catgcat 1077 <210> SEQ ID NO
196 <211> LENGTH: 1155 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 196 ctcgagatgt
ccggggtggt gcggaccctc agccgctgcc tgctgccggc cgaggccggc 60
ggggcccgcg agcgcagggc gggcagcggc gcgcgcgacg cggagcgcga ggcccggagg
120 cgtagccgcg acatcgacgc gctgctggcc cgcgagcggc gcgcggtccg
gcgcctggtg 180 aagatcctgc tgctgggcgc gggcgagagc ggcaagtcca
cgttcctcaa gcagatgcgc 240 atcatccacg gccgcgagtt cgaccagaag
gcgctgctgg agttccgcga caccatcttc 300 gacaacatcc tcaagggctc
aagggttctt gttgatgcac gagataagct tggcattcct 360 tggcagtatt
ctgaaaatga gaagcatggg atgttcctga tggccttcga gaacaaggcg 420
gggctgcctg tggagccggc caccttccag ctgtacgtcc cggccctgag cgcactctgg
480 agggattctg gcatcaggga ggctttcagc cggagaagcg agtttcagct
gggggagtcg 540 gtgaagtact tcctggacaa cttggaccgg atcggccagc
tgaattactt tcctagtaag 600 caagatatcc tgctggctag gaaagccacc
aagggaattg tggagcatga cttcgttatt 660 aagaagatcc cctttaagat
ggtggatgtg ggcggccagc ggtcccagcg ccagaagtgg 720 ttccagtgct
tcgacgggat cacgtccatc ctgttcatgg tctcctccag cgagtacgac 780
caggtcctca tggaggacag gcgcaccaac cggctggtgg agtccatgaa catcttcgag
840 accatcgtca acaacaagct cttcttcaac gtctccatca ttctcttcct
caacaagatg 900 gacctcctgg tggagaaggt gaagaccgtg agcatcaaga
agcacttccc ggacttcagg 960 ggcgacccgc accagctgga ggacgtccag
cgctacctgg tccagtgctt cgacaggaag 1020 agacggaacc gcagcaagcc
actcttccac cacttcacca ccgccatcga caccgagaac 1080 gtccgcttcg
tgttccatgc tgtgaaagac accatcctgc aggagaacct gaaggacatc 1140
atgctgcaga tcgat 1155 <210> SEQ ID NO 197 <400>
SEQUENCE: 197 000 <210> SEQ ID NO 198 <400> SEQUENCE:
198 000 <210> SEQ ID NO 199 <400> SEQUENCE: 199 000
<210> SEQ ID NO 200 <400> SEQUENCE: 200 000 <210>
SEQ ID NO 201 <400> SEQUENCE: 201 000 <210> SEQ ID NO
202 <400> SEQUENCE: 202 000 <210> SEQ ID NO 203
<400> SEQUENCE: 203 000 <210> SEQ ID NO 204 <400>
SEQUENCE: 204 000 <210> SEQ ID NO 205 <211> LENGTH:
3307 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleic acid sequence <400> SEQUENCE: 205
gtcgacatgg ggcaacccgg gaacggcagc gccttcttgc tggcacccaa tggaagccat
60 gcgccggacc acgacgtcac gcagcaaagg gacgaggtgt gggtggtggg
catgggcatc 120 gtcatgtctc tcatcgtcct ggccatcgtg tttggcaatg
tgctggtcat cacagccatt 180 gccaagttcg agcgtctgca gacggtcacc
aactacttca tcacttcact ggcctgtgct 240 gatctggtca tgggcctagc
agtggtgccc tttggggccg cccatattct tatgaaaatg 300 tggacttttg
gcaacttctg gtgcgagttt tggacttcca ttgatgtgct gtgcgtcacg 360
gccagcattg agaccctgtg cgtgatcgca gtggatcgct actttgccat tacttcacct
420 ttcaagtacc agagcctgct gaccaagaat aaggcccggg tgatcattct
gatggtgtgg 480 attgtgtcag gccttayctc cttcttgccc attcagatgc
actggtacag ggccacccac 540
caggaagcca tcaactgcta tgccaatgag acctgctgtg acttcttcac gaaccaagcc
600 tatgccattg cctcttccat cgtgtccttc tacgttcccc tggtgatcat
ggtcttcgtc 660 tactccaggg tctttcagga ggccaaaagg cagctccaga
agattgacaa atctgagggc 720 cgcttccatg tccagaacct tagccaggtg
gagcaggatg ggcggacggg gcatggactc 780 cgcagatctt ccaagttctg
cttgaaggag cacaaagccc tcaagacgtt aggcatcatc 840 atgggcactt
tcaccctctg ctggctgccc ttcttcatcg ttaacattgt gcatgtgatc 900
caggataacc tcatccgtaa ggaagtttac atcctcctaa attggatagg ctatgtcaat
960 tctggtttca atccccttat ctactgccgg agcccagatt tcaggattgc
cttccaggag 1020 cttctgtgcc tgcgcaggtc ttctttgaag gcctatggca
atggctactc cagcaacggc 1080 aacacagggg agcagagtgg atatcacgtg
gaacaggaga aagaaaataa actgctgtgt 1140 gaagacctcc caggcacgga
agactttgtg ggccatcaag gtactgtgcc tagcgataac 1200 attgattcac
aagggaggaa ttgtagtaca aatgactcac tgctagagcg tggccagacg 1260
gtcaccaacc tgcagggaca caactcaaaa gagatatcga tgagtcatat tggtactaaa
1320 ttcattcttg ctgaaaaatt taccttcgat cccctaagca atactctgat
tgacaaagaa 1380 gatagtgaag agatcattcg attaggcagc aacgaaagcc
gaattctttg gctgctggcc 1440 caacgtccaa acgaggtaat ttctcgcaat
gatttgcatg actttgtttg gcgagagcaa 1500 ggttttgaag tcgatgattc
cagcttaacc caagccattt cgactctgcg caaaatgctc 1560 aaagattcga
caaagtcccc acaatacgtc aaaacggttc cgaagcgcgg ttaccaattg 1620
atcgcccgag tggaaacggt tgaagaagag atggctcgcg aaaacgaagc tgctcatgac
1680 atctcttaat aatcaaggag gccctcgaga tgggctgcct cgggaacagt
aagaccgagg 1740 accagcgcaa cgaggagaag gcgcagcgtg aggccaacaa
aaagatcgag aagcagctgc 1800 agaaggacaa gcaggtctac cgggccacgc
accgcctgct gctgctgggt gctggagaat 1860 ctggtaaaag caccattgtg
aagcagatga ggatcctgca tgttaatggg tttaatggag 1920 acagtgagaa
ggcaaccaaa gtgcaggaca tcaaaaacaa cctgaaagag gcgattgaaa 1980
ccattgtggc cgccatgagc aacctggtgc cccccgtgga gctggccaac cccgagaacc
2040 agttcagagt ggactacatc ctgagtgtga tgaacgtgcc tgactttgac
ttccctcccg 2100 aattctatga gcatgccaag gctctgtggg aggatgaagg
agtgcgtgcc tgctacgaac 2160 gctccaacga gtaccagctg attgactgtg
cccagtactt cctggacaag atcgacgtga 2220 tcaagcaggc tgactatgtg
ccgagcgatc aggacctgct tcgctgccgt gtcctgactt 2280 ctggaatctt
tgagaccaag ttccaggtgg acaaagtcaa cttccacatg tttgacgtgg 2340
gtggccagcg cgatgaacgc cgcaagtgga tccagtgctt caacgatgtg actgccatca
2400 tcttcgtggt ggccagcagc agctacaaca tggtcatccg ggaggacaac
cagaccaacc 2460 gcctgcagga ggctctgaac ctcttcaaga gcatctggaa
caacagatgg ctgcgcacca 2520 tctctgtgat cctgttcctc aacaagcaag
atctgctcgc tgagaaagtc cttgctggga 2580 aatcgaagat tgaggactac
tttccagaat ttgctcgcta cactactcct gaggatgcta 2640 ctcccgagcc
cggagaggac ccacgcgtga cccgggccaa gtacttcatt cgagatgagt 2700
ttctgaggat cagcactgcc agtggagatg ggcgtcacta ctgctaccct catttcacct
2760 gcgctgtgga cactgagaac atccgccgtg tgttcaacga ctgccgtgac
atcattcagc 2820 gcatgcacct tcgtcagtac gagctgctca tcgatggaca
caactcaaaa gagatatcga 2880 tgagtcatat tggtactaaa ttcattcttg
ctgaaaaatt taccttcgat cccctaagca 2940 atactctgat tgacaaagaa
gatagtgaag agatcattcg attaggcagc aacgaaagcc 3000 gaattctttg
gctgctggcc caacgtccaa acgaggtaat ttctcgcaat gatttgcatg 3060
actttgtttg gcgagagcaa ggttttgaag tcgatgattc cagcttaacc caagccattt
3120 cgactctgcg caaaatgctc aaagattcga caaagtcccc acaatacgtc
aaaacggttc 3180 cgaagcgcgg ttaccaattg atcgcccgag tggaaacggt
tgaagaagag atggctcgcg 3240 aaaacgaagc tgctcatgac atctcttaat
aatctagagg atccccgcgc cctcatccga 3300 aagggcg 3307 <210> SEQ
ID NO 206 <400> SEQUENCE: 206 000 <210> SEQ ID NO 207
<400> SEQUENCE: 207 000 <210> SEQ ID NO 208 <211>
LENGTH: 3284 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleic acid sequence <400> SEQUENCE: 208
tctagaggct gtgggtagaa gtgaaacggg gtttaccgat aaaaacagaa aatgataaaa
60 aaggactaaa tagtatattt tgatttttga tttttgattt caaataatac
aaatttattt 120 acttatttaa ttgttttgat caattatttt tctgttaaac
aaagggagca ttatatggta 180 aagaccatga ttacggattc actggccgtc
gttttacaac gtcgtgactg ggaaaaccct 240 ggcgttaccc aacttaatcg
ccttgcagca catccccctt tcgccagctg gcgtaatagc 300 gaagaggccc
gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc 360
tttgcctggt ttccggcacc agaagcggtg ccggaaagct ggctggagtg cgatcttcct
420 gaggccgata ctgtcgtcgt cccctcaaac tggcagatgc acggttacga
tgcgcccatc 480 tacaccaacg tgacctatcc cattacggtc aatccgccgt
ttgttcccac ggagaatccg 540 acgggttgtt actcgctcac atttaatgtt
gatgaaagct ggctacagga aggccagacg 600 cgaattattt ttgatggcgt
taactcggcg tttcatctgt ggtgcaacgg gcgctgggtc 660 ggttacggcc
aggacagtcg tttgccgtct gaatttgacc tgagcgcatt tttacgcgcc 720
ggagaaaacc gcctcgcggt gatggtgctg cgctggagtg acggcagtta tctggaagat
780 caggatatgt ggcggatgag cggcattttc cgtgacgtct cgttgctgca
taaaccgact 840 acacaaatca gcgatttcca tgttgccact cgctttaatg
atgatttcag ccgcgctgta 900 ctggaggctg aagttcagat gtgcggcgag
ttgcgtgact acctacgggt aacagtttct 960 ttatggcagg gtgaaacgca
ggtcgccagc ggcaccgcgc ctttcggcgg tgaaattatc 1020 gatgagcgtg
gtggttatgc cgatcgcgtc acactacgtc tgaacgtcga aaacccgaaa 1080
ctgtggagcg ccgaaatccc gaatctctat cgtgcggtgg ttgaactgca caccgccgac
1140 ggcacgctga ttgaagcaga agcctgcgat gtcggtttcc gcgaggtgcg
gattgaaaat 1200 ggtctgctgc tgctgaacgg caagccgttg ctgattcgag
gcgttaaccg tcacgagcat 1260 catcctctgc atggtcaggt catggatgag
cagacgatgg tgcaggatat cctgctgatg 1320 aagcagaaca actttaacgc
cgtgcgctgt tcgcattatc cgaaccatcc gctgtggtac 1380 acgctgtgcg
accgctacgg cctgtatgtg gtggatgaag ccaatattga aacccacggc 1440
atggtgccaa tgaatcgtct gaccgatgat ccgcgctggc taccggcgat gagcgaacgc
1500 gtaacgcgaa tggtgcagcg cgatcgtaat cacccgagtg tgatcatctg
gtcgctgggg 1560 aatgaatcag gccacggcgc taatcacgac gcgctgtatc
gctggatcaa atctgtcgat 1620 ccttcccgcc cggtgcagta tgaaggcggc
ggagccgaca ccacggccac cgatattatt 1680 tgcccgatgt acgcgcgcgt
ggatgaagac cagcccttcc cggctgtgcc gaaatggtcc 1740 atcaaaaaat
ggctttcgct acctggagag acgcgcccgc tgatcctttg cgaatacgcc 1800
cacgcgatgg gtaacagtct tggcggtttc gctaaatact ggcaggcgtt tcgtcagtat
1860 ccccgtttac agggcggctt cgtctgggac tgggtggatc agtcgctgat
taaatatgat 1920 gaaaacggca acccgtggtc ggcttacggc ggtgattttg
gcgatacgcc gaacgatcgc 1980 cagttctgta tgaacggtct ggtctttgcc
gaccgcacgc cgcatccagc gctgacggaa 2040 gcaaaacacc agcagcagtt
tttccagttc cgtttatccg ggcaaaccat cgaagtgacc 2100 agcgaatacc
tgttccgtca tagcgataac gagctcctgc actggatggt ggcgctggat 2160
ggtaagccgc tggcaagcgg tgaagtgcct ctggatgtcg ctccacaagg taaacagttg
2220 attgaactgc ctgaactacc gcagccggag agcgccgggc aactctggct
cacagtacgc 2280 gtagtgcaac cgaacgcgac cgcatggtca gaagccgggc
acatcagcgc ctggcagcag 2340 tggcgtctgg cggaaaacct cagtgtgacg
ctccccgccg cgtcccacgc catcccgcat 2400 ctgaccacca gcgaaatgga
tttttgcatc gagctgggta ataagcgttg gcaatttaac 2460 cgccagtcag
gctttctttc acagatgtgg attggcgata aaaaacaact gctgacgccg 2520
ctgcgcgatc agttcacccg tgcaccgctg gataacgaca ttggcgtaag tgaagcgacc
2580 cgcattgacc ctaacgcctg ggtcgaacgc tggaaggcgg cgggccatta
ccaggccgaa 2640 gcagcgttgt tgcagtgcac ggcagataca cttgctgatg
cggtgctgat tacgaccgct 2700 cacgcgtggc agcatcaggg gaaaacctta
tttatcagcc ggaaaaccta ccggattgat 2760 ggtagtggtc aaatggcgat
taccgttgat gttgaagtgg cgagcgatac accgcatccg 2820 gcgcggattg
gcctgaactg ccagctggcg caggtagcag agcgggtaaa ctggctcgga 2880
ttagggccgc aagaaaacta tcccgaccgc cttactgccg cctgttttga ccgctgggat
2940 ctgccattgt cagacatgta taccccgtac gtcttcccga gcgaaaacgg
tctgcgctgc 3000 gggacgcgcg aattgaatta tggcccacac cagtggcgcg
gcgacttcca gttcaacatc 3060 agccgctaca gtcaacagca actgatggaa
accagccatc gccatctgct gcacgcggaa 3120 gaaggcacat ggctgaatat
cgacggtttc catatgggga ttggtggcga cgactcctgg 3180 agcccgtcag
tatcggcgga attccagctg agcgccggtc gctaccatta ccagttggtc 3240
tggtgtcaaa aataataacg ccctcatccg aaagggcgtc taga 3284 <210>
SEQ ID NO 209 <211> LENGTH: 25 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 209 ggggcaaccc gggaacggca gcgcc 25
<210> SEQ ID NO 210 <211> LENGTH: 30 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 210
gcagtgagtc atttgtacta caattcctcc 30 <210> SEQ ID NO 211
<211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 211 cgcggtcgac atggggcaac ccgggaacgg cagcgcc 37
<210> SEQ ID NO 212 <211> LENGTH: 67 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 212 ggctcgagct gcaggttggt gaccgtctgg
ccacgctcta gcagtgagtc atttgtacta 60 caattcc 67 <210> SEQ ID
NO 213 <211> LENGTH: 29 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 213 gggctgcctc gggaacagta agaccgagg 29
<210> SEQ ID NO 214 <211> LENGTH: 29 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 214 gagcagctcg tactgacgaa ggtgcatgc
29 <210> SEQ ID NO 215 <211> LENGTH: 44 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 215 ggaggccctc gagatgggct
gcctcgggaa cagtaagacc gagg 44 <210> SEQ ID NO 216 <211>
LENGTH: 48 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 216
cctctagatt attatcgatg agcagctcgt actgacgaag gtgcatgc 48 <210>
SEQ ID NO 217 <211> LENGTH: 37 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 217 ccatcgatga gcagctcgta ctgacgaagg
tgcatgc 37 <210> SEQ ID NO 218 <211> LENGTH: 26
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 218
ccggggtggt gcggaccctc agccgc 26 <210> SEQ ID NO 219
<211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 219 ctgcagcatg atgtccttca ggttctcc 28 <210> SEQ ID
NO 220 <211> LENGTH: 41 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 220 gcgggctcga gatgtccggg gtggtgcgga
ccctcagccg c 41 <210> SEQ ID NO 221 <211> LENGTH: 39
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 221
gcgccatcga tctgcagcat gatgtccttc aggttctcc 39 <210> SEQ ID NO
222 <211> LENGTH: 28 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 222 gactctggag tccatcatgg cgtgctgc 28
<210> SEQ ID NO 223 <211> LENGTH: 29 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 223 ccagattgta ctccttcagg ttcaactgg
29 <210> SEQ ID NO 224 <211> LENGTH: 30 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 224 atgactctgg agtccatcat
ggcgtgctgc 30 <210> SEQ ID NO 225 <211> LENGTH: 42
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 225
gcgccatcga tgaccagatt gtactccttc aggttcaact gg 42 <210> SEQ
ID NO 226 <211> LENGTH: 29 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 226 gggctgcacc gtgagcgccg aggacaagg 29
<210> SEQ ID NO 227 <211> LENGTH: 30 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 227 ccttcaggtt gttcttgatg atgacatcgg
30 <210> SEQ ID NO 228 <211> LENGTH: 31 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 228 atgggctgca ccgtgagcgc
cgaggacaag g 31 <210> SEQ ID NO 229 <211> LENGTH: 55
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 229
gcgccatcga tgaagaggcc gcagtccttc aggttgttct tgatgatgac atcgg 55
<210> SEQ ID NO 230 <211> LENGTH: 29 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 230 gggctgcctc gggaacagta agaccgagg
29 <210> SEQ ID NO 231 <211> LENGTH: 29 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence
<400> SEQUENCE: 231 gagcagctcg tactgacgaa ggtgcatgc 29
<210> SEQ ID NO 232 <211> LENGTH: 31 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 232 atgggctgcc tcgggaacag taagaccgag
g 31 <210> SEQ ID NO 233 <211> LENGTH: 40 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 233 gcgccatcga tgagcagctc
gtactgacga aggtgcatgc 40 <210> SEQ ID NO 234 <211>
LENGTH: 107 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic oligonucleotide primer sequence <400> SEQUENCE: 234
ggctcgaggg cctccttgat tattactcga gggcctcctt gattattact gcaggttggt
60 gaccgtctgg ccacgctcta gcagtgagtc atttgtacta caattcc 107
<210> SEQ ID NO 235 <211> LENGTH: 61 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 235 ccctgcaggt tggtgaccgt ctggccacgc
tctagcagtg agtcatttgt actacaattc 60 c 61 <210> SEQ ID NO 236
<211> LENGTH: 38 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 236 ggacacaact caaaagagat atcgatgagt catattgg 38
<210> SEQ ID NO 237 <211> LENGTH: 29 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 237 gagatgtcat gagcagcttc gttttcgcg
29 <210> SEQ ID NO 238 <211> LENGTH: 52 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 238 gcgtggccag acggtcacca
acctgcaggg acacaactca aaagagatat cg 52 <210> SEQ ID NO 239
<211> LENGTH: 51 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 239 cggggatcct ctagattatt aagagatgtc atgagcagct
tcgttttcgc g 51 <210> SEQ ID NO 240 <211> LENGTH: 32
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 240
ggctgtgggt agaagtgaaa cggggtttac cg 32 <210> SEQ ID NO 241
<211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 241 ctttaccata taatgctccc tttgtttaac ag 32 <210>
SEQ ID NO 242 <211> LENGTH: 43 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 242 cgcggtctag aggctgtggg tagaagtgaa
acggggttta ccg 43 <210> SEQ ID NO 243 <211> LENGTH: 60
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 243
cgacggccag tgaatccgta atcatggtct ttaccatata atgctccctt tgtttaacag
60 <210> SEQ ID NO 244 <211> LENGTH: 27 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 244 ccatgattac ggattcactg
gccgtcg 27 <210> SEQ ID NO 245 <211> LENGTH: 27
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 245
ccagaccaac tggtaatggt agcgacc 27 <210> SEQ ID NO 246
<211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 246 ggtaaagacc atgattacgg attcactggc cgtcg 35 <210>
SEQ ID NO 247 <211> LENGTH: 77 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 247 gcgcctctag aaatacgccc tttcggatga
gggcgttatt atttttgaca ccagaccaac 60 tggtaatggt agcgacc 77
<210> SEQ ID NO 248 <400> SEQUENCE: 248 000 <210>
SEQ ID NO 249 <400> SEQUENCE: 249 000 <210> SEQ ID NO
250 <400> SEQUENCE: 250 000 <210> SEQ ID NO 251
<400> SEQUENCE: 251 000 <210> SEQ ID NO 252 <400>
SEQUENCE: 252 000 <210> SEQ ID NO 253 <211> LENGTH: 89
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 253
cgcggatgca tatgaaaata aaaacaggtg cacgcatcct cgcattatcc gcattaacga
60 cgatgatgtt ttccgcctcg gctctcgcc 89
<210> SEQ ID NO 254 <211> LENGTH: 69 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 254 cgtcgaccga ggcctgcagg cgggcttcga
tgattttggc gagagccgag gcggaaaaca 60 tcatcgtcg 69 <210> SEQ ID
NO 255 <211> LENGTH: 81 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 255 cgaagcccgc ctgcaggcct cggtcgacgc
cgaatctaga gattataaag atgacgatga 60 caaataataa gctagcggcg c 81
<210> SEQ ID NO 256 <211> LENGTH: 27 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 256 gcgccgctag cttattattt gtcatcg 27
<210> SEQ ID NO 257 <211> LENGTH: 26 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 257 ggtgcacgca tcctcgcatt atccgc 26
<210> SEQ ID NO 258 <211> LENGTH: 30 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 258 ggcgttttcc atggtggcgg caatacgtgg
30 <210> SEQ ID NO 259 <211> LENGTH: 52 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 259 cgcggatgca tatgaaaata
aaaacaggtg cacgcatcct cgcattatcc gc 52 <210> SEQ ID NO 260
<211> LENGTH: 82 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 260 ccgaggcctg caggcgggct tcgatacgca cggcatacca
gaaagcggac tgggcgtttt 60 ccatggtggc ggcaatacgt gg 82 <210>
SEQ ID NO 261 <211> LENGTH: 87 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic oligonucleotide primer
sequence <400> SEQUENCE: 261 gcgccgctag cttattattt gtcatcgtca
tctttataat ctctagattc ggcgtcgacc 60 gaggcctgca ggcgggcttc gatacgc
87 <210> SEQ ID NO 262 <211> LENGTH: 26 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic oligonucleotide
primer sequence <400> SEQUENCE: 262 cctgactgac gacagttttg
acacgg 26 <210> SEQ ID NO 263 <211> LENGTH: 32
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
oligonucleotide primer sequence <400> SEQUENCE: 263
cctttagaca gtgcacccac tttggttgcc gc 32 <210> SEQ ID NO 264
<211> LENGTH: 78 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic oligonucleotide primer sequence <400>
SEQUENCE: 264 cgcggctgca ggcctcggtc gacgccgaat ctagaagcga
taaaattatt cacctgactg 60 acgacagttt tgacacgg 78 <210> SEQ ID
NO 265 <211> LENGTH: 95 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic oligonucleotide primer sequence
<400> SEQUENCE: 265 gcgccgctag cttattattt gtcatcgtca
tctttataat ccgccaggtt ctctttcaac 60 tgacctttag acagtgcacc
cactttggtt gccgc 95 <210> SEQ ID NO 266 <211> LENGTH:
216 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleic acid sequence <400> SEQUENCE: 266 gaattcaggc
gctttttaga ctggtcgtaa tgaaattcag gaggttctgc atatgaaaat 60
aaaaacaggt gcacgcatcc tcgcattatc cgcattaacg acgatgatgt tttccgcctc
120 ggctctcgcc aaaatcatcg aagcccgcct gcaggcctcg gtcgacgccg
aatctagaga 180 ttataaagat gacgatgaca aataataagc tagagg 216
<210> SEQ ID NO 267 <211> LENGTH: 202 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleic acid sequence
<400> SEQUENCE: 267 ccatacccgt ttttttgggc tagcaggagg
ccctgcatat gaaaataaaa acaggtgcac 60 gcatcctcgc attatccgca
ttaacgacga tgatgttttc cgcctcggct ctcgccaaaa 120 tcatcgaagc
ccgcctgcag gcctcggtcg acgccgaatc tagagattat aaagatgacg 180
atgacaaata ataagctaga gg 202 <210> SEQ ID NO 268 <211>
LENGTH: 182 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleic acid sequence <400> SEQUENCE: 268
aggaggttct gcatatgaaa ataaaaacag gtgcacgcat cctcgcatta tccgcattaa
60 cgacgatgat gttttccgcc tcggctctcg ccaaaatcat cgaagcccgc
ctgcaggcct 120 cggtcgacgc cgaatctaga gattataaag atgacgatga
caaataataa gctagaggta 180 cc 182 <210> SEQ ID NO 269
<211> LENGTH: 182 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic nucleic acid sequence <400> SEQUENCE:
269 aggaggttct gcatatgaaa ataaaaacag gtgcacgcat cctcgcatta
tccgcattaa 60 cgacgatgat gttttccgcc tcggctctcg ccaaaatcat
cgaagcccgc ctgcaggcct 120 cggtcgacgc cgaatctaga gattataaag
atgacgatga caaataataa gctagaggta 180 cc 182 <210> SEQ ID NO
270 <211> LENGTH: 1080 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic nucleic acid sequence <400>
SEQUENCE: 270 gaattcaggc gctttttaga ctggtcgtaa tgaaattcag
gaggttctgc atatgaaaat 60 aaaaacaggt gcacgcatcc tcgcattatc
cgcattaacg acgatgatgt tttccgcctc 120 ggctctcgcc aaaatcgaag
aaggtaaact ggtaatctgg attaacggcg ataaaggcta 180 taacggtctc
gctgaagtcg gtaagaaatt cgagaaagat accggaatta aagtcaccgt 240
tgagcatccg gataaactgg aagagaaatt cccacaggtt gcggcaactg gcgatggccc
300 tgacattatc ttctgggcac acgaccgctt tggtggctac gctcaatctg
gcctgttggc 360
tgaaatcacc ccggacaaag cgttccagga caagctgtat ccgtttacct gggatgccgt
420 acgttacaac ggcaagctga ttgcttaccc gatcgctgtt gaagcgttat
cgctgattta 480 taacaaagat ctgctgccga acccgccaaa aacctgggaa
gagatcccgg cgctggataa 540 agaactgaaa gcgaaaggta agagcgcgct
gatgttcaac ctgcaagaac cgtacttcac 600 ctggccgctg attgctgctg
acgggggtta tgcgttcaag tatgaaaacg gcaagtacga 660 cattaaagac
gtgggcgtgg ataacgctgg cgcgaaagcg ggtctgacct tcctggttga 720
cctgattaaa aacaaacaca tgaatgcaga caccgattac tccatcgcag aagctgcctt
780 taataaaggc gaaacagcga tgaccatcaa cggcccgtgg gcatggtcca
acatcgacac 840 cagcaaagtg aattatggtg taacggtact gccgaccttc
aagggtcaac catccaaacc 900 gttcgttggc gtgctgagcg caggtattaa
cgccgccagt ccgaacaaag agctggcgaa 960 agagttcctc gaaaactatc
tgctgactga tgaaggtctg gaagcggtta ataaagacaa 1020 accgctgggt
gccgtagcgc tgaagtctta cgaggaagag ttggcgaaag atccacgtat 1080
<210> SEQ ID NO 271 <211> LENGTH: 1196 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic nucleic acid
sequence <400> SEQUENCE: 271 ccatacccgt ttttttgggc tagcaggagg
ccctgcatat gaaaataaaa acaggtgcac 60 gcatcctcgc attatccgca
ttaacgacga tgatgttttc cgcctcggct ctcgccaaaa 120 tcgaagaagg
taaactggta atctggatta acggcgataa aggctataac ggtctcgctg 180
aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag catccggata
240 aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac
attatcttct 300 gggcacacga ccgctttggt ggctacgctc aatctggcct
gttggctgaa atcaccccgg 360 acaaagcgtt ccaggacaag ctgtatccgt
ttacctggga tgccgtacgt tacaacggca 420 agctgattgc ttacccgatc
gctgttgaag cgttatcgct gatttataac aaagatctgc 480 tgccgaaccc
gccaaaaacc tgggaagaga tcccggcgct ggataaagaa ctgaaagcga 540
aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg ccgctgattg
600 ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt
aaagacgtgg 660 gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct
ggttgacctg attaaaaaca 720 aacacatgaa tgcagacacc gattactcca
tcgcagaagc tgcctttaat aaaggcgaaa 780 cagcgatgac catcaacggc
ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt 840 atggtgtaac
ggtactgccg accttcaagg gtcaaccatc caaaccgttc gttggcgtgc 900
tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcgaaagag ttcctcgaaa
960 actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg
ctgggtgccg 1020 tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc
acgtattgcc gccaccatgg 1080 aaaacgccca gtccgctttc tggtatgccg
tgcgtatcga agcccgcctg caggcctcgg 1140 tcgacgccga atctagagat
tataaagatg acgatgacaa ataataagct agagga 1196 <210> SEQ ID NO
272 <211> LENGTH: 1171 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic nucleic acid sequence <400>
SEQUENCE: 272 aggaggttct gcatatgaaa ataaaaacag gtgcacgcat
cctcgcatta tccgcattaa 60 cgacgatgat gttttccgcc tcggctctcg
ccaaaatcga agaaggtaaa ctggtaatct 120 ggattaacgg cgataaaggc
tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag 180 ataccggaat
taaagtcacc gttgagcatc cggataaact ggaagagaaa ttcccacagg 240
ttgcggcaac tggcgatggc cctgacatta tcttctgggc acacgaccgc tttggtggct
300 acgctcaatc tggcctgttg gctgaaatca ccccggacaa agcgttccag
gacaagctgt 360 atccgtttac ctgggatgcc gtacgttaca acggcaagct
gattgcttac ccgatcgctg 420 ttgaagcgtt atcgctgatt tataacaaag
atctgctgcc gaacccgcca aaaacctggg 480 aagagatccc ggcgctggat
aaagaactga aagcgaaagg taagagcgcg ctgatgttca 540 acctgcaaga
accgtacttc acctggccgc tgattgctgc tgacgggggt tatgcgttca 600
agtatgaaaa cggcaagtac gacattaaag acgtgggcgt ggataacgct ggcgcgaaag
660 cgggtctgac cttcctggtt gacctgatta aaaacaaaca catgaatgca
gacaccgatt 720 actccatcgc agaagctgcc tttaataaag gcgaaacagc
gatgaccatc aacggcccgt 780 gggcatggtc caacatcgac accagcaaag
tgaattatgg tgtaacggta ctgccgacct 840 tcaagggtca accatccaaa
ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca 900 gtccgaacaa
agagctggcg aaagagttcc tcgaaaacta tctgctgact gatgaaggtc 960
tggaagcggt taataaagac aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag
1020 agttggcgaa agatccacgt attgccgcca ccatggaaaa cgcccagtcc
gctttctggt 1080 atgccgtgcg tatcgaagcc cgcctgcagg cctcggtcga
cgccgaatct agagattata 1140 aagatgacga tgacaaataa taagctagag g 1171
<210> SEQ ID NO 273 <211> LENGTH: 1171 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic nucleic acid
sequence <400> SEQUENCE: 273 aggaggttct gcatatgaaa ataaaaacag
gtgcacgcat cctcgcatta tccgcattaa 60 cgacgatgat gttttccgcc
tcggctctcg ccaaaatcga agaaggtaaa ctggtaatct 120 ggattaacgg
cgataaaggc tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag 180
ataccggaat taaagtcacc gttgagcatc cggataaact ggaagagaaa ttcccacagg
240 ttgcggcaac tggcgatggc cctgacatta tcttctgggc acacgaccgc
tttggtggct 300 acgctcaatc tggcctgttg gctgaaatca ccccggacaa
agcgttccag gacaagctgt 360 atccgtttac ctgggatgcc gtacgttaca
acggcaagct gattgcttac ccgatcgctg 420 ttgaagcgtt atcgctgatt
tataacaaag atctgctgcc gaacccgcca aaaacctggg 480 aagagatccc
ggcgctggat aaagaactga aagcgaaagg taagagcgcg ctgatgttca 540
acctgcaaga accgtacttc acctggccgc tgattgctgc tgacgggggt tatgcgttca
600 agtatgaaaa cggcaagtac gacattaaag acgtgggcgt ggataacgct
ggcgcgaaag 660 cgggtctgac cttcctggtt gacctgatta aaaacaaaca
catgaatgca gacaccgatt 720 actccatcgc agaagctgcc tttaataaag
gcgaaacagc gatgaccatc aacggcccgt 780 gggcatggtc caacatcgac
accagcaaag tgaattatgg tgtaacggta ctgccgacct 840 tcaagggtca
accatccaaa ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca 900
gtccgaacaa agagctggcg aaagagttcc tcgaaaacta tctgctgact gatgaaggtc
960 tggaagcggt taataaagac aaaccgctgg gtgccgtagc gctgaagtct
tacgaggaag 1020 agttggcgaa agatccacgt attgccgcca ccatggaaaa
cgcccagtcc gctttctggt 1080 atgccgtgcg tatcgaagcc cgcctgcagg
cctcggtcga cgccgaatct agagattata 1140 aagatgacga tgacaaataa
taagctagag g 1171 <210> SEQ ID NO 274 <211> LENGTH: 392
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleic acid sequence <400> SEQUENCE: 274 tagcaggagg
ccctgcaggc ctcggtcgac gccgaatcta gaagcgataa aattattcac 60
ctgactgacg acagttttga cacggatgta ctcaaagcgg acggggcgat cctcgtcgat
120 ttctgggcag agtggtgcgg tccgtgcaaa atgatcgccc cgattctgga
tgaaatcgct 180 gacgaatatc agggcaaact gaccgttgca aaactgaaca
tcgatcaaaa ccctggcact 240 gcgccgaaat atggcatccg tggtatcccg
actctgctgc tgttcaaaaa cggtgaagtg 300 gcggcaacca aagtgggtgc
actgtctaaa ggtcagttga aagagaacct ggcggattat 360 aaagatgacg
atgacaaata ataagctaga gg 392 <210> SEQ ID NO 275 <211>
LENGTH: 426 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleic acid sequence <400> SEQUENCE: 275
gaattcaggc gctttttaga ctggtcgtaa tgaaattcag gaggttctgc aggcctcggt
60 cgacgccgaa tctagaagcg ataaaattat tcacctgact gacgacagtt
ttgacacgga 120 tgtactcaaa gcggacgggg cgatcctcgt cgatttctgg
gcagagtggt gcggtccgtg 180 caaaatgatc gccccgattc tggatgaaat
cgctgacgaa tatcagggca aactgaccgt 240 tgcaaaactg aacatcgatc
aaaaccctgg cactgcgccg aaatatggca tccgtggtat 300 cccgactctg
ctgctgttca aaaacggtga agtggcggca accaaagtgg gtgcactgtc 360
taaaggtcag ttgaaagaga acctggcgga ttataaagat gacgatgaca aataataagc
420 tagagg 426 <210> SEQ ID NO 276 <211> LENGTH: 392
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleic acid sequence <400> SEQUENCE: 276 aggaggttct
gcaggcctcg gtcgacgccg aatctagaag cgataaaatt attcacctga 60
ctgacgacag ttttgacacg gatgtactca aagcggacgg ggcgatcctc gtcgatttct
120 gggcagagtg gtgcggtccg tgcaaaatga tcgccccgat tctggatgaa
atcgctgacg 180 aatatcaggg caaactgacc gttgcaaaac tgaacatcga
tcaaaaccct ggcactgcgc 240 cgaaatatgg catccgtggt atcccgactc
tgctgctgtt caaaaacggt gaagtggcgg 300 caaccaaagt gggtgcactg
tctaaaggtc agttgaaaga gaacctggcg gattataaag 360 atgacgatga
caaataataa gctagaggta cc 392 <210> SEQ ID NO 277 <211>
LENGTH: 392
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
nucleic acid sequence <400> SEQUENCE: 277 aggaggttct
gcaggcctcg gtcgacgccg aatctagaag cgataaaatt attcacctga 60
ctgacgacag ttttgacacg gatgtactca aagcggacgg ggcgatcctc gtcgatttct
120 gggcagagtg gtgcggtccg tgcaaaatga tcgccccgat tctggatgaa
atcgctgacg 180 aatatcaggg caaactgacc gttgcaaaac tgaacatcga
tcaaaaccct ggcactgcgc 240 cgaaatatgg catccgtggt atcccgactc
tgctgctgtt caaaaacggt gaagtggcgg 300 caaccaaagt gggtgcactg
tctaaaggtc agttgaaaga gaacctggcg gattataaag 360 atgacgatga
caaataataa gctagaggta cc 392 <210> SEQ ID NO 278 <211>
LENGTH: 528 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleic acid sequence <400> SEQUENCE: 278
gaattcaggc gctttttaga ctggtcgtaa tgaaattcag gaggttctgc atatgaaaat
60 aaaaacaggt gcacgcatcc tcgcattatc cgcattaacg acgatgatgt
tttccgcctc 120 ggctctcgcc aaaatcatcg aagcccgcct gcaggcctcg
gtcgacgccg aatctagaag 180 cgataaaatt attcacctga ctgacgacag
ttttgacacg gatgtactca aagcggacgg 240 ggcgatcctc gtcgatttct
gggcagagtg gtgcggtccg tgcaaaatga tcgccccgat 300 tctggatgaa
atcgctgacg aatatcaggg caaactgacc gttgcaaaac tgaacatcga 360
tcaaaaccct ggcactgcgc cgaaatatgg catccgtggt atcccgactc tgctgctgtt
420 caaaaacggt gaagtggcgg caaccaaagt gggtgcactg tctaaaggtc
agttgaaaga 480 gaacctggcg gattataaag atgacgatga caaataataa gctagagg
528 <210> SEQ ID NO 279 <211> LENGTH: 514 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic nucleic acid
sequence <400> SEQUENCE: 279 ccatacccgt ttttttgggc tagcaggagg
ccctgcatat gaaaataaaa acaggtgcac 60 gcatcctcgc attatccgca
ttaacgacga tgatgttttc cgcctcggct ctcgccaaaa 120 tcatcgaagc
ccgcctgcag gcctcggtcg acgccgaatc tagaagcgat aaaattattc 180
acctgactga cgacagtttt gacacggatg tactcaaagc ggacggggcg atcctcgtcg
240 atttctgggc agagtggtgc ggtccgtgca aaatgatcgc cccgattctg
gatgaaatcg 300 ctgacgaata tcagggcaaa ctgaccgttg caaaactgaa
catcgatcaa aaccctggca 360 ctgcgccgaa atatggcatc cgtggtatcc
cgactctgct gctgttcaaa aacggtgaag 420 tggcggcaac caaagtgggt
gcactgtcta aaggtcagtt gaaagagaac ctggcggatt 480 ataaagatga
cgatgacaaa taataagcta gagg 514 <210> SEQ ID NO 280
<211> LENGTH: 494 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic nucleic acid sequence <400> SEQUENCE:
280 aggaggttct gcatatgaaa ataaaaacag gtgcacgcat cctcgcatta
tccgcattaa 60 cgacgatgat gttttccgcc tcggctctcg ccaaaatcat
cgaagcccgc ctgcaggcct 120 cggtcgacgc cgaatctaga agcgataaaa
ttattcacct gactgacgac agttttgaca 180 cggatgtact caaagcggac
ggggcgatcc tcgtcgattt ctgggcagag tggtgcggtc 240 cgtgcaaaat
gatcgccccg attctggatg aaatcgctga cgaatatcag ggcaaactga 300
ccgttgcaaa actgaacatc gatcaaaacc ctggcactgc gccgaaatat ggcatccgtg
360 gtatcccgac tctgctgctg ttcaaaaacg gtgaagtggc ggcaaccaaa
gtgggtgcac 420 tgtctaaagg tcagttgaaa gagaacctgg cggattataa
agatgacgat gacaaataat 480 aagctagagg tacc 494 <210> SEQ ID NO
281 <211> LENGTH: 494 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic nucleic acid sequence <400>
SEQUENCE: 281 aggaggttct gcatatgaaa ataaaaacag gtgcacgcat
cctcgcatta tccgcattaa 60 cgacgatgat gttttccgcc tcggctctcg
ccaaaatcat cgaagcccgc ctgcaggcct 120 cggtcgacgc cgaatctaga
agcgataaaa ttattcacct gactgacgac agttttgaca 180 cggatgtact
caaagcggac ggggcgatcc tcgtcgattt ctgggcagag tggtgcggtc 240
cgtgcaaaat gatcgccccg attctggatg aaatcgctga cgaatatcag ggcaaactga
300 ccgttgcaaa actgaacatc gatcaaaacc ctggcactgc gccgaaatat
ggcatccgtg 360 gtatcccgac tctgctgctg ttcaaaaacg gtgaagtggc
ggcaaccaaa gtgggtgcac 420 tgtctaaagg tcagttgaaa gagaacctgg
cggattataa agatgacgat gacaaataat 480 aagctagagg tacc 494
<210> SEQ ID NO 282 <211> LENGTH: 1521 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic nucleic acid
sequence <400> SEQUENCE: 282 gaattcaggc gctttttaga ctggtcgtaa
tgaaattcag gaggttctgc atatgaaaat 60 aaaaacaggt gcacgcatcc
tcgcattatc cgcattaacg acgatgatgt tttccgcctc 120 ggctctcgcc
aaaatcgaag aaggtaaact ggtaatctgg attaacggcg ataaaggcta 180
taacggtctc gctgaagtcg gtaagaaatt cgagaaagat accggaatta aagtcaccgt
240 tgagcatccg gataaactgg aagagaaatt cccacaggtt gcggcaactg
gcgatggccc 300 tgacattatc ttctgggcac acgaccgctt tggtggctac
gctcaatctg gcctgttggc 360 tgaaatcacc ccggacaaag cgttccagga
caagctgtat ccgtttacct gggatgccgt 420 acgttacaac ggcaagctga
ttgcttaccc gatcgctgtt gaagcgttat cgctgattta 480 taacaaagat
ctgctgccga acccgccaaa aacctgggaa gagatcccgg cgctggataa 540
agaactgaaa gcgaaaggta agagcgcgct gatgttcaac ctgcaagaac cgtacttcac
600 ctggccgctg attgctgctg acgggggtta tgcgttcaag tatgaaaacg
gcaagtacga 660 cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg
ggtctgacct tcctggttga 720 cctgattaaa aacaaacaca tgaatgcaga
caccgattac tccatcgcag aagctgcctt 780 taataaaggc gaaacagcga
tgaccatcaa cggcccgtgg gcatggtcca acatcgacac 840 cagcaaagtg
aattatggtg taacggtact gccgaccttc aagggtcaac catccaaacc 900
gttcgttggc gtgctgagcg caggtattaa cgccgccagt ccgaacaaag agctggcgaa
960 agagttcctc gaaaactatc tgctgactga tgaaggtctg gaagcggtta
ataaagacaa 1020 accgctgggt gccgtagcgc tgaagtctta cgaggaagag
ttggcgaaag atccacgtat 1080 tgccgccacc atggaaaacg cccagtccgc
tttctggtat gccgtgcgta tcgaagcccg 1140 cctgcaggcc tcggtcgacg
ccgaatctag aagcgataaa attattcacc tgactgacga 1200 cagttttgac
acggatgtac tcaaagcgga cggggcgatc ctcgtcgatt tctgggcaga 1260
gtggtgcggt ccgtgcaaaa tgatcgcccc gattctggat gaaatcgctg acgaatatca
1320 gggcaaactg accgttgcaa aactgaacat cgatcaaaac cctggcactg
cgccgaaata 1380 tggcatccgt ggtatcccga ctctgctgct gttcaaaaac
ggtgaagtgg cggcaaccaa 1440 agtgggtgca ctgtctaaag gtcagttgaa
agagaacctg gcggattata aagatgacga 1500 tgacaaataa taagctagag g 1521
<210> SEQ ID NO 283 <211> LENGTH: 1507 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic nucleic acid
sequence <400> SEQUENCE: 283 ccatacccgt ttttttgggc tagcaggagg
ccctgcatat gaaaataaaa acaggtgcac 60 gcatcctcgc attatccgca
ttaacgacga tgatgttttc cgcctcggct ctcgccaaaa 120 tcgaagaagg
taaactggta atctggatta acggcgataa aggctataac ggtctcgctg 180
aagtcggtaa gaaattcgag aaagataccg gaattaaagt caccgttgag catccggata
240 aactggaaga gaaattccca caggttgcgg caactggcga tggccctgac
attatcttct 300 gggcacacga ccgctttggt ggctacgctc aatctggcct
gttggctgaa atcaccccgg 360 acaaagcgtt ccaggacaag ctgtatccgt
ttacctggga tgccgtacgt tacaacggca 420 agctgattgc ttacccgatc
gctgttgaag cgttatcgct gatttataac aaagatctgc 480 tgccgaaccc
gccaaaaacc tgggaagaga tcccggcgct ggataaagaa ctgaaagcga 540
aaggtaagag cgcgctgatg ttcaacctgc aagaaccgta cttcacctgg ccgctgattg
600 ctgctgacgg gggttatgcg ttcaagtatg aaaacggcaa gtacgacatt
aaagacgtgg 660 gcgtggataa cgctggcgcg aaagcgggtc tgaccttcct
ggttgacctg attaaaaaca 720 aacacatgaa tgcagacacc gattactcca
tcgcagaagc tgcctttaat aaaggcgaaa 780 cagcgatgac catcaacggc
ccgtgggcat ggtccaacat cgacaccagc aaagtgaatt 840 atggtgtaac
ggtactgccg accttcaagg gtcaaccatc caaaccgttc gttggcgtgc 900
tgagcgcagg tattaacgcc gccagtccga acaaagagct ggcgaaagag ttcctcgaaa
960 actatctgct gactgatgaa ggtctggaag cggttaataa agacaaaccg
ctgggtgccg 1020 tagcgctgaa gtcttacgag gaagagttgg cgaaagatcc
acgtattgcc gccaccatgg 1080 aaaacgccca gtccgctttc tggtatgccg
tgcgtatcga agcccgcctg caggcctcgg 1140 tcgacgccga atctagaagc
gataaaatta ttcacctgac tgacgacagt tttgacacgg 1200 atgtactcaa
agcggacggg gcgatcctcg tcgatttctg ggcagagtgg tgcggtccgt 1260
gcaaaatgat cgccccgatt ctggatgaaa tcgctgacga atatcagggc aaactgaccg
1320 ttgcaaaact gaacatcgat caaaaccctg gcactgcgcc gaaatatggc
atccgtggta 1380
tcccgactct gctgctgttc aaaaacggtg aagtggcggc aaccaaagtg ggtgcactgt
1440 ctaaaggtca gttgaaagag aacctggcgg attataaaga tgacgatgac
aaataataag 1500 ctagagg 1507 <210> SEQ ID NO 284 <211>
LENGTH: 1476 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleic acid sequence <400> SEQUENCE: 284
aggaggttct gcatatgaaa ataaaaacag gtgcacgcat cctcgcatta tccgcattaa
60 cgacgatgat gttttccgcc tcggctctcg ccaaaatcga agaaggtaaa
ctggtaatct 120 ggattaacgg cgataaaggc tataacggtc tcgctgaagt
cggtaagaaa ttcgagaaag 180 ataccggaat taaagtcacc gttgagcatc
cggataaact ggaagagaaa ttcccacagg 240 ttgcggcaac tggcgatggc
cctgacatta tcttctgggc acacgaccgc tttggtggct 300 acgctcaatc
tggcctgttg gctgaaatca ccccggacaa agcgttccag gacaagctgt 360
atccgtttac ctgggatgcc gtacgttaca acggcaagct gattgcttac ccgatcgctg
420 ttgaagcgtt atcgctgatt tataacaaag atctgctgcc gaacccgcca
aaaacctggg 480 aagagatccc ggcgctggat aaagaactga aagcgaaagg
taagagcgcg ctgatgttca 540 acctgcaaga accgtacttc acctggccgc
tgattgctgc tgacgggggt tatgcgttca 600 agtatgaaaa cggcaagtac
gacattaaag acgtgggcgt ggataacgct ggcgcgaaag 660 cgggtctgac
cttcctggtt gacctgatta aaaacaaaca catgaatgca gacaccgatt 720
actccatcgc agaagctgcc tttaataaag gcgaaacagc gatgaccatc aacggcccgt
780 gggcatggtc caacatcgac accagcaaag tgaattatgg tgtaacggta
ctgccgacct 840 tcaagggtca accatccaaa ccgttcgttg gcgtgctgag
cgcaggtatt aacgccgcca 900 gtccgaacaa agagctggcg aaagagttcc
tcgaaaacta tctgctgact gatgaaggtc 960 tggaagcggt taataaagac
aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag 1020 agttggcgaa
agatccacgt attgccgcca ccatggaaaa cgcccagtcc gctttctggt 1080
atgccgtgcg tatcgaagcc cgcctgcagg cctcggtcga cgccgaatct agaagcgata
1140 aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg
gacggggcga 1200 tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaa
aatgatcgcc ccgattctgg 1260 atgaaatcgc tgacgaatat cagggcaaac
tgaccgttgc aaaactgaac atcgatcaaa 1320 accctggcac tgcgccgaaa
tatggcatcc gtggtatccc gactctgctg ctgttcaaaa 1380 acggtgaagt
ggcggcaacc aaagtgggtg cactgtctaa aggtcagttg aaagagaacc 1440
tggcggatta taaagatgac gatgacaaat aataag 1476 <210> SEQ ID NO
285 <211> LENGTH: 1476 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic nucleic acid sequence <400>
SEQUENCE: 285 aggaggttct gcatatgaaa ataaaaacag gtgcacgcat
cctcgcatta tccgcattaa 60 cgacgatgat gttttccgcc tcggctctcg
ccaaaatcga agaaggtaaa ctggtaatct 120 ggattaacgg cgataaaggc
tataacggtc tcgctgaagt cggtaagaaa ttcgagaaag 180 ataccggaat
taaagtcacc gttgagcatc cggataaact ggaagagaaa ttcccacagg 240
ttgcggcaac tggcgatggc cctgacatta tcttctgggc acacgaccgc tttggtggct
300 acgctcaatc tggcctgttg gctgaaatca ccccggacaa agcgttccag
gacaagctgt 360 atccgtttac ctgggatgcc gtacgttaca acggcaagct
gattgcttac ccgatcgctg 420 ttgaagcgtt atcgctgatt tataacaaag
atctgctgcc gaacccgcca aaaacctggg 480 aagagatccc ggcgctggat
aaagaactga aagcgaaagg taagagcgcg ctgatgttca 540 acctgcaaga
accgtacttc acctggccgc tgattgctgc tgacgggggt tatgcgttca 600
agtatgaaaa cggcaagtac gacattaaag acgtgggcgt ggataacgct ggcgcgaaag
660 cgggtctgac cttcctggtt gacctgatta aaaacaaaca catgaatgca
gacaccgatt 720 actccatcgc agaagctgcc tttaataaag gcgaaacagc
gatgaccatc aacggcccgt 780 gggcatggtc caacatcgac accagcaaag
tgaattatgg tgtaacggta ctgccgacct 840 tcaagggtca accatccaaa
ccgttcgttg gcgtgctgag cgcaggtatt aacgccgcca 900 gtccgaacaa
agagctggcg aaagagttcc tcgaaaacta tctgctgact gatgaaggtc 960
tggaagcggt taataaagac aaaccgctgg gtgccgtagc gctgaagtct tacgaggaag
1020 agttggcgaa agatccacgt attgccgcca ccatggaaaa cgcccagtcc
gctttctggt 1080 atgccgtgcg tatcgaagcc cgcctgcagg cctcggtcga
cgccgaatct agaagcgata 1140 aaattattca cctgactgac gacagttttg
acacggatgt actcaaagcg gacggggcga 1200 tcctcgtcga tttctgggca
gagtggtgcg gtccgtgcaa aatgatcgcc ccgattctgg 1260 atgaaatcgc
tgacgaatat cagggcaaac tgaccgttgc aaaactgaac atcgatcaaa 1320
accctggcac tgcgccgaaa tatggcatcc gtggtatccc gactctgctg ctgttcaaaa
1380 acggtgaagt ggcggcaacc aaagtgggtg cactgtctaa aggtcagttg
aaagagaacc 1440 tggcggatta taaagatgac gatgacaaat aataag 1476
<210> SEQ ID NO 286 <211> LENGTH: 383 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 286 Met Phe Glu Pro Met Glu Leu Thr Asn Asp
Ala Val Ile Lys Val Ile 1 5 10 15 Gly Val Gly Gly Gly Gly Gly Asn
Ala Val Glu His Met Val Arg Glu 20 25 30 Arg Ile Glu Gly Val Glu
Phe Phe Ala Val Asn Thr Asp Ala Gln Ala 35 40 45 Leu Arg Lys Thr
Ala Val Gly Gln Thr Ile Gln Ile Gly Ser Gly Ile 50 55 60 Thr Lys
Gly Leu Gly Ala Gly Ala Asn Pro Glu Val Gly Arg Asn Ala 65 70 75 80
Ala Asp Glu Asp Arg Asp Ala Leu Arg Ala Ala Leu Glu Gly Ala Asp 85
90 95 Met Val Phe Ile Ala Ala Gly Met Gly Gly Gly Thr Gly Thr Gly
Ala 100 105 110 Ala Pro Val Val Ala Glu Val Ala Lys Asp Leu Gly Ile
Leu Thr Val 115 120 125 Ala Val Val Thr Lys Pro Phe Asn Phe Glu Gly
Lys Lys Arg Met Ala 130 135 140 Phe Ala Glu Gln Gly Ile Thr Glu Leu
Ser Lys His Val Asp Ser Leu 145 150 155 160 Ile Thr Ile Pro Asn Asp
Lys Leu Leu Lys Val Leu Gly Arg Gly Ile 165 170 175 Ser Leu Leu Asp
Ala Phe Gly Ala Ala Asn Asp Val Leu Lys Gly Ala 180 185 190 Val Gln
Gly Ile Ala Glu Leu Ile Thr Arg Pro Gly Leu Met Asn Val 195 200 205
Asp Phe Ala Asp Val Arg Thr Val Met Ser Glu Met Gly Tyr Ala Met 210
215 220 Met Gly Ser Gly Val Ala Ser Gly Glu Asp Arg Ala Glu Glu Ala
Ala 225 230 235 240 Glu Met Ala Ile Ser Ser Pro Leu Leu Glu Asp Ile
Asp Leu Ser Gly 245 250 255 Ala Arg Gly Val Leu Val Asn Ile Thr Ala
Gly Phe Asp Leu Arg Leu 260 265 270 Asp Glu Phe Glu Thr Val Gly Asn
Thr Ile Arg Ala Phe Ala Ser Asp 275 280 285 Asn Ala Thr Val Val Ile
Gly Thr Ser Leu Asp Pro Asp Met Asn Asp 290 295 300 Glu Leu Arg Val
Thr Val Val Ala Thr Gly Ile Gly Met Asp Lys Arg 305 310 315 320 Pro
Glu Ile Thr Leu Val Thr Asn Lys Gln Val Gln Gln Pro Val Met 325 330
335 Asp Arg Tyr Gln Gln His Gly Met Ala Pro Leu Thr Gln Glu Gln Lys
340 345 350 Pro Val Ala Lys Val Val Asn Asp Asn Ala Pro Gln Thr Ala
Lys Glu 355 360 365 Pro Asp Tyr Leu Asp Ile Pro Ala Phe Leu Arg Lys
Gln Ala Asp 370 375 380 <210> SEQ ID NO 287 <211>
LENGTH: 383 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic amino acid sequence <400> SEQUENCE: 287 Met Phe Glu
Pro Met Glu Leu Thr Asn Asp Ala Val Ile Lys Val Ile 1 5 10 15 Gly
Val Gly Gly Gly Gly Gly Asn Ala Val Glu His Met Val Arg Glu 20 25
30 Arg Ile Glu Gly Val Glu Phe Phe Ala Val Asn Thr Asp Ala Gln Ala
35 40 45 Leu Arg Lys Thr Ala Val Gly Gln Thr Ile Gln Ile Gly Ser
Gly Ile 50 55 60 Thr Lys Gly Leu Gly Ala Gly Ala Asn Pro Glu Val
Gly Arg Asn Ala 65 70 75 80 Ala Asp Glu Asp Arg Asp Ala Leu Arg Ala
Ala Leu Glu Gly Ala Asp 85 90 95 Met Val Phe Ile Ala Ala Gly Met
Gly Gly Gly Thr Gly Thr Gly Ala 100 105 110 Ala Pro Val Val Ala Glu
Val Ala Lys Asp Leu Gly Ile Leu Thr Val 115 120 125 Ala Val Val Thr
Lys Pro Phe Asn Phe Glu Gly Lys Lys Arg Met Ala 130 135 140 Phe Ala
Glu Gln Gly Ile Thr Glu Leu Ser Lys His Val Asp Ser Leu 145 150 155
160 Ile Thr Ile Pro Asn Asp Lys Leu Leu Lys Val Leu Gly Arg Gly Ile
165 170 175
Ser Leu Leu Asp Ala Phe Gly Ala Ala Asn Asp Val Leu Lys Gly Ala 180
185 190 Val Gln Gly Ile Ala Glu Leu Ile Thr Arg Pro Gly Leu Met Asn
Val 195 200 205 Asp Phe Ala Asp Val Arg Thr Val Met Ser Glu Met Gly
Tyr Ala Met 210 215 220 Met Gly Ser Gly Val Ala Ser Gly Glu Asp Arg
Ala Glu Glu Ala Ala 225 230 235 240 Glu Met Ala Ile Ser Ser Pro Leu
Leu Glu Asp Ile Asp Leu Ser Gly 245 250 255 Ala Arg Gly Val Leu Val
Asn Ile Thr Ala Gly Phe Asp Leu Arg Leu 260 265 270 Asp Glu Phe Glu
Thr Val Gly Asn Thr Ile Arg Ala Phe Ala Ser Asp 275 280 285 Asn Ala
Thr Val Val Ile Gly Thr Ser Leu Asp Pro Asp Met Asn Asp 290 295 300
Glu Leu Arg Val Thr Val Val Ala Thr Gly Ile Gly Met Asp Lys Arg 305
310 315 320 Pro Glu Ile Thr Leu Val Thr Asn Lys Gln Val Gln Gln Pro
Val Met 325 330 335 Asp Arg Tyr Gln Gln His Gly Met Ala Pro Leu Thr
Gln Glu Gln Lys 340 345 350 Pro Val Ala Lys Val Val Asn Asp Asn Ala
Pro Gln Thr Ala Lys Glu 355 360 365 Pro Asp Tyr Leu Asp Ile Pro Ala
Phe Leu Arg Lys Gln Ala Asp 370 375 380 <210> SEQ ID NO 288
<211> LENGTH: 446 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic amino acid sequence <400> SEQUENCE:
288 Met Pro Val Leu Glu Asn Arg Ala Ala Gln Gly Asp Ile Thr Ala Pro
1 5 10 15 Gly Gly Ala Arg Arg Leu Thr Gly Asp Gln Thr Ala Ala Leu
Arg Asp 20 25 30 Ser Leu Ser Asp Lys Pro Ala Lys Asn Ile Ile Leu
Leu Ile Gly Asp 35 40 45 Gly Met Gly Asp Ser Glu Ile Thr Ala Ala
Arg Asn Tyr Ala Glu Gly 50 55 60 Ala Gly Gly Phe Phe Lys Gly Ile
Asp Ala Leu Pro Leu Thr Gly Gln 65 70 75 80 Tyr Thr His Tyr Ala Leu
Asn Lys Lys Thr Gly Lys Pro Asp Tyr Val 85 90 95 Thr Asp Ser Ala
Ala Ser Ala Thr Ala Trp Ser Thr Gly Val Lys Thr 100 105 110 Tyr Asn
Gly Ala Leu Gly Val Asp Ile His Glu Lys Asp His Pro Thr 115 120 125
Ile Leu Glu Met Ala Lys Ala Ala Gly Leu Ala Thr Gly Asn Val Ser 130
135 140 Thr Ala Glu Leu Gln Asp Ala Thr Pro Ala Ala Leu Val Ala His
Val 145 150 155 160 Thr Ser Arg Lys Cys Tyr Gly Pro Ser Ala Thr Ser
Glu Lys Cys Pro 165 170 175 Gly Asn Ala Leu Glu Lys Gly Gly Lys Gly
Ser Ile Thr Glu Gln Leu 180 185 190 Leu Asn Ala Arg Ala Asp Val Thr
Leu Gly Gly Gly Ala Lys Thr Phe 195 200 205 Ala Glu Thr Ala Thr Ala
Gly Glu Trp Gln Gly Lys Thr Leu Arg Glu 210 215 220 Gln Ala Gln Ala
Arg Gly Tyr Gln Leu Val Ser Asp Ala Ala Ser Leu 225 230 235 240 Asn
Ser Val Thr Glu Ala Asn Gln Gln Lys Pro Leu Leu Gly Leu Phe 245 250
255 Ala Asp Gly Asn Met Pro Val Arg Trp Leu Gly Pro Lys Ala Thr Tyr
260 265 270 His Gly Asn Ile Asp Lys Pro Ala Val Thr Cys Thr Pro Asn
Pro Gln 275 280 285 Arg Asn Asp Ser Val Pro Thr Leu Ala Gln Met Thr
Asp Lys Ala Ile 290 295 300 Glu Leu Leu Ser Lys Asn Glu Lys Gly Phe
Phe Leu Gln Val Glu Gly 305 310 315 320 Ala Ser Ile Asp Lys Gln Asp
His Ala Ala Asn Pro Cys Gly Gln Ile 325 330 335 Gly Glu Thr Val Asp
Leu Asp Glu Ala Val Gln Arg Ala Leu Glu Phe 340 345 350 Ala Lys Lys
Glu Gly Asn Thr Leu Val Ile Val Thr Ala Asp His Ala 355 360 365 His
Ala Ser Gln Ile Val Ala Pro Asp Thr Lys Ala Pro Gly Leu Thr 370 375
380 Gln Ala Leu Asn Thr Lys Asp Gly Ala Val Met Val Met Ser Tyr Gly
385 390 395 400 Asn Ser Glu Glu Asp Ser Gln Glu His Thr Gly Ser Gln
Leu Arg Ile 405 410 415 Ala Ala Tyr Gly Pro His Ala Ala Asn Val Val
Gly Leu Thr Asp Gln 420 425 430 Thr Asp Leu Phe Tyr Thr Met Lys Ala
Ala Leu Gly Leu Lys 435 440 445 <210> SEQ ID NO 289
<211> LENGTH: 494 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic amino acid sequence <400> SEQUENCE:
289 Met Ser Arg Pro Arg Leu Ile Val Ala Leu Phe Leu Phe Phe Asn Val
1 5 10 15 Phe Val His Gly Glu Asn Lys Val Lys Gln Ser Thr Ile Ala
Leu Ala 20 25 30 Leu Leu Pro Leu Leu Phe Thr Pro Val Thr Lys Ala
Arg Thr Pro Glu 35 40 45 Met Pro Val Leu Glu Asn Arg Ala Ala Gln
Gly Asp Ile Thr Ala Pro 50 55 60 Gly Gly Ala Arg Arg Leu Thr Gly
Asp Gln Thr Ala Ala Leu Arg Asp 65 70 75 80 Ser Leu Ser Asp Lys Pro
Ala Lys Asn Ile Ile Leu Leu Ile Gly Asp 85 90 95 Gly Met Gly Asp
Ser Glu Ile Thr Ala Ala Arg Asn Tyr Ala Glu Gly 100 105 110 Ala Gly
Gly Phe Phe Lys Gly Ile Asp Ala Leu Pro Leu Thr Gly Gln 115 120 125
Tyr Thr His Tyr Ala Leu Asn Lys Lys Thr Gly Lys Pro Asp Tyr Val 130
135 140 Thr Asp Ser Ala Ala Ser Ala Thr Ala Trp Ser Thr Gly Val Lys
Thr 145 150 155 160 Tyr Asn Gly Ala Leu Gly Val Asp Ile His Glu Lys
Asp His Pro Thr 165 170 175 Ile Leu Glu Met Ala Lys Ala Ala Gly Leu
Ala Thr Gly Asn Val Ser 180 185 190 Thr Ala Glu Leu Gln Asp Ala Thr
Pro Ala Ala Leu Val Ala His Val 195 200 205 Thr Ser Arg Lys Cys Tyr
Gly Pro Ser Ala Thr Ser Glu Lys Cys Pro 210 215 220 Gly Asn Ala Leu
Glu Lys Gly Gly Lys Gly Ser Ile Thr Glu Gln Leu 225 230 235 240 Leu
Asn Ala Arg Ala Asp Val Thr Leu Gly Gly Gly Ala Lys Thr Phe 245 250
255 Ala Glu Thr Ala Thr Ala Gly Glu Trp Gln Gly Lys Thr Leu Arg Glu
260 265 270 Gln Ala Gln Ala Arg Gly Tyr Gln Leu Val Ser Asp Ala Ala
Ser Leu 275 280 285 Asn Ser Val Thr Glu Ala Asn Gln Gln Lys Pro Leu
Leu Gly Leu Phe 290 295 300 Ala Asp Gly Asn Met Pro Val Arg Trp Leu
Gly Pro Lys Ala Thr Tyr 305 310 315 320 His Gly Asn Ile Asp Lys Pro
Ala Val Thr Cys Thr Pro Asn Pro Gln 325 330 335 Arg Asn Asp Ser Val
Pro Thr Leu Ala Gln Met Thr Asp Lys Ala Ile 340 345 350 Glu Leu Leu
Ser Lys Asn Glu Lys Gly Phe Phe Leu Gln Val Glu Gly 355 360 365 Ala
Ser Ile Asp Lys Gln Asp His Ala Ala Asn Pro Cys Gly Gln Ile 370 375
380 Gly Glu Thr Val Asp Leu Asp Glu Ala Val Gln Arg Ala Leu Glu Phe
385 390 395 400 Ala Lys Lys Glu Gly Asn Thr Leu Val Ile Val Thr Ala
Asp His Ala 405 410 415 His Ala Ser Gln Ile Val Ala Pro Asp Thr Lys
Ala Pro Gly Leu Thr 420 425 430 Gln Ala Leu Asn Thr Lys Asp Gly Ala
Val Met Val Met Ser Tyr Gly 435 440 445 Asn Ser Glu Glu Asp Ser Gln
Glu His Thr Gly Ser Gln Leu Arg Ile 450 455 460 Ala Ala Tyr Gly Pro
His Ala Ala Asn Val Val Gly Leu Thr Asp Gln 465 470 475 480 Thr Asp
Leu Phe Tyr Thr Met Lys Ala Ala Leu Gly Leu Lys 485 490 <210>
SEQ ID NO 290 <211> LENGTH: 468 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic chimeric amino acid
sequence <400> SEQUENCE: 290 Met Asn Leu Gly Asn Arg Leu Phe
Ile Leu Ile Ala Val Leu Leu Pro 1 5 10 15 Leu Ala Val Leu Leu Leu
Met Pro Val Leu Glu Asn Arg Ala Ala Gln 20 25 30
Gly Asp Ile Thr Ala Pro Gly Gly Ala Arg Arg Leu Thr Gly Asp Gln 35
40 45 Thr Ala Ala Leu Arg Asp Ser Leu Ser Asp Lys Pro Ala Lys Asn
Ile 50 55 60 Ile Leu Leu Ile Gly Asp Gly Met Gly Asp Ser Glu Ile
Thr Ala Ala 65 70 75 80 Arg Asn Tyr Ala Glu Gly Ala Gly Gly Phe Phe
Lys Gly Ile Asp Ala 85 90 95 Leu Pro Leu Thr Gly Gln Tyr Thr His
Tyr Ala Leu Asn Lys Lys Thr 100 105 110 Gly Lys Pro Asp Tyr Val Thr
Asp Ser Ala Ala Ser Ala Thr Ala Trp 115 120 125 Ser Thr Gly Val Lys
Thr Tyr Asn Gly Ala Leu Gly Val Asp Ile His 130 135 140 Glu Lys Asp
His Pro Thr Ile Leu Glu Met Ala Lys Ala Ala Gly Leu 145 150 155 160
Ala Thr Gly Asn Val Ser Thr Ala Glu Leu Gln Asp Ala Thr Pro Ala 165
170 175 Ala Leu Val Ala His Val Thr Ser Arg Lys Cys Tyr Gly Pro Ser
Ala 180 185 190 Thr Ser Glu Lys Cys Pro Gly Asn Ala Leu Glu Lys Gly
Gly Lys Gly 195 200 205 Ser Ile Thr Glu Gln Leu Leu Asn Ala Arg Ala
Asp Val Thr Leu Gly 210 215 220 Gly Gly Ala Lys Thr Phe Ala Glu Thr
Ala Thr Ala Gly Glu Trp Gln 225 230 235 240 Gly Lys Thr Leu Arg Glu
Gln Ala Gln Ala Arg Gly Tyr Gln Leu Val 245 250 255 Ser Asp Ala Ala
Ser Leu Asn Ser Val Thr Glu Ala Asn Gln Gln Lys 260 265 270 Pro Leu
Leu Gly Leu Phe Ala Asp Gly Asn Met Pro Val Arg Trp Leu 275 280 285
Gly Pro Lys Ala Thr Tyr His Gly Asn Ile Asp Lys Pro Ala Val Thr 290
295 300 Cys Thr Pro Asn Pro Gln Arg Asn Asp Ser Val Pro Thr Leu Ala
Gln 305 310 315 320 Met Thr Asp Lys Ala Ile Glu Leu Leu Ser Lys Asn
Glu Lys Gly Phe 325 330 335 Phe Leu Gln Val Glu Gly Ala Ser Ile Asp
Lys Gln Asp His Ala Ala 340 345 350 Asn Pro Cys Gly Gln Ile Gly Glu
Thr Val Asp Leu Asp Glu Ala Val 355 360 365 Gln Arg Ala Leu Glu Phe
Ala Lys Lys Glu Gly Asn Thr Leu Val Ile 370 375 380 Val Thr Ala Asp
His Ala His Ala Ser Gln Ile Val Ala Pro Asp Thr 385 390 395 400 Lys
Ala Pro Gly Leu Thr Gln Ala Leu Asn Thr Lys Asp Gly Ala Val 405 410
415 Met Val Met Ser Tyr Gly Asn Ser Glu Glu Asp Ser Gln Glu His Thr
420 425 430 Gly Ser Gln Leu Arg Ile Ala Ala Tyr Gly Pro His Ala Ala
Asn Val 435 440 445 Val Gly Leu Thr Asp Gln Thr Asp Leu Phe Tyr Thr
Met Lys Ala Ala 450 455 460 Leu Gly Leu Lys 465 <210> SEQ ID
NO 291 <211> LENGTH: 424 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 291 Met Ala Thr Thr His Ala Gln Gly His Pro Pro Val Leu
Gly Asn Asp 1 5 10 15 Thr Leu Arg Glu His Tyr Asp Tyr Val Gly Lys
Leu Ala Gly Arg Leu 20 25 30 Arg Asp Pro Pro Glu Gly Ser Thr Leu
Ile Thr Thr Ile Leu Phe Leu 35 40 45 Val Thr Cys Ser Phe Ile Val
Leu Glu Asn Leu Met Val Leu Ile Ala 50 55 60 Ile Trp Lys Asn Asn
Lys Phe His Asn Arg Met Tyr Phe Phe Ile Gly 65 70 75 80 Asn Leu Ala
Leu Cys Asp Leu Leu Ala Gly Ile Ala Tyr Lys Val Asn 85 90 95 Ile
Leu Met Ser Gly Arg Lys Thr Phe Ser Leu Ser Pro Thr Val Trp 100 105
110 Phe Leu Arg Glu Gly Ser Met Phe Val Ala Leu Gly Ala Ser Thr Cys
115 120 125 Ser Leu Leu Ala Ile Ala Ile Glu Arg His Leu Thr Met Ile
Lys Met 130 135 140 Arg Pro Tyr Asp Ala Asn Lys Lys His Arg Val Phe
Leu Leu Ile Gly 145 150 155 160 Met Cys Trp Leu Ile Ala Phe Ser Leu
Gly Ala Leu Pro Ile Leu Gly 165 170 175 Trp Asn Cys Leu Glu Asn Phe
Pro Asp Cys Ser Thr Ile Leu Pro Leu 180 185 190 Tyr Ser Lys Lys Tyr
Ile Ala Phe Leu Ile Ser Ile Phe Ile Ala Ile 195 200 205 Leu Val Thr
Ile Val Ile Leu Tyr Ala Arg Ile Tyr Phe Leu Val Lys 210 215 220 Ser
Ser Ser Arg Arg Val Ala Asn His Asn Ser Glu Arg Ser Met Ala 225 230
235 240 Leu Leu Arg Thr Val Val Ile Val Val Ser Val Phe Ile Ala Cys
Trp 245 250 255 Ser Pro Leu Phe Ile Leu Phe Leu Ile Asp Val Ala Cys
Arg Ala Lys 260 265 270 Glu Cys Ser Ile Leu Phe Lys Ser Gln Trp Phe
Ile Met Leu Ala Val 275 280 285 Leu Asn Ser Ala Met Asn Pro Val Ile
Tyr Thr Leu Ala Ser Lys Glu 290 295 300 Met Arg Arg Ala Phe Phe Arg
Leu Val Cys Gly Cys Leu Val Lys Gly 305 310 315 320 Lys Gly Thr Gln
Ala Ser Pro Met Gln Pro Ala Leu Asp Pro Ser Arg 325 330 335 Ser Lys
Ser Ser Ser Ser Asn Asn Ser Ser Ser His Ser Pro Lys Val 340 345 350
Lys Glu Asp Leu Pro His Val Ala Thr Ser Ser Cys Val Thr Asp Lys 355
360 365 Thr Arg Ser Leu Gln Asn Gly Val Leu Cys Lys Lys Gly Asn Ser
Ala 370 375 380 Asp Ile Gln His Ser Gly Gly Arg Ser Ser Leu Glu Gly
Pro Arg Phe 385 390 395 400 Glu Gly Lys Pro Ile Pro Asn Pro Leu Leu
Gly Leu Asp Ser Thr Arg 405 410 415 Thr Gly His His His His His His
420 <210> SEQ ID NO 292 <211> LENGTH: 413 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: UNSURE <222> LOCATION: 164 <223>
OTHER INFORMATION: Xaa = unknown amino acid <400> SEQUENCE:
292 Met Gly Gln Pro Gly Asn Gly Ser Ala Phe Leu Leu Ala Pro Asn Gly
1 5 10 15 Ser His Ala Pro Asp His Asp Val Thr Gln Gln Arg Asp Glu
Val Trp 20 25 30 Val Val Gly Met Gly Ile Val Met Ser Leu Ile Val
Leu Ala Ile Val 35 40 45 Phe Gly Asn Val Leu Val Ile Thr Ala Ile
Ala Lys Phe Glu Arg Leu 50 55 60 Gln Thr Val Thr Asn Tyr Phe Ile
Thr Ser Leu Ala Cys Ala Asp Leu 65 70 75 80 Val Met Gly Leu Ala Val
Val Pro Phe Gly Ala Ala His Ile Leu Met 85 90 95 Lys Met Trp Thr
Phe Gly Asn Phe Trp Cys Glu Phe Trp Thr Ser Ile 100 105 110 Asp Val
Leu Cys Val Thr Ala Ser Ile Glu Thr Leu Cys Val Ile Ala 115 120 125
Val Asp Arg Tyr Phe Ala Ile Thr Ser Pro Phe Lys Tyr Gln Ser Leu 130
135 140 Leu Thr Lys Asn Lys Ala Arg Val Ile Ile Leu Met Val Trp Ile
Val 145 150 155 160 Ser Gly Leu Xaa Ser Phe Leu Pro Ile Gln Met His
Trp Tyr Arg Ala 165 170 175 Thr His Gln Glu Ala Ile Asn Cys Tyr Ala
Asn Glu Thr Cys Cys Asp 180 185 190 Phe Phe Thr Asn Gln Ala Tyr Ala
Ile Ala Ser Ser Ile Val Ser Phe 195 200 205 Tyr Val Pro Leu Val Ile
Met Val Phe Val Tyr Ser Arg Val Phe Gln 210 215 220 Glu Ala Lys Arg
Gln Leu Gln Lys Ile Asp Lys Ser Glu Gly Arg Phe 225 230 235 240 His
Val Gln Asn Leu Ser Gln Val Glu Gln Asp Gly Arg Thr Gly His 245 250
255 Gly Leu Arg Arg Ser Ser Lys Phe Cys Leu Lys Glu His Lys Ala Leu
260 265 270 Lys Thr Leu Gly Ile Ile Met Gly Thr Phe Thr Leu Cys Trp
Leu Pro 275 280 285 Phe Phe Ile Val Asn Ile Val His Val Ile Gln Asp
Asn Leu Ile Arg 290 295 300 Lys Glu Val Tyr Ile Leu Leu Asn Trp Ile
Gly Tyr Val Asn Ser Gly 305 310 315 320 Phe Asn Pro Leu Ile Tyr Cys
Arg Ser Pro Asp Phe Arg Ile Ala Phe 325 330 335
Gln Glu Leu Leu Cys Leu Arg Arg Ser Ser Leu Lys Ala Tyr Gly Asn 340
345 350 Gly Tyr Ser Ser Asn Gly Asn Thr Gly Glu Gln Ser Gly Tyr His
Val 355 360 365 Glu Gln Glu Lys Glu Asn Lys Leu Leu Cys Glu Asp Leu
Pro Gly Thr 370 375 380 Glu Asp Phe Val Gly His Gln Gly Thr Val Pro
Ser Asp Asn Ile Asp 385 390 395 400 Ser Gln Gly Arg Asn Cys Ser Thr
Asn Asp Ser Leu Leu 405 410 <210> SEQ ID NO 293 <211>
LENGTH: 416 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic amino acid sequence <400> SEQUENCE: 293 Met Asp Ser
Val Cys Pro Gln Gly Lys Tyr Ile His Pro Gln Asn Asn 1 5 10 15 Ser
Ile Cys Cys Thr Lys Cys His Lys Gly Thr Tyr Leu Tyr Asn Asp 20 25
30 Cys Pro Gly Pro Gly Gln Asp Thr Asp Cys Arg Glu Cys Glu Ser Gly
35 40 45 Ser Phe Thr Ala Ser Glu Asn His Leu Arg His Cys Leu Ser
Cys Ser 50 55 60 Lys Cys Arg Lys Glu Met Gly Gln Val Glu Ile Ser
Ser Cys Thr Val 65 70 75 80 Asp Arg Asp Thr Val Cys Gly Cys Arg Lys
Asn Gln Tyr Arg His Tyr 85 90 95 Trp Ser Glu Asn Leu Phe Gln Cys
Phe Asn Cys Ser Leu Cys Leu Asn 100 105 110 Gly Thr Val His Leu Ser
Cys Gln Glu Lys Gln Asn Thr Val Cys Thr 115 120 125 Cys His Ala Gly
Phe Phe Leu Arg Glu Asn Glu Cys Val Ser Cys Ser 130 135 140 Asn Cys
Lys Lys Ser Leu Glu Cys Thr Lys Leu Cys Leu Pro Gln Ile 145 150 155
160 Glu Asn Val Lys Gly Thr Glu Asp Ser Gly Thr Thr Val Leu Leu Pro
165 170 175 Leu Val Ile Phe Phe Gly Leu Cys Leu Leu Ser Leu Leu Phe
Ile Gly 180 185 190 Leu Met Tyr Arg Tyr Gln Arg Trp Lys Ser Lys Leu
Tyr Ser Ile Val 195 200 205 Cys Gly Lys Ser Thr Pro Glu Lys Glu Gly
Glu Leu Glu Gly Thr Thr 210 215 220 Thr Lys Pro Leu Ala Pro Asn Pro
Ser Phe Ser Pro Thr Pro Gly Phe 225 230 235 240 Thr Pro Thr Leu Gly
Phe Ser Pro Val Pro Ser Ser Thr Phe Thr Ser 245 250 255 Ser Ser Thr
Tyr Thr Pro Gly Asp Cys Pro Asn Phe Ala Ala Pro Arg 260 265 270 Arg
Glu Val Ala Pro Pro Tyr Gln Gly Ala Asp Pro Ile Leu Ala Thr 275 280
285 Ala Leu Ala Ser Asp Pro Ile Pro Asn Pro Leu Gln Lys Trp Glu Asp
290 295 300 Ser Ala His Lys Pro Gln Ser Leu Asp Thr Asp Asp Pro Ala
Thr Leu 305 310 315 320 Tyr Ala Val Val Glu Asn Val Pro Pro Leu Arg
Trp Lys Glu Phe Val 325 330 335 Arg Arg Leu Gly Leu Ser Asp His Glu
Ile Asp Arg Leu Glu Leu Gln 340 345 350 Asn Gly Arg Cys Leu Arg Glu
Ala Gln Tyr Ser Met Leu Ala Thr Trp 355 360 365 Arg Arg Arg Thr Pro
Arg Arg Glu Ala Thr Leu Glu Leu Leu Gly Arg 370 375 380 Val Leu Arg
Asp Met Asp Leu Leu Gly Cys Leu Glu Asp Ile Glu Glu 385 390 395 400
Ala Leu Cys Gly Pro Ala Ala Leu Pro Pro Ala Pro Ser Leu Leu Arg 405
410 415 <210> SEQ ID NO 294 <211> LENGTH: 428
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 294 Met Gly Leu Val Pro His Leu
Gly Asp Arg Glu Lys Arg Asp Ser Val 1 5 10 15 Cys Pro Gln Gly Lys
Tyr Ile His Pro Gln Asn Asn Ser Ile Cys Cys 20 25 30 Thr Lys Cys
His Lys Gly Thr Tyr Leu Tyr Asn Asp Cys Pro Gly Pro 35 40 45 Gly
Gln Asp Thr Asp Cys Arg Glu Cys Glu Ser Gly Ser Phe Thr Ala 50 55
60 Ser Glu Asn His Leu Arg His Cys Leu Ser Cys Ser Lys Cys Arg Lys
65 70 75 80 Glu Met Gly Gln Val Glu Ile Ser Ser Cys Thr Val Asp Arg
Asp Thr 85 90 95 Val Cys Gly Cys Arg Lys Asn Gln Tyr Arg His Tyr
Trp Ser Glu Asn 100 105 110 Leu Phe Gln Cys Phe Asn Cys Ser Leu Cys
Leu Asn Gly Thr Val His 115 120 125 Leu Ser Cys Gln Glu Lys Gln Asn
Thr Val Cys Thr Cys His Ala Gly 130 135 140 Phe Phe Leu Arg Glu Asn
Glu Cys Val Ser Cys Ser Asn Cys Lys Lys 145 150 155 160 Ser Leu Glu
Cys Thr Lys Leu Cys Leu Pro Gln Ile Glu Asn Val Lys 165 170 175 Gly
Thr Glu Asp Ser Gly Thr Thr Val Leu Leu Pro Leu Val Ile Phe 180 185
190 Phe Gly Leu Cys Leu Leu Ser Leu Leu Phe Ile Gly Leu Met Tyr Arg
195 200 205 Tyr Gln Arg Trp Lys Ser Lys Leu Tyr Ser Ile Val Cys Gly
Lys Ser 210 215 220 Thr Pro Glu Lys Glu Gly Glu Leu Glu Gly Thr Thr
Thr Lys Pro Leu 225 230 235 240 Ala Pro Asn Pro Ser Phe Ser Pro Thr
Pro Gly Phe Thr Pro Thr Leu 245 250 255 Gly Phe Ser Pro Val Pro Ser
Ser Thr Phe Thr Ser Ser Ser Thr Tyr 260 265 270 Thr Pro Gly Asp Cys
Pro Asn Phe Ala Ala Pro Arg Arg Glu Val Ala 275 280 285 Pro Pro Tyr
Gln Gly Ala Asp Pro Ile Leu Ala Thr Ala Leu Ala Ser 290 295 300 Asp
Pro Ile Pro Asn Pro Leu Gln Lys Trp Glu Asp Ser Ala His Lys 305 310
315 320 Pro Gln Ser Leu Asp Thr Asp Asp Pro Ala Thr Leu Tyr Ala Val
Val 325 330 335 Glu Asn Val Pro Pro Leu Arg Trp Lys Glu Phe Val Arg
Arg Leu Gly 340 345 350 Leu Ser Asp His Glu Ile Asp Arg Leu Glu Leu
Gln Asn Gly Arg Cys 355 360 365 Leu Arg Glu Ala Gln Tyr Ser Met Leu
Ala Thr Trp Arg Arg Arg Thr 370 375 380 Pro Arg Arg Glu Ala Thr Leu
Glu Leu Leu Gly Arg Val Leu Arg Asp 385 390 395 400 Met Asp Leu Leu
Gly Cys Leu Glu Asp Ile Glu Glu Ala Leu Cys Gly 405 410 415 Pro Ala
Ala Leu Pro Pro Ala Pro Ser Leu Leu Arg 420 425 <210> SEQ ID
NO 295 <211> LENGTH: 233 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 295 Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala
Glu Glu Ala 1 5 10 15 Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser
Arg Arg Cys Leu Phe 20 25 30 Leu Ser Leu Phe Ser Phe Leu Ile Val
Ala Gly Ala Thr Thr Leu Phe 35 40 45 Cys Leu Leu His Phe Gly Val
Ile Gly Pro Gln Arg Glu Glu Phe Pro 50 55 60 Arg Asp Leu Ser Leu
Ile Ser Pro Leu Ala Gln Ala Val Arg Ser Ser 65 70 75 80 Ser Arg Thr
Pro Ser Asp Lys Pro Val Ala His Val Val Ala Asn Pro 85 90 95 Gln
Ala Glu Gly Gln Leu Gln Trp Leu Asn Arg Arg Ala Asn Ala Leu 100 105
110 Leu Ala Asn Gly Val Glu Leu Arg Asp Asn Gln Leu Val Val Pro Ser
115 120 125 Glu Gly Leu Tyr Leu Ile Tyr Ser Gln Val Leu Phe Lys Gly
Gln Gly 130 135 140 Cys Pro Ser Thr His Val Leu Leu Thr His Thr Ile
Ser Arg Ile Ala 145 150 155 160 Val Ser Tyr Gln Thr Lys Val Asn Leu
Leu Ser Ala Ile Lys Ser Pro 165 170 175 Cys Gln Arg Glu Thr Pro Glu
Gly Ala Glu Ala Lys Pro Trp Tyr Glu 180 185 190 Pro Ile Tyr Leu Gly
Gly Val Phe Gln Leu Glu Lys Gly Asp Arg Leu 195 200 205 Ser Ala Glu
Ile Asn Arg Pro Asp Tyr Leu Asp Phe Ala Glu Ser Gly 210 215 220 Gln
Val Tyr Phe Gly Ile Ile Ala Leu 225 230 <210> SEQ ID NO
296
<211> LENGTH: 75 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic amino acid sequence <400> SEQUENCE:
296 Met Asn Leu Gly Asn Arg Leu Phe Ile Leu Ile Ala Val Leu Leu Pro
1 5 10 15 Leu Ala Val Leu Leu Leu Asn Ser Asp Ser Glu Cys Pro Leu
Ser His 20 25 30 Asp Gly Tyr Cys Leu His Asp Gly Val Cys Met Tyr
Ile Glu Ala Leu 35 40 45 Asp Lys Tyr Ala Cys Asn Cys Val Val Gly
Tyr Ile Gly Glu Arg Cys 50 55 60 Gln Tyr Arg Asp Leu Lys Trp Trp
Glu Leu Arg 65 70 75 <210> SEQ ID NO 297 <211> LENGTH:
518 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 297 Met Asn Leu Gly Asn Arg Leu
Phe Ile Leu Ile Ala Val Leu Leu Pro 1 5 10 15 Leu Ala Val Leu Leu
Leu Ser Phe Thr Leu Ser Val Thr Val Gln Gln 20 25 30 Pro Gln Leu
Thr Leu Thr Ala Ala Val Ile Gly Asp Gly Ala Pro Ala 35 40 45 Asn
Gly Lys Thr Ala Ile Thr Val Glu Phe Thr Val Ala Asp Phe Glu 50 55
60 Gly Lys Pro Leu Ala Gly Gln Glu Val Val Ile Thr Thr Asn Asn Gly
65 70 75 80 Ala Leu Pro Asn Lys Ile Thr Glu Lys Thr Asp Ala Asn Gly
Val Ala 85 90 95 Arg Ile Ala Leu Thr Asn Thr Thr Asp Gly Val Thr
Val Val Thr Ala 100 105 110 Glu Val Glu Gly Gln Arg Gln Ser Val Asp
Thr His Phe Val Lys Gly 115 120 125 Thr Ile Ala Ala Asp Lys Ser Thr
Leu Ala Ala Val Pro Thr Ser Ile 130 135 140 Ile Ala Asp Gly Leu Met
Ala Ser Thr Ile Thr Leu Glu Leu Lys Asp 145 150 155 160 Thr Tyr Gly
Asp Pro Gln Ala Gly Ala Asn Val Ala Phe Asp Thr Thr 165 170 175 Leu
Gly Asn Met Gly Val Ile Thr Asp His Asn Asp Gly Thr Tyr Ser 180 185
190 Ala Pro Leu Thr Ser Thr Thr Leu Gly Val Ala Thr Val Thr Val Lys
195 200 205 Val Asp Gly Ala Ala Phe Ser Val Pro Ser Val Thr Val Asn
Phe Thr 210 215 220 Ala Asp Pro Ile Pro Asp Ala Gly Arg Ser Ser Phe
Thr Val Ser Thr 225 230 235 240 Pro Asp Ile Leu Ala Asp Gly Thr Met
Ser Ser Thr Leu Ser Phe Val 245 250 255 Pro Val Asp Lys Asn Gly His
Phe Ile Ser Gly Met Gln Gly Leu Ser 260 265 270 Phe Thr Gln Asn Gly
Val Pro Val Ser Ile Ser Pro Ile Thr Glu Gln 275 280 285 Pro Asp Ser
Tyr Thr Ala Thr Val Val Gly Asn Ser Val Gly Asp Val 290 295 300 Thr
Ile Thr Pro Gln Val Asp Thr Leu Ile Leu Ser Thr Leu Gln Lys 305 310
315 320 Lys Ile Ser Leu Phe Pro Val Pro Thr Leu Thr Gly Ile Leu Val
Asn 325 330 335 Gly Gln Asn Phe Ala Thr Asp Lys Gly Phe Pro Lys Thr
Ile Phe Lys 340 345 350 Asn Ala Thr Phe Gln Leu Gln Met Asp Asn Asp
Val Ala Asn Asn Thr 355 360 365 Gln Tyr Glu Trp Ser Ser Ser Phe Thr
Pro Asn Val Ser Val Asn Asp 370 375 380 Gln Gly Gln Val Thr Ile Thr
Tyr Gln Thr Tyr Ser Glu Val Ala Val 385 390 395 400 Thr Ala Lys Ser
Lys Lys Phe Pro Ser Tyr Ser Val Ser Tyr Arg Phe 405 410 415 Tyr Pro
Asn Arg Trp Ile Tyr Asp Gly Gly Arg Ser Leu Val Ser Ser 420 425 430
Leu Glu Ala Ser Arg Gln Cys Gln Gly Ser Asp Met Ser Ala Val Leu 435
440 445 Glu Ser Ser Arg Ala Thr Asn Gly Thr Arg Ala Pro Asp Gly Thr
Leu 450 455 460 Trp Gly Glu Trp Gly Ser Leu Thr Ala Tyr Ser Ser Asp
Trp Gln Ser 465 470 475 480 Gly Glu Tyr Trp Val Lys Lys Thr Ser Thr
Asp Phe Glu Thr Met Asn 485 490 495 Met Asp Thr Gly Ala Leu Gln Pro
Gly Pro Ala Tyr Leu Ala Phe Pro 500 505 510 Leu Cys Ala Leu Ser Ile
515 <210> SEQ ID NO 298 <211> LENGTH: 50 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic amino acid
sequence <400> SEQUENCE: 298 Met Ser Arg Pro Arg Leu Ile Val
Ala Leu Phe Leu Phe Phe Asn Val 1 5 10 15 Phe Val His Gly Glu Asn
Lys Val Lys Gln Ser Thr Ile Ala Leu Ala 20 25 30 Leu Leu Pro Leu
Leu Phe Thr Pro Val Thr Lys Ala Arg Thr Pro Glu 35 40 45 Ser Arg 50
<210> SEQ ID NO 299 <211> LENGTH: 496 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 299 Met Ser Arg Pro Arg Leu Ile Val Ala Leu
Phe Leu Phe Phe Asn Val 1 5 10 15 Phe Val His Gly Glu Asn Lys Val
Lys Gln Ser Thr Ile Ala Leu Ala 20 25 30 Leu Leu Pro Leu Leu Phe
Thr Pro Val Thr Lys Ala Arg Thr Pro Glu 35 40 45 Met Pro Val Leu
Glu Asn Arg Ala Ala Gln Gly Asp Ile Thr Ala Pro 50 55 60 Gly Gly
Ala Arg Arg Leu Thr Gly Asp Gln Thr Ala Ala Leu Arg Asp 65 70 75 80
Ser Leu Ser Asp Lys Pro Ala Lys Asn Ile Ile Leu Leu Ile Gly Asp 85
90 95 Gly Met Gly Asp Ser Glu Ile Thr Ala Ala Arg Asn Tyr Ala Glu
Gly 100 105 110 Ala Gly Gly Phe Phe Lys Gly Ile Asp Ala Leu Pro Leu
Thr Gly Gln 115 120 125 Tyr Thr His Tyr Ala Leu Asn Lys Lys Thr Gly
Lys Pro Asp Tyr Val 130 135 140 Thr Asp Ser Ala Ala Ser Ala Thr Ala
Trp Ser Thr Gly Val Lys Thr 145 150 155 160 Tyr Asn Gly Ala Leu Gly
Val Asp Ile His Glu Lys Asp His Pro Thr 165 170 175 Ile Leu Glu Met
Ala Lys Ala Ala Gly Leu Ala Thr Gly Asn Val Ser 180 185 190 Thr Ala
Glu Leu Gln Asp Ala Thr Pro Ala Ala Leu Val Ala His Val 195 200 205
Thr Ser Arg Lys Cys Tyr Gly Pro Ser Ala Thr Ser Glu Lys Cys Pro 210
215 220 Gly Asn Ala Leu Glu Lys Gly Gly Lys Gly Ser Ile Thr Glu Gln
Leu 225 230 235 240 Leu Asn Ala Arg Ala Asp Val Thr Leu Gly Gly Gly
Ala Lys Thr Phe 245 250 255 Ala Glu Thr Ala Thr Ala Gly Glu Trp Gln
Gly Lys Thr Leu Arg Glu 260 265 270 Gln Ala Gln Ala Arg Gly Tyr Gln
Leu Val Ser Asp Ala Ala Ser Leu 275 280 285 Asn Ser Val Thr Glu Ala
Asn Gln Gln Lys Pro Leu Leu Gly Leu Phe 290 295 300 Ala Asp Gly Asn
Met Pro Val Arg Trp Leu Gly Pro Lys Ala Thr Tyr 305 310 315 320 His
Gly Asn Ile Asp Lys Pro Ala Val Thr Cys Thr Pro Asn Pro Gln 325 330
335 Arg Asn Asp Ser Val Pro Thr Leu Ala Gln Met Thr Asp Lys Ala Ile
340 345 350 Glu Leu Leu Ser Lys Asn Glu Lys Gly Phe Phe Leu Gln Val
Glu Gly 355 360 365 Ala Ser Ile Asp Lys Gln Asp His Ala Ala Asn Pro
Cys Gly Gln Ile 370 375 380 Gly Glu Thr Val Asp Leu Asp Glu Ala Val
Gln Arg Ala Leu Glu Phe 385 390 395 400 Ala Lys Lys Glu Gly Asn Thr
Leu Val Ile Val Thr Ala Asp His Ala 405 410 415 His Ala Ser Gln Ile
Val Ala Pro Asp Thr Lys Ala Pro Gly Leu Thr 420 425 430 Gln Ala Leu
Asn Thr Lys Asp Gly Ala Val Met Val Met Ser Tyr Gly 435 440 445 Asn
Ser Glu Glu Asp Ser Gln Glu His Thr Gly Ser Gln Leu Arg Ile 450 455
460
Ala Ala Tyr Gly Pro His Ala Ala Asn Val Val Gly Leu Thr Asp Gln 465
470 475 480 Thr Asp Leu Phe Tyr Thr Met Lys Ala Ala Leu Gly Leu Lys
Ser Arg 485 490 495 <210> SEQ ID NO 300 <211> LENGTH:
30 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 300 Met Lys Ile Lys Thr Gly Ala
Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser
Ala Ser Ala Leu Ala Lys Ile Ser Arg 20 25 30 <210> SEQ ID NO
301 <211> LENGTH: 372 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 301 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser
Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys
Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile Trp Ile Asn Gly Asp Lys
Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val Gly Lys Lys Phe Glu Lys
Asp Thr Gly Ile Lys Val Thr Val Glu 50 55 60 His Pro Asp Lys Leu
Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly 65 70 75 80 Asp Gly Pro
Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85 90 95 Ala
Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln 100 105
110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys
115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile
Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu
Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys Glu Leu Lys Ala Lys Gly
Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu Gln Glu Pro Tyr Phe Thr
Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185 190 Tyr Ala Phe Lys Tyr
Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly 195 200 205 Val Asp Asn
Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210 215 220 Ile
Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu 225 230
235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile Asn Gly Pro
Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly
Val Thr Val 260 265 270 Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro
Phe Val Gly Val Leu 275 280 285 Ser Ala Gly Ile Asn Ala Ala Ser Pro
Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe Leu Glu Asn Tyr Leu Leu
Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310 315 320 Lys Asp Lys Pro
Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 330 335 Leu Ala
Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Lys 340 345 350
Gly Glu Ile Met Pro Asn Ile Pro Gln Met Ser Ala Phe Trp Tyr Ala 355
360 365 Val Arg Ser Arg 370 <210> SEQ ID NO 302 <211>
LENGTH: 133 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic amino acid sequence <400> SEQUENCE: 302 Met Ile Glu
Ala Arg Ser Arg Leu Glu Ser Asp Lys Ile Ile His Leu 1 5 10 15 Thr
Asp Asp Ser Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala Ile 20 25
30 Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile Ala
35 40 45 Pro Ile Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu
Thr Val 50 55 60 Ala Lys Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala
Pro Lys Tyr Gly 65 70 75 80 Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe
Lys Asn Gly Glu Val Ala 85 90 95 Ala Thr Lys Val Gly Ala Leu Ser
Lys Gly Gln Leu Lys Glu Phe Leu 100 105 110 Asp Ala Asn Leu Ala Leu
Glu Asp Tyr Lys Asp His Asp Gly Asp Tyr 115 120 125 Lys Asp His Asp
Asp 130 <210> SEQ ID NO 303 <211> LENGTH: 767
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 303 Met Lys Ile Lys Thr Gly Ala
Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser
Ala Ser Ala Leu Ala Lys Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile
Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val
Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu 50 55
60 His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly
65 70 75 80 Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly
Gly Tyr 85 90 95 Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp
Lys Ala Phe Gln 100 105 110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala
Val Arg Tyr Asn Gly Lys 115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val
Glu Ala Leu Ser Leu Ile Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn
Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys
Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu
Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185
190 Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly
195 200 205 Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val
Asp Leu 210 215 220 Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr
Ser Ile Ala Glu 225 230 235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala
Met Thr Ile Asn Gly Pro Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr
Ser Lys Val Asn Tyr Gly Val Thr Val 260 265 270 Leu Pro Thr Phe Lys
Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu 275 280 285 Ser Ala Gly
Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe
Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310
315 320 Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu
Glu 325 330 335 Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn
Ala Gln Lys 340 345 350 Gly Glu Ile Met Pro Asn Ile Pro Gln Met Ser
Ala Phe Trp Tyr Ala 355 360 365 Val Leu Ile Glu Ala Arg Thr Ser Glu
Ser Asp Thr Ala Gly Pro Asn 370 375 380 Ser Asp Leu Asp Val Asn Thr
Asp Ile Tyr Ser Lys Val Leu Val Thr 385 390 395 400 Ala Ile Tyr Leu
Ala Leu Phe Val Val Gly Thr Val Gly Asn Ser Val 405 410 415 Thr Ala
Phe Thr Leu Ala Arg Lys Lys Ser Leu Gln Ser Leu Gln Ser 420 425 430
Thr Val His Tyr His Leu Gly Ser Leu Ala Leu Ser Asp Leu Leu Ile 435
440 445 Leu Leu Leu Ala Met Pro Val Glu Leu Tyr Asn Phe Ile Trp Val
His 450 455 460 His Pro Trp Ala Phe Gly Asp Ala Gly Cys Arg Gly Tyr
Tyr Phe Leu 465 470 475 480 Arg Asp Ala Cys Thr Tyr Ala Thr Ala Leu
Asn Val Ala Ser Leu Ser 485 490 495 Val Glu Arg Tyr Leu Ala Ile Cys
His Pro Phe Lys Ala Lys Thr Leu 500 505 510 Met Ser Arg Ser Arg Thr
Lys Lys Phe Ile Ser Ala Ile Trp Leu Ala 515 520 525 Ser Ala Leu Leu
Ala Ile Pro Met Leu Phe Thr Met Gly Leu Gln Asn
530 535 540 Arg Ser Gly Asp Gly Thr His Pro Gly Gly Leu Val Cys Thr
Pro Ile 545 550 555 560 Val Asp Thr Ala Thr Val Lys Val Val Ile Gln
Val Asn Thr Phe Met 565 570 575 Ser Phe Leu Phe Pro Met Leu Val Ile
Ser Ile Leu Asn Thr Val Ile 580 585 590 Ala Asn Lys Leu Thr Val Met
Val His Gln Ala Ala Glu Gln Gly Arg 595 600 605 Val Cys Thr Val Gly
Thr His Asn Gly Leu Glu His Ser Thr Phe Asn 610 615 620 Met Thr Ile
Glu Pro Gly Arg Val Gln Ala Leu Arg His Gly Val Leu 625 630 635 640
Val Leu Arg Ala Val Val Ile Ala Phe Val Val Cys Trp Leu Pro Tyr 645
650 655 His Val Arg Arg Leu Met Phe Cys Tyr Ile Ser Asp Glu Gln Trp
Thr 660 665 670 Thr Phe Leu Phe Asp Phe Tyr His Tyr Phe Tyr Met Leu
Thr Asn Ala 675 680 685 Leu Phe Tyr Val Ser Ser Ala Ile Asn Pro Ile
Leu Tyr Asn Leu Val 690 695 700 Ser Ala Asn Phe Arg Gln Val Phe Leu
Ser Thr Leu Ala Cys Leu Cys 705 710 715 720 Pro Gly Trp Arg His Arg
Arg Lys Lys Arg Pro Thr Phe Ser Arg Lys 725 730 735 Pro Asn Ser Met
Ser Ser Asn His Ala Phe Ser Thr Ser Ala Thr Arg 740 745 750 Glu Thr
Leu Tyr Ala Ala Ala Asp Tyr Lys Asp Asp Asp Asp Lys 755 760 765
<210> SEQ ID NO 304 <211> LENGTH: 425 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 304 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu
Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala
Leu Ala Lys Ile Ile Glu Ala Arg 20 25 30 Thr Ser Glu Ser Asp Thr
Ala Gly Pro Asn Ser Asp Leu Asp Val Asn 35 40 45 Thr Asp Ile Tyr
Ser Lys Val Leu Val Thr Ala Ile Tyr Leu Ala Leu 50 55 60 Phe Val
Val Gly Thr Val Gly Asn Ser Val Thr Ala Phe Thr Leu Ala 65 70 75 80
Arg Lys Lys Ser Leu Gln Ser Leu Gln Ser Thr Val His Tyr His Leu 85
90 95 Gly Ser Leu Ala Leu Ser Asp Leu Leu Ile Leu Leu Leu Ala Met
Pro 100 105 110 Val Glu Leu Tyr Asn Phe Ile Trp Val His His Pro Trp
Ala Phe Gly 115 120 125 Asp Ala Gly Cys Arg Gly Tyr Tyr Phe Leu Arg
Asp Ala Cys Thr Tyr 130 135 140 Ala Thr Ala Leu Asn Val Ala Ser Leu
Ser Val Glu Arg Tyr Leu Ala 145 150 155 160 Ile Cys His Pro Phe Lys
Ala Lys Thr Leu Met Ser Arg Ser Arg Thr 165 170 175 Lys Lys Phe Ile
Ser Ala Ile Trp Leu Ala Ser Ala Leu Leu Ala Ile 180 185 190 Pro Met
Leu Phe Thr Met Gly Leu Gln Asn Arg Ser Gly Asp Gly Thr 195 200 205
His Pro Gly Gly Leu Val Cys Thr Pro Ile Val Asp Thr Ala Thr Val 210
215 220 Lys Val Val Ile Gln Val Asn Thr Phe Met Ser Phe Leu Phe Pro
Met 225 230 235 240 Leu Val Ile Ser Ile Leu Asn Thr Val Ile Ala Asn
Lys Leu Thr Val 245 250 255 Met Val His Gln Ala Ala Glu Gln Gly Arg
Val Cys Thr Val Gly Thr 260 265 270 His Asn Gly Leu Glu His Ser Thr
Phe Asn Met Thr Ile Glu Pro Gly 275 280 285 Arg Val Gln Ala Leu Arg
His Gly Val Leu Val Leu Arg Ala Val Val 290 295 300 Ile Ala Phe Val
Val Cys Trp Leu Pro Tyr His Val Arg Arg Leu Met 305 310 315 320 Phe
Cys Tyr Ile Ser Asp Glu Gln Trp Thr Thr Phe Leu Phe Asp Phe 325 330
335 Tyr His Tyr Phe Tyr Met Leu Thr Asn Ala Leu Phe Tyr Val Ser Ser
340 345 350 Ala Ile Asn Pro Ile Leu Tyr Asn Leu Val Ser Ala Asn Phe
Arg Gln 355 360 365 Val Phe Leu Ser Thr Leu Ala Cys Leu Cys Pro Gly
Trp Arg His Arg 370 375 380 Arg Lys Lys Arg Pro Thr Phe Ser Arg Lys
Pro Asn Ser Met Ser Ser 385 390 395 400 Asn His Ala Phe Ser Thr Ser
Ala Thr Arg Glu Thr Leu Tyr Ala Ala 405 410 415 Ala Asp Tyr Lys Asp
Asp Asp Asp Lys 420 425 <210> SEQ ID NO 305 <211>
LENGTH: 878 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic amino acid sequence <400> SEQUENCE: 305 Met Lys Ile
Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr
Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Glu Glu Gly Lys 20 25
30 Leu Val Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu
35 40 45 Val Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr
Val Glu 50 55 60 His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val
Ala Ala Thr Gly 65 70 75 80 Asp Gly Pro Asp Ile Ile Phe Trp Ala His
Asp Arg Phe Gly Gly Tyr 85 90 95 Ala Gln Ser Gly Leu Leu Ala Glu
Ile Thr Pro Asp Lys Ala Phe Gln 100 105 110 Asp Lys Leu Tyr Pro Phe
Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys 115 120 125 Leu Ile Ala Tyr
Pro Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn 130 135 140 Lys Asp
Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala 145 150 155
160 Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn
165 170 175 Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp
Gly Gly 180 185 190 Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile
Lys Asp Val Gly 195 200 205 Val Asp Asn Ala Gly Ala Lys Ala Gly Leu
Thr Phe Leu Val Asp Leu 210 215 220 Ile Lys Asn Lys His Met Asn Ala
Asp Thr Asp Tyr Ser Ile Ala Glu 225 230 235 240 Ala Ala Phe Asn Lys
Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp 245 250 255 Ala Trp Ser
Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val 260 265 270 Leu
Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu 275 280
285 Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu
290 295 300 Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala
Val Asn 305 310 315 320 Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys
Ser Tyr Glu Glu Glu 325 330 335 Leu Ala Lys Asp Pro Arg Ile Ala Ala
Thr Met Glu Asn Ala Gln Lys 340 345 350 Gly Glu Ile Met Pro Asn Ile
Pro Gln Met Ser Ala Phe Trp Tyr Ala 355 360 365 Val Leu Ile Glu Ala
Arg Thr Ser Glu Ser Asp Thr Ala Gly Pro Asn 370 375 380 Ser Asp Leu
Asp Val Asn Thr Asp Ile Tyr Ser Lys Val Leu Val Thr 385 390 395 400
Ala Ile Tyr Leu Ala Leu Phe Val Val Gly Thr Val Gly Asn Ser Val 405
410 415 Thr Ala Phe Thr Leu Ala Arg Lys Lys Ser Leu Gln Ser Leu Gln
Ser 420 425 430 Thr Val His Tyr His Leu Gly Ser Leu Ala Leu Ser Asp
Leu Leu Ile 435 440 445 Leu Leu Leu Ala Met Pro Val Glu Leu Tyr Asn
Phe Ile Trp Val His 450 455 460 His Pro Trp Ala Phe Gly Asp Ala Gly
Cys Arg Gly Tyr Tyr Phe Leu 465 470 475 480 Arg Asp Ala Cys Thr Tyr
Ala Thr Ala Leu Asn Val Ala Ser Leu Ser 485 490 495 Val Glu Arg Tyr
Leu Ala Ile Cys His Pro Phe Lys Ala Lys Thr Leu 500 505 510 Met Ser
Arg Ser Arg Thr Lys Lys Phe Ile Ser Ala Ile Trp Leu Ala 515 520 525
Ser Ala Leu Leu Ala Ile Pro Met Leu Phe Thr Met Gly Leu Gln Asn 530
535 540 Arg Ser Gly Asp Gly Thr His Pro Gly Gly Leu Val Cys Thr Pro
Ile 545 550 555 560 Val Asp Thr Ala Thr Val Lys Val Val Ile Gln Val
Asn Thr Phe Met
565 570 575 Ser Phe Leu Phe Pro Met Leu Val Ile Ser Ile Leu Asn Thr
Val Ile 580 585 590 Ala Asn Lys Leu Thr Val Met Val His Gln Ala Ala
Glu Gln Gly Arg 595 600 605 Val Cys Thr Val Gly Thr His Asn Gly Leu
Glu His Ser Thr Phe Asn 610 615 620 Met Thr Ile Glu Pro Gly Arg Val
Gln Ala Leu Arg His Gly Val Leu 625 630 635 640 Val Leu Arg Ala Val
Val Ile Ala Phe Val Val Cys Trp Leu Pro Tyr 645 650 655 His Val Arg
Arg Leu Met Phe Cys Tyr Ile Ser Asp Glu Gln Trp Thr 660 665 670 Thr
Phe Leu Phe Asp Phe Tyr His Tyr Phe Tyr Met Leu Thr Asn Ala 675 680
685 Leu Phe Tyr Val Ser Ser Ala Ile Asn Pro Ile Leu Tyr Asn Leu Val
690 695 700 Ser Ala Asn Phe Arg Gln Val Phe Leu Ser Thr Leu Ala Cys
Leu Cys 705 710 715 720 Pro Gly Trp Arg His Arg Arg Lys Lys Arg Pro
Thr Phe Ser Arg Lys 725 730 735 Pro Asn Ser Met Ser Ser Asn His Ala
Phe Ser Thr Ser Ala Thr Arg 740 745 750 Glu Thr Leu Tyr Ala Ala Ala
Ser Asp Lys Ile Ile His Leu Thr Asp 755 760 765 Asp Ser Phe Asp Thr
Asp Val Leu Lys Ala Asp Gly Ala Ile Leu Val 770 775 780 Asp Phe Trp
Ala Glu Trp Cys Gly Pro Cys Lys Met Ile Ala Pro Ile 785 790 795 800
Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys 805
810 815 Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile
Arg 820 825 830 Gly Ile Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val
Ala Ala Thr 835 840 845 Lys Val Gly Ala Leu Ser Lys Gly Gln Leu Lys
Glu Phe Leu Asp Ala 850 855 860 Asn Leu Ala Ala Ala Ala Asp Tyr Lys
Asp Asp Asp Asp Lys 865 870 875 <210> SEQ ID NO 306
<211> LENGTH: 536 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic amino acid sequence <400> SEQUENCE:
306 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr
1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Ile Glu
Ala Arg 20 25 30 Thr Ser Glu Ser Asp Thr Ala Gly Pro Asn Ser Asp
Leu Asp Val Asn 35 40 45 Thr Asp Ile Tyr Ser Lys Val Leu Val Thr
Ala Ile Tyr Leu Ala Leu 50 55 60 Phe Val Val Gly Thr Val Gly Asn
Ser Val Thr Ala Phe Thr Leu Ala 65 70 75 80 Arg Lys Lys Ser Leu Gln
Ser Leu Gln Ser Thr Val His Tyr His Leu 85 90 95 Gly Ser Leu Ala
Leu Ser Asp Leu Leu Ile Leu Leu Leu Ala Met Pro 100 105 110 Val Glu
Leu Tyr Asn Phe Ile Trp Val His His Pro Trp Ala Phe Gly 115 120 125
Asp Ala Gly Cys Arg Gly Tyr Tyr Phe Leu Arg Asp Ala Cys Thr Tyr 130
135 140 Ala Thr Ala Leu Asn Val Ala Ser Leu Ser Val Glu Arg Tyr Leu
Ala 145 150 155 160 Ile Cys His Pro Phe Lys Ala Lys Thr Leu Met Ser
Arg Ser Arg Thr 165 170 175 Lys Lys Phe Ile Ser Ala Ile Trp Leu Ala
Ser Ala Leu Leu Ala Ile 180 185 190 Pro Met Leu Phe Thr Met Gly Leu
Gln Asn Arg Ser Gly Asp Gly Thr 195 200 205 His Pro Gly Gly Leu Val
Cys Thr Pro Ile Val Asp Thr Ala Thr Val 210 215 220 Lys Val Val Ile
Gln Val Asn Thr Phe Met Ser Phe Leu Phe Pro Met 225 230 235 240 Leu
Val Ile Ser Ile Leu Asn Thr Val Ile Ala Asn Lys Leu Thr Val 245 250
255 Met Val His Gln Ala Ala Glu Gln Gly Arg Val Cys Thr Val Gly Thr
260 265 270 His Asn Gly Leu Glu His Ser Thr Phe Asn Met Thr Ile Glu
Pro Gly 275 280 285 Arg Val Gln Ala Leu Arg His Gly Val Leu Val Leu
Arg Ala Val Val 290 295 300 Ile Ala Phe Val Val Cys Trp Leu Pro Tyr
His Val Arg Arg Leu Met 305 310 315 320 Phe Cys Tyr Ile Ser Asp Glu
Gln Trp Thr Thr Phe Leu Phe Asp Phe 325 330 335 Tyr His Tyr Phe Tyr
Met Leu Thr Asn Ala Leu Phe Tyr Val Ser Ser 340 345 350 Ala Ile Asn
Pro Ile Leu Tyr Asn Leu Val Ser Ala Asn Phe Arg Gln 355 360 365 Val
Phe Leu Ser Thr Leu Ala Cys Leu Cys Pro Gly Trp Arg His Arg 370 375
380 Arg Lys Lys Arg Pro Thr Phe Ser Arg Lys Pro Asn Ser Met Ser Ser
385 390 395 400 Asn His Ala Phe Ser Thr Ser Ala Thr Arg Glu Thr Leu
Tyr Ala Ala 405 410 415 Ala Ser Asp Lys Ile Ile His Leu Thr Asp Asp
Ser Phe Asp Thr Asp 420 425 430 Val Leu Lys Ala Asp Gly Ala Ile Leu
Val Asp Phe Trp Ala Glu Trp 435 440 445 Cys Gly Pro Cys Lys Met Ile
Ala Pro Ile Leu Asp Glu Ile Ala Asp 450 455 460 Glu Tyr Gln Gly Lys
Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn 465 470 475 480 Pro Gly
Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu 485 490 495
Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser 500
505 510 Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Ala Ala
Ala 515 520 525 Asp Tyr Lys Asp Asp Asp Asp Lys 530 535 <210>
SEQ ID NO 307 <211> LENGTH: 380 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 307 Met Gly Cys Leu Gly Asn Ser Lys Thr Glu
Asp Gln Arg Asn Glu Glu 1 5 10 15 Lys Ala Gln Arg Glu Ala Asn Lys
Lys Ile Glu Lys Gln Leu Gln Lys 20 25 30 Asp Lys Gln Val Tyr Arg
Ala Thr His Arg Leu Leu Leu Leu Gly Ala 35 40 45 Gly Glu Ser Gly
Lys Ser Thr Ile Val Lys Gln Met Arg Ile Leu His 50 55 60 Val Asn
Gly Phe Asn Gly Asp Ser Glu Lys Ala Thr Lys Val Gln Asp 65 70 75 80
Ile Lys Asn Asn Leu Lys Glu Ala Ile Glu Thr Ile Val Ala Ala Met 85
90 95 Ser Asn Leu Val Pro Pro Val Glu Leu Ala Asn Pro Glu Asn Gln
Phe 100 105 110 Arg Val Asp Tyr Ile Leu Ser Val Met Asn Val Pro Asp
Phe Asp Phe 115 120 125 Pro Pro Glu Phe Tyr Glu His Ala Lys Ala Leu
Trp Glu Asp Glu Gly 130 135 140 Val Arg Ala Cys Tyr Glu Arg Ser Asn
Glu Tyr Gln Leu Ile Asp Cys 145 150 155 160 Ala Gln Tyr Phe Leu Asp
Lys Ile Asp Val Ile Lys Gln Ala Asp Tyr 165 170 175 Val Pro Ser Asp
Gln Asp Leu Leu Arg Cys Arg Val Leu Thr Ser Gly 180 185 190 Ile Phe
Glu Thr Lys Phe Gln Val Asp Lys Val Asn Phe His Met Phe 195 200 205
Asp Val Gly Gly Gln Arg Asp Glu Arg Arg Lys Trp Ile Gln Cys Phe 210
215 220 Asn Asp Val Thr Ala Ile Ile Phe Val Val Ala Ser Ser Ser Tyr
Asn 225 230 235 240 Met Val Ile Arg Glu Asp Asn Gln Thr Asn Arg Leu
Gln Glu Ala Leu 245 250 255 Asn Leu Phe Lys Ser Ile Trp Asn Asn Arg
Trp Leu Arg Thr Ile Ser 260 265 270 Val Ile Leu Phe Leu Asn Lys Gln
Asp Leu Leu Ala Glu Lys Val Leu 275 280 285 Ala Gly Lys Ser Lys Ile
Glu Asp Tyr Phe Pro Glu Phe Ala Arg Tyr 290 295 300 Thr Thr Pro Glu
Asp Ala Thr Pro Glu Pro Gly Glu Asp Pro Arg Val 305 310 315 320 Thr
Arg Ala Lys Tyr Phe Ile Arg Asp Glu Phe Leu Arg Ile Ser Thr 325 330
335 Ala Ser Gly Asp Gly Arg His Tyr Cys Tyr Pro His Phe Thr Cys Ala
340 345 350 Val Asp Thr Glu Asn Ile Arg Arg Val Phe Asn Asp Cys Arg
Asp Ile 355 360 365 Ile Gln Arg Met His Leu Arg Gln Tyr Glu Leu
Leu
370 375 380 <210> SEQ ID NO 308 <211> LENGTH: 394
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 308 Met Gly Cys Leu Gly Asn Ser
Lys Thr Glu Asp Gln Arg Asn Glu Glu 1 5 10 15 Lys Ala Gln Arg Glu
Ala Asn Lys Lys Ile Glu Lys Gln Leu Gln Lys 20 25 30 Asp Lys Gln
Val Tyr Arg Ala Thr His Arg Leu Leu Leu Leu Gly Ala 35 40 45 Gly
Glu Ser Gly Lys Ser Thr Ile Val Lys Gln Met Arg Ile Leu His 50 55
60 Val Asn Gly Phe Asn Gly Glu Gly Gly Glu Glu Asp Pro Gln Ala Ala
65 70 75 80 Arg Ser Asn Ser Asp Gly Glu Lys Ala Thr Lys Val Gln Asp
Ile Lys 85 90 95 Asn Asn Leu Lys Glu Ala Ile Glu Thr Ile Val Ala
Ala Met Ser Asn 100 105 110 Leu Val Pro Pro Val Glu Leu Ala Asn Pro
Glu Asn Gln Phe Arg Val 115 120 125 Asp Tyr Ile Leu Ser Val Met Asn
Val Pro Asp Phe Asp Phe Pro Pro 130 135 140 Glu Phe Tyr Glu His Ala
Lys Ala Leu Trp Glu Asp Glu Gly Val Arg 145 150 155 160 Ala Cys Tyr
Glu Arg Ser Asn Glu Tyr Gln Leu Ile Asp Cys Ala Gln 165 170 175 Tyr
Phe Leu Asp Lys Ile Asp Val Ile Lys Gln Ala Asp Tyr Val Pro 180 185
190 Ser Asp Gln Asp Leu Leu Arg Cys Arg Val Leu Thr Ser Gly Ile Phe
195 200 205 Glu Thr Lys Phe Gln Val Asp Lys Val Asn Phe His Met Phe
Asp Val 210 215 220 Gly Gly Gln Arg Asp Glu Arg Arg Lys Trp Ile Gln
Cys Phe Asn Asp 225 230 235 240 Val Thr Ala Ile Ile Phe Val Val Ala
Ser Ser Ser Tyr Asn Met Val 245 250 255 Ile Arg Glu Asp Asn Gln Thr
Asn Arg Leu Gln Glu Ala Leu Asn Leu 260 265 270 Phe Lys Ser Ile Trp
Asn Asn Arg Trp Leu Arg Thr Ile Ser Val Ile 275 280 285 Leu Phe Leu
Asn Lys Gln Asp Leu Leu Ala Glu Lys Val Leu Ala Gly 290 295 300 Lys
Ser Lys Ile Glu Asp Tyr Phe Pro Glu Phe Ala Arg Tyr Thr Thr 305 310
315 320 Pro Glu Asp Ala Thr Pro Glu Pro Gly Glu Asp Pro Arg Val Thr
Arg 325 330 335 Ala Lys Tyr Phe Ile Arg Asp Glu Phe Leu Arg Ile Ser
Thr Ala Ser 340 345 350 Gly Asp Gly Arg His Tyr Cys Tyr Pro His Phe
Thr Cys Ala Val Asp 355 360 365 Thr Glu Asn Ile Arg Arg Val Phe Asn
Asp Cys Arg Asp Ile Ile Gln 370 375 380 Arg Met His Leu Arg Gln Tyr
Glu Leu Leu 385 390 <210> SEQ ID NO 309 <211> LENGTH:
359 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 309 Met Thr Leu Glu Ser Ile Met
Ala Cys Cys Leu Ser Glu Glu Ala Lys 1 5 10 15 Glu Ala Arg Arg Ile
Asn Asp Glu Ile Glu Arg Gln Leu Arg Arg Asp 20 25 30 Lys Arg Asp
Ala Arg Arg Glu Leu Lys Leu Leu Leu Leu Gly Thr Gly 35 40 45 Glu
Ser Gly Lys Ser Thr Phe Ile Lys Gln Met Arg Ile Ile His Gly 50 55
60 Ser Gly Tyr Ser Asp Glu Asp Lys Arg Gly Phe Thr Lys Leu Val Tyr
65 70 75 80 Gln Asn Ile Phe Thr Ala Met Gln Ala Met Ile Arg Ala Met
Asp Thr 85 90 95 Leu Lys Ile Pro Tyr Lys Tyr Glu His Asn Lys Ala
His Ala Gln Leu 100 105 110 Val Arg Glu Val Asp Val Glu Lys Val Ser
Ala Phe Glu Asn Pro Tyr 115 120 125 Val Asp Ala Ile Lys Ser Leu Trp
Asn Asp Pro Gly Ile Gln Glu Cys 130 135 140 Tyr Asp Arg Arg Arg Glu
Tyr Gln Leu Ser Asp Ser Thr Lys Tyr Tyr 145 150 155 160 Leu Asn Asp
Leu Asp Arg Val Ala Asp Pro Ala Tyr Leu Pro Thr Gln 165 170 175 Gln
Asp Val Leu Arg Val Arg Val Pro Thr Thr Gly Ile Ile Glu Tyr 180 185
190 Pro Phe Asp Leu Gln Ser Val Ile Phe Arg Met Val Asp Val Gly Gly
195 200 205 Gln Arg Ser Glu Arg Arg Lys Trp Ile His Cys Phe Glu Asn
Val Thr 210 215 220 Ser Ile Met Phe Leu Val Ala Leu Ser Glu Tyr Asp
Gln Val Leu Val 225 230 235 240 Glu Ser Asp Asn Glu Asn Arg Met Glu
Glu Ser Lys Ala Leu Phe Arg 245 250 255 Thr Ile Ile Thr Tyr Pro Trp
Phe Gln Asn Ser Ser Val Ile Leu Phe 260 265 270 Leu Asn Lys Lys Asp
Leu Leu Glu Glu Lys Ile Met Tyr Ser His Leu 275 280 285 Val Asp Tyr
Phe Pro Glu Tyr Asp Gly Pro Gln Arg Asp Ala Gln Ala 290 295 300 Ala
Arg Glu Phe Ile Leu Lys Met Phe Val Asp Leu Asn Pro Asp Ser 305 310
315 320 Asp Lys Ile Ile Tyr Ser His Phe Thr Cys Ala Thr Asp Thr Glu
Asn 325 330 335 Ile Arg Phe Val Phe Ala Ala Val Lys Asp Thr Ile Leu
Gln Leu Asn 340 345 350 Leu Lys Glu Tyr Asn Leu Val 355 <210>
SEQ ID NO 310 <211> LENGTH: 355 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 310 Met Gly Cys Thr Val Ser Ala Glu Asp Lys
Ala Ala Ala Glu Arg Ser 1 5 10 15 Lys Met Ile Asp Lys Asn Leu Arg
Glu Asp Gly Glu Lys Ala Ala Arg 20 25 30 Glu Val Lys Leu Leu Leu
Leu Gly Ala Gly Glu Ser Gly Lys Ser Thr 35 40 45 Ile Val Lys Gln
Met Lys Ile Ile His Glu Asp Gly Tyr Ser Glu Glu 50 55 60 Glu Cys
Arg Gln Tyr Arg Ala Val Val Tyr Ser Asn Thr Ile Gln Ser 65 70 75 80
Ile Met Ala Ile Val Lys Ala Met Gly Asn Leu Gln Ile Asp Phe Ala 85
90 95 Asp Pro Ser Arg Ala Asp Asp Ala Arg Gln Leu Phe Ala Leu Ser
Cys 100 105 110 Thr Ala Glu Glu Gln Gly Val Leu Pro Asp Asp Leu Ser
Gly Val Ile 115 120 125 Arg Arg Leu Trp Ala Asp His Gly Val Gln Ala
Cys Phe Gly Arg Ser 130 135 140 Arg Glu Tyr Gln Leu Asn Asp Ser Ala
Ala Tyr Tyr Leu Asn Asp Leu 145 150 155 160 Glu Arg Ile Ala Gln Ser
Asp Tyr Ile Pro Thr Gln Gln Asp Val Leu 165 170 175 Arg Thr Arg Val
Lys Thr Thr Gly Ile Val Glu Thr His Phe Thr Phe 180 185 190 Lys Asp
Leu His Phe Lys Met Phe Asp Val Gly Gly Gln Arg Ser Glu 195 200 205
Arg Lys Lys Trp Ile His Cys Phe Glu Gly Val Thr Ala Ile Ile Phe 210
215 220 Cys Val Ala Leu Ser Ala Tyr Asp Leu Val Leu Ala Glu Asp Glu
Glu 225 230 235 240 Met Asn Arg Met His Glu Ser Met Lys Leu Phe Asp
Ser Ile Cys Asn 245 250 255 Asn Lys Trp Phe Thr Asp Thr Ser Ile Ile
Leu Phe Leu Asn Lys Lys 260 265 270 Asp Leu Phe Glu Glu Lys Ile Thr
His Ser Pro Leu Thr Ile Cys Phe 275 280 285 Pro Glu Tyr Thr Gly Ala
Asn Lys Tyr Asp Glu Ala Ala Ser Tyr Ile 290 295 300 Gln Ser Lys Phe
Glu Asp Leu Asn Lys Arg Lys Asp Thr Lys Glu Ile 305 310 315 320 Tyr
Thr His Phe Thr Cys Ala Thr Asp Thr Lys Asn Val Gln Phe Val 325 330
335 Phe Asp Ala Val Thr Asp Val Ile Ile Lys Asn Asn Leu Lys Asp Cys
340 345 350 Gly Leu Phe 355 <210> SEQ ID NO 311 <211>
LENGTH: 381 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 311 Met Ser Gly Val Val Arg Thr Leu Ser Arg
Cys Leu Leu Pro Ala Glu 1 5 10 15 Ala Gly Gly Ala Arg Glu Arg Arg
Ala Gly Ser Gly Ala Arg Asp Ala 20 25 30 Glu Arg Glu Ala Arg Arg
Arg Ser Arg Asp Ile Asp Ala Leu Leu Ala 35 40 45 Arg Glu Arg Arg
Ala Val Arg Arg Leu Val Lys Ile Leu Leu Leu Gly 50 55 60 Ala Gly
Glu Ser Gly Lys Ser Thr Phe Leu Lys Gln Met Arg Ile Ile 65 70 75 80
His Gly Arg Glu Phe Asp Gln Lys Ala Leu Leu Glu Phe Arg Asp Thr 85
90 95 Ile Phe Asp Asn Ile Leu Lys Gly Ser Arg Val Leu Val Asp Ala
Arg 100 105 110 Asp Lys Leu Gly Ile Pro Trp Gln Tyr Ser Glu Asn Glu
Lys His Gly 115 120 125 Met Phe Leu Met Ala Phe Glu Asn Lys Ala Gly
Leu Pro Val Glu Pro 130 135 140 Ala Thr Phe Gln Leu Tyr Val Pro Ala
Leu Ser Ala Leu Trp Arg Asp 145 150 155 160 Ser Gly Ile Arg Glu Ala
Phe Ser Arg Arg Ser Glu Phe Gln Leu Gly 165 170 175 Glu Ser Val Lys
Tyr Phe Leu Asp Asn Leu Asp Arg Ile Gly Gln Leu 180 185 190 Asn Tyr
Phe Pro Ser Lys Gln Asp Ile Leu Leu Ala Arg Lys Ala Thr 195 200 205
Lys Gly Ile Val Glu His Asp Phe Val Ile Lys Lys Ile Pro Phe Lys 210
215 220 Met Val Asp Val Gly Gly Gln Arg Ser Gln Arg Gln Lys Trp Phe
Gln 225 230 235 240 Cys Phe Asp Gly Ile Thr Ser Ile Leu Phe Met Val
Ser Ser Ser Glu 245 250 255 Tyr Asp Gln Val Leu Met Glu Asp Arg Arg
Thr Asn Arg Leu Val Glu 260 265 270 Ser Met Asn Ile Phe Glu Thr Ile
Val Asn Asn Lys Leu Phe Phe Asn 275 280 285 Val Ser Ile Ile Leu Phe
Leu Asn Lys Met Asp Leu Leu Val Glu Lys 290 295 300 Val Lys Thr Val
Ser Ile Lys Lys His Phe Pro Asp Phe Arg Gly Asp 305 310 315 320 Pro
His Gln Leu Glu Asp Val Gln Arg Tyr Leu Val Gln Cys Phe Asp 325 330
335 Arg Lys Arg Arg Asn Arg Ser Lys Pro Leu Phe His His Phe Thr Thr
340 345 350 Ala Ile Asp Thr Glu Asn Val Arg Phe Val Phe His Ala Val
Lys Asp 355 360 365 Thr Ile Leu Gln Glu Asn Leu Lys Asp Ile Met Leu
Gln 370 375 380 <210> SEQ ID NO 312 <211> LENGTH: 50
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 312 Met Lys Ile Lys Thr Gly Ala
Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser
Ala Ser Ala Leu Ala Lys Ile Ile Glu Ala Arg 20 25 30 Leu Gln Ala
Ser Val Asp Ala Glu Ser Arg Asp Tyr Lys Asp Asp Asp 35 40 45 Asp
Lys 50 <210> SEQ ID NO 313 <211> LENGTH: 27 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic amino acid
sequence <400> SEQUENCE: 313 Met Lys Ile Lys Thr Gly Ala Arg
Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala
Ser Ala Leu Ala Lys 20 25 <210> SEQ ID NO 314 <211>
LENGTH: 50 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic amino acid sequence <400> SEQUENCE: 314 Met Lys Ile
Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr
Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Ile Glu Ala Arg 20 25
30 Leu Gln Ala Ser Val Asp Ala Glu Ser Arg Asp Tyr Lys Asp Asp Asp
35 40 45 Asp Lys 50 <210> SEQ ID NO 315 <211> LENGTH:
50 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 315 Met Lys Ile Lys Thr Gly Ala
Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser
Ala Ser Ala Leu Ala Lys Ile Ile Glu Ala Arg 20 25 30 Leu Gln Ala
Ser Val Asp Ala Glu Ser Arg Asp Tyr Lys Asp Asp Asp 35 40 45 Asp
Lys 50 <210> SEQ ID NO 316 <211> LENGTH: 343
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 316 Met Lys Ile Lys Thr Gly Ala
Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser
Ala Ser Ala Leu Ala Lys Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile
Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val
Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu 50 55
60 His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly
65 70 75 80 Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly
Gly Tyr 85 90 95 Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp
Lys Ala Phe Gln 100 105 110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala
Val Arg Tyr Asn Gly Lys 115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val
Glu Ala Leu Ser Leu Ile Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn
Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys
Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu
Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185
190 Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly
195 200 205 Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val
Asp Leu 210 215 220 Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr
Ser Ile Ala Glu 225 230 235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala
Met Thr Ile Asn Gly Pro Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr
Ser Lys Val Asn Tyr Gly Val Thr Val 260 265 270 Leu Pro Thr Phe Lys
Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu 275 280 285 Ser Ala Gly
Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe
Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310
315 320 Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu
Glu 325 330 335 Leu Ala Lys Asp Pro Arg Ile 340 <210> SEQ ID
NO 317 <211> LENGTH: 381 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 317 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser
Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys
Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile Trp Ile Asn Gly Asp Lys
Gly Tyr Asn Gly Leu Ala Glu
35 40 45 Val Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr
Val Glu 50 55 60 His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val
Ala Ala Thr Gly 65 70 75 80 Asp Gly Pro Asp Ile Ile Phe Trp Ala His
Asp Arg Phe Gly Gly Tyr 85 90 95 Ala Gln Ser Gly Leu Leu Ala Glu
Ile Thr Pro Asp Lys Ala Phe Gln 100 105 110 Asp Lys Leu Tyr Pro Phe
Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys 115 120 125 Leu Ile Ala Tyr
Pro Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn 130 135 140 Lys Asp
Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala 145 150 155
160 Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn
165 170 175 Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp
Gly Gly 180 185 190 Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile
Lys Asp Val Gly 195 200 205 Val Asp Asn Ala Gly Ala Lys Ala Gly Leu
Thr Phe Leu Val Asp Leu 210 215 220 Ile Lys Asn Lys His Met Asn Ala
Asp Thr Asp Tyr Ser Ile Ala Glu 225 230 235 240 Ala Ala Phe Asn Lys
Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp 245 250 255 Ala Trp Ser
Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val 260 265 270 Leu
Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu 275 280
285 Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu
290 295 300 Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala
Val Asn 305 310 315 320 Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys
Ser Tyr Glu Glu Glu 325 330 335 Leu Ala Lys Asp Pro Arg Ile Ala Ala
Thr Met Glu Asn Ala Gln Ser 340 345 350 Ala Phe Trp Tyr Ala Val Arg
Ile Glu Ala Arg Leu Gln Ala Ser Val 355 360 365 Asp Ala Glu Ser Arg
Asp Tyr Lys Asp Asp Asp Asp Lys 370 375 380 <210> SEQ ID NO
318 <211> LENGTH: 381 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 318 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser
Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys
Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile Trp Ile Asn Gly Asp Lys
Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val Gly Lys Lys Phe Glu Lys
Asp Thr Gly Ile Lys Val Thr Val Glu 50 55 60 His Pro Asp Lys Leu
Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly 65 70 75 80 Asp Gly Pro
Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85 90 95 Ala
Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln 100 105
110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys
115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile
Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu
Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys Glu Leu Lys Ala Lys Gly
Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu Gln Glu Pro Tyr Phe Thr
Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185 190 Tyr Ala Phe Lys Tyr
Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly 195 200 205 Val Asp Asn
Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210 215 220 Ile
Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu 225 230
235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile Asn Gly Pro
Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly
Val Thr Val 260 265 270 Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro
Phe Val Gly Val Leu 275 280 285 Ser Ala Gly Ile Asn Ala Ala Ser Pro
Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe Leu Glu Asn Tyr Leu Leu
Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310 315 320 Lys Asp Lys Pro
Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 330 335 Leu Ala
Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Ser 340 345 350
Ala Phe Trp Tyr Ala Val Arg Ile Glu Ala Arg Leu Gln Ala Ser Val 355
360 365 Asp Ala Glu Ser Arg Asp Tyr Lys Asp Asp Asp Asp Lys 370 375
380 <210> SEQ ID NO 319 <211> LENGTH: 381 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic amino acid
sequence <400> SEQUENCE: 319 Met Lys Ile Lys Thr Gly Ala Arg
Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala
Ser Ala Leu Ala Lys Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile Trp
Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val Gly
Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu 50 55 60
His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly 65
70 75 80 Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly
Gly Tyr 85 90 95 Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp
Lys Ala Phe Gln 100 105 110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala
Val Arg Tyr Asn Gly Lys 115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val
Glu Ala Leu Ser Leu Ile Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn
Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys
Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu
Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185
190 Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly
195 200 205 Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val
Asp Leu 210 215 220 Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr
Ser Ile Ala Glu 225 230 235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala
Met Thr Ile Asn Gly Pro Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr
Ser Lys Val Asn Tyr Gly Val Thr Val 260 265 270 Leu Pro Thr Phe Lys
Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu 275 280 285 Ser Ala Gly
Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe
Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310
315 320 Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu
Glu 325 330 335 Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn
Ala Gln Ser 340 345 350 Ala Phe Trp Tyr Ala Val Arg Ile Glu Ala Arg
Leu Gln Ala Ser Val 355 360 365 Asp Ala Glu Ser Arg Asp Tyr Lys Asp
Asp Asp Asp Lys 370 375 380 <210> SEQ ID NO 320 <211>
LENGTH: 120 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic amino acid sequence <400> SEQUENCE: 320 Ala Ser Val
Asp Ala Glu Ser Arg Ser Asp Lys Ile Ile His Leu Thr 1 5 10 15 Asp
Asp Ser Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala Ile Leu 20 25
30 Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile Ala Pro
35 40 45 Ile Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr
Val Ala 50 55 60
Lys Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile 65
70 75 80 Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val
Ala Ala 85 90 95 Thr Lys Val Gly Ala Leu Ser Lys Gly Gln Leu Lys
Glu Asn Leu Ala 100 105 110 Asp Tyr Lys Asp Asp Asp Asp Lys 115 120
<210> SEQ ID NO 321 <211> LENGTH: 120 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 321 Ala Ser Val Asp Ala Glu Ser Arg Ser Asp
Lys Ile Ile His Leu Thr 1 5 10 15 Asp Asp Ser Phe Asp Thr Asp Val
Leu Lys Ala Asp Gly Ala Ile Leu 20 25 30 Val Asp Phe Trp Ala Glu
Trp Cys Gly Pro Cys Lys Met Ile Ala Pro 35 40 45 Ile Leu Asp Glu
Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala 50 55 60 Lys Leu
Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile 65 70 75 80
Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala 85
90 95 Thr Lys Val Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Asn Leu
Ala 100 105 110 Asp Tyr Lys Asp Asp Asp Asp Lys 115 120 <210>
SEQ ID NO 322 <211> LENGTH: 120 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 322 Ala Ser Val Asp Ala Glu Ser Arg Ser Asp
Lys Ile Ile His Leu Thr 1 5 10 15 Asp Asp Ser Phe Asp Thr Asp Val
Leu Lys Ala Asp Gly Ala Ile Leu 20 25 30 Val Asp Phe Trp Ala Glu
Trp Cys Gly Pro Cys Lys Met Ile Ala Pro 35 40 45 Ile Leu Asp Glu
Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala 50 55 60 Lys Leu
Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile 65 70 75 80
Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala 85
90 95 Thr Lys Val Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Asn Leu
Ala 100 105 110 Asp Tyr Lys Asp Asp Asp Asp Lys 115 120 <210>
SEQ ID NO 323 <211> LENGTH: 120 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 323 Ala Ser Val Asp Ala Glu Ser Arg Ser Asp
Lys Ile Ile His Leu Thr 1 5 10 15 Asp Asp Ser Phe Asp Thr Asp Val
Leu Lys Ala Asp Gly Ala Ile Leu 20 25 30 Val Asp Phe Trp Ala Glu
Trp Cys Gly Pro Cys Lys Met Ile Ala Pro 35 40 45 Ile Leu Asp Glu
Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala 50 55 60 Lys Leu
Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile 65 70 75 80
Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala 85
90 95 Thr Lys Val Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Asn Leu
Ala 100 105 110 Asp Tyr Lys Asp Asp Asp Asp Lys 115 120 <210>
SEQ ID NO 324 <211> LENGTH: 154 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 324 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu
Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala
Leu Ala Lys Ile Ile Glu Ala Arg 20 25 30 Leu Gln Ala Ser Val Asp
Ala Glu Ser Arg Ser Asp Lys Ile Ile His 35 40 45 Leu Thr Asp Asp
Ser Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala 50 55 60 Ile Leu
Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile 65 70 75 80
Ala Pro Ile Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr 85
90 95 Val Ala Lys Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys
Tyr 100 105 110 Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys Asn
Gly Glu Val 115 120 125 Ala Ala Thr Lys Val Gly Ala Leu Ser Lys Gly
Gln Leu Lys Glu Asn 130 135 140 Leu Ala Asp Tyr Lys Asp Asp Asp Asp
Lys 145 150 <210> SEQ ID NO 325 <211> LENGTH: 154
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 325 Met Lys Ile Lys Thr Gly Ala
Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser
Ala Ser Ala Leu Ala Lys Ile Ile Glu Ala Arg 20 25 30 Leu Gln Ala
Ser Val Asp Ala Glu Ser Arg Ser Asp Lys Ile Ile His 35 40 45 Leu
Thr Asp Asp Ser Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala 50 55
60 Ile Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile
65 70 75 80 Ala Pro Ile Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys
Leu Thr 85 90 95 Val Ala Lys Leu Asn Ile Asp Gln Asn Pro Gly Thr
Ala Pro Lys Tyr 100 105 110 Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu
Phe Lys Asn Gly Glu Val 115 120 125 Ala Ala Thr Lys Val Gly Ala Leu
Ser Lys Gly Gln Leu Lys Glu Asn 130 135 140 Leu Ala Asp Tyr Lys Asp
Asp Asp Asp Lys 145 150 <210> SEQ ID NO 326 <211>
LENGTH: 154 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic amino acid sequence <400> SEQUENCE: 326 Met Lys Ile
Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr
Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Ile Glu Ala Arg 20 25
30 Leu Gln Ala Ser Val Asp Ala Glu Ser Arg Ser Asp Lys Ile Ile His
35 40 45 Leu Thr Asp Asp Ser Phe Asp Thr Asp Val Leu Lys Ala Asp
Gly Ala 50 55 60 Ile Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro
Cys Lys Met Ile 65 70 75 80 Ala Pro Ile Leu Asp Glu Ile Ala Asp Glu
Tyr Gln Gly Lys Leu Thr 85 90 95 Val Ala Lys Leu Asn Ile Asp Gln
Asn Pro Gly Thr Ala Pro Lys Tyr 100 105 110 Gly Ile Arg Gly Ile Pro
Thr Leu Leu Leu Phe Lys Asn Gly Glu Val 115 120 125 Ala Ala Thr Lys
Val Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Asn 130 135 140 Leu Ala
Asp Tyr Lys Asp Asp Asp Asp Lys 145 150 <210> SEQ ID NO 327
<211> LENGTH: 154 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic amino acid sequence <400> SEQUENCE:
327 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr
1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Ile Glu
Ala Arg 20 25 30 Leu Gln Ala Ser Val Asp Ala Glu Ser Arg Ser Asp
Lys Ile Ile His
35 40 45 Leu Thr Asp Asp Ser Phe Asp Thr Asp Val Leu Lys Ala Asp
Gly Ala 50 55 60 Ile Leu Val Asp Phe Trp Ala Glu Trp Cys Gly Pro
Cys Lys Met Ile 65 70 75 80 Ala Pro Ile Leu Asp Glu Ile Ala Asp Glu
Tyr Gln Gly Lys Leu Thr 85 90 95 Val Ala Lys Leu Asn Ile Asp Gln
Asn Pro Gly Thr Ala Pro Lys Tyr 100 105 110 Gly Ile Arg Gly Ile Pro
Thr Leu Leu Leu Phe Lys Asn Gly Glu Val 115 120 125 Ala Ala Thr Lys
Val Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Asn 130 135 140 Leu Ala
Asp Tyr Lys Asp Asp Asp Asp Lys 145 150 <210> SEQ ID NO 328
<211> LENGTH: 485 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic amino acid sequence <400> SEQUENCE:
328 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr
1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Glu Glu
Gly Lys 20 25 30 Leu Val Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn
Gly Leu Ala Glu 35 40 45 Val Gly Lys Lys Phe Glu Lys Asp Thr Gly
Ile Lys Val Thr Val Glu 50 55 60 His Pro Asp Lys Leu Glu Glu Lys
Phe Pro Gln Val Ala Ala Thr Gly 65 70 75 80 Asp Gly Pro Asp Ile Ile
Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85 90 95 Ala Gln Ser Gly
Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln 100 105 110 Asp Lys
Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys 115 120 125
Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn 130
135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro
Ala 145 150 155 160 Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala
Leu Met Phe Asn 165 170 175 Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu
Ile Ala Ala Asp Gly Gly 180 185 190 Tyr Ala Phe Lys Tyr Glu Asn Gly
Lys Tyr Asp Ile Lys Asp Val Gly 195 200 205 Val Asp Asn Ala Gly Ala
Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210 215 220 Ile Lys Asn Lys
His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu 225 230 235 240 Ala
Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp 245 250
255 Ala Trp Ser Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val
260 265 270 Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly
Val Leu 275 280 285 Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu
Leu Ala Lys Glu 290 295 300 Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu
Gly Leu Glu Ala Val Asn 305 310 315 320 Lys Asp Lys Pro Leu Gly Ala
Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 330 335 Leu Ala Lys Asp Pro
Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Ser 340 345 350 Ala Phe Trp
Tyr Ala Val Arg Ile Glu Ala Arg Leu Gln Ala Ser Val 355 360 365 Asp
Ala Glu Ser Arg Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser 370 375
380 Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe
385 390 395 400 Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile Ala Pro
Ile Leu Asp 405 410 415 Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr
Val Ala Lys Leu Asn 420 425 430 Ile Asp Gln Asn Pro Gly Thr Ala Pro
Lys Tyr Gly Ile Arg Gly Ile 435 440 445 Pro Thr Leu Leu Leu Phe Lys
Asn Gly Glu Val Ala Ala Thr Lys Val 450 455 460 Gly Ala Leu Ser Lys
Gly Gln Leu Lys Glu Asn Leu Ala Asp Tyr Lys 465 470 475 480 Asp Asp
Asp Asp Lys 485 <210> SEQ ID NO 329 <211> LENGTH: 485
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 329 Met Lys Ile Lys Thr Gly Ala
Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser
Ala Ser Ala Leu Ala Lys Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile
Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val
Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu 50 55
60 His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly
65 70 75 80 Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly
Gly Tyr 85 90 95 Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp
Lys Ala Phe Gln 100 105 110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala
Val Arg Tyr Asn Gly Lys 115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val
Glu Ala Leu Ser Leu Ile Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn
Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys
Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu
Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185
190 Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly
195 200 205 Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val
Asp Leu 210 215 220 Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr
Ser Ile Ala Glu 225 230 235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala
Met Thr Ile Asn Gly Pro Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr
Ser Lys Val Asn Tyr Gly Val Thr Val 260 265 270 Leu Pro Thr Phe Lys
Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu 275 280 285 Ser Ala Gly
Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe
Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310
315 320 Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu
Glu 325 330 335 Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn
Ala Gln Ser 340 345 350 Ala Phe Trp Tyr Ala Val Arg Ile Glu Ala Arg
Leu Gln Ala Ser Val 355 360 365 Asp Ala Glu Ser Arg Ser Asp Lys Ile
Ile His Leu Thr Asp Asp Ser 370 375 380 Phe Asp Thr Asp Val Leu Lys
Ala Asp Gly Ala Ile Leu Val Asp Phe 385 390 395 400 Trp Ala Glu Trp
Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp 405 410 415 Glu Ile
Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn 420 425 430
Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile 435
440 445 Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys
Val 450 455 460 Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Asn Leu Ala
Asp Tyr Lys 465 470 475 480 Asp Asp Asp Asp Lys 485 <210> SEQ
ID NO 330 <211> LENGTH: 485 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 330 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser
Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys
Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile Trp Ile Asn Gly Asp Lys
Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val Gly Lys Lys Phe Glu Lys
Asp Thr Gly Ile Lys Val Thr Val Glu 50 55 60
His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly 65
70 75 80 Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly
Gly Tyr 85 90 95 Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp
Lys Ala Phe Gln 100 105 110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala
Val Arg Tyr Asn Gly Lys 115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val
Glu Ala Leu Ser Leu Ile Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn
Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys
Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu
Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185
190 Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly
195 200 205 Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val
Asp Leu 210 215 220 Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr
Ser Ile Ala Glu 225 230 235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala
Met Thr Ile Asn Gly Pro Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr
Ser Lys Val Asn Tyr Gly Val Thr Val 260 265 270 Leu Pro Thr Phe Lys
Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu 275 280 285 Ser Ala Gly
Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe
Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310
315 320 Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu
Glu 325 330 335 Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn
Ala Gln Ser 340 345 350 Ala Phe Trp Tyr Ala Val Arg Ile Glu Ala Arg
Leu Gln Ala Ser Val 355 360 365 Asp Ala Glu Ser Arg Ser Asp Lys Ile
Ile His Leu Thr Asp Asp Ser 370 375 380 Phe Asp Thr Asp Val Leu Lys
Ala Asp Gly Ala Ile Leu Val Asp Phe 385 390 395 400 Trp Ala Glu Trp
Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp 405 410 415 Glu Ile
Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn 420 425 430
Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile 435
440 445 Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys
Val 450 455 460 Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Asn Leu Ala
Asp Tyr Lys 465 470 475 480 Asp Asp Asp Asp Lys 485 <210> SEQ
ID NO 331 <211> LENGTH: 485 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 331 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser
Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys
Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile Trp Ile Asn Gly Asp Lys
Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val Gly Lys Lys Phe Glu Lys
Asp Thr Gly Ile Lys Val Thr Val Glu 50 55 60 His Pro Asp Lys Leu
Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly 65 70 75 80 Asp Gly Pro
Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85 90 95 Ala
Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln 100 105
110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys
115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile
Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu
Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys Glu Leu Lys Ala Lys Gly
Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu Gln Glu Pro Tyr Phe Thr
Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185 190 Tyr Ala Phe Lys Tyr
Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly 195 200 205 Val Asp Asn
Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210 215 220 Ile
Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu 225 230
235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile Asn Gly Pro
Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly
Val Thr Val 260 265 270 Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro
Phe Val Gly Val Leu 275 280 285 Ser Ala Gly Ile Asn Ala Ala Ser Pro
Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe Leu Glu Asn Tyr Leu Leu
Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310 315 320 Lys Asp Lys Pro
Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 330 335 Leu Ala
Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Ser 340 345 350
Ala Phe Trp Tyr Ala Val Arg Ile Glu Ala Arg Leu Gln Ala Ser Val 355
360 365 Asp Ala Glu Ser Arg Ser Asp Lys Ile Ile His Leu Thr Asp Asp
Ser 370 375 380 Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala Ile Leu
Val Asp Phe 385 390 395 400 Trp Ala Glu Trp Cys Gly Pro Cys Lys Met
Ile Ala Pro Ile Leu Asp 405 410 415 Glu Ile Ala Asp Glu Tyr Gln Gly
Lys Leu Thr Val Ala Lys Leu Asn 420 425 430 Ile Asp Gln Asn Pro Gly
Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile 435 440 445 Pro Thr Leu Leu
Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val 450 455 460 Gly Ala
Leu Ser Lys Gly Gln Leu Lys Glu Asn Leu Ala Asp Tyr Lys 465 470 475
480 Asp Asp Asp Asp Lys 485 <210> SEQ ID NO 332 <211>
LENGTH: 767 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic amino acid sequence <400> SEQUENCE: 332 Met Lys Ile
Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr
Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Glu Glu Gly Lys 20 25
30 Leu Val Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu
35 40 45 Val Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr
Val Glu 50 55 60 His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val
Ala Ala Thr Gly 65 70 75 80 Asp Gly Pro Asp Ile Ile Phe Trp Ala His
Asp Arg Phe Gly Gly Tyr 85 90 95 Ala Gln Ser Gly Leu Leu Ala Glu
Ile Thr Pro Asp Lys Ala Phe Gln 100 105 110 Asp Lys Leu Tyr Pro Phe
Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys 115 120 125 Leu Ile Ala Tyr
Pro Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn 130 135 140 Lys Asp
Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala 145 150 155
160 Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn
165 170 175 Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp
Gly Gly 180 185 190 Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile
Lys Asp Val Gly 195 200 205 Val Asp Asn Ala Gly Ala Lys Ala Gly Leu
Thr Phe Leu Val Asp Leu 210 215 220 Ile Lys Asn Lys His Met Asn Ala
Asp Thr Asp Tyr Ser Ile Ala Glu 225 230 235 240 Ala Ala Phe Asn Lys
Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp 245 250 255 Ala Trp Ser
Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val 260 265 270 Leu
Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu 275 280
285 Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu
290 295 300
Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305
310 315 320 Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu
Glu Glu 325 330 335 Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr Met Glu
Asn Ala Gln Lys 340 345 350 Gly Glu Ile Met Pro Asn Ile Pro Gln Met
Ser Ala Phe Trp Tyr Ala 355 360 365 Val Leu Ile Glu Ala Arg Thr Ser
Glu Ser Asp Thr Ala Gly Pro Asn 370 375 380 Ser Asp Leu Asp Val Asn
Thr Asp Ile Tyr Ser Lys Val Leu Val Thr 385 390 395 400 Ala Ile Tyr
Leu Ala Leu Phe Val Val Gly Thr Val Gly Asn Ser Val 405 410 415 Thr
Ala Phe Thr Leu Ala Arg Lys Lys Ser Leu Gln Ser Leu Gln Ser 420 425
430 Thr Val His Tyr His Leu Gly Ser Leu Ala Leu Ser Asp Leu Leu Ile
435 440 445 Leu Leu Leu Ala Met Pro Val Glu Leu Tyr Asn Phe Ile Trp
Val His 450 455 460 His Pro Trp Ala Phe Gly Asp Ala Gly Cys Arg Gly
Tyr Tyr Phe Leu 465 470 475 480 Arg Asp Ala Cys Thr Tyr Ala Thr Ala
Leu Asn Val Ala Ser Leu Ser 485 490 495 Val Glu Arg Tyr Leu Ala Ile
Cys His Pro Phe Lys Ala Lys Thr Leu 500 505 510 Met Ser Arg Ser Arg
Thr Lys Lys Phe Ile Ser Ala Ile Trp Leu Ala 515 520 525 Ser Ala Leu
Leu Ala Ile Pro Met Leu Phe Thr Met Gly Leu Gln Asn 530 535 540 Arg
Ser Gly Asp Gly Thr His Pro Gly Gly Leu Val Cys Thr Pro Ile 545 550
555 560 Val Asp Thr Ala Thr Val Lys Val Val Ile Gln Val Asn Thr Phe
Met 565 570 575 Ser Phe Leu Phe Pro Met Leu Val Ile Ser Ile Leu Asn
Thr Val Ile 580 585 590 Ala Asn Lys Leu Thr Val Met Val His Gln Ala
Ala Glu Gln Gly Arg 595 600 605 Val Cys Thr Val Gly Thr His Asn Gly
Leu Glu His Ser Thr Phe Asn 610 615 620 Met Thr Ile Glu Pro Gly Arg
Val Gln Ala Leu Arg His Gly Val Leu 625 630 635 640 Val Leu Arg Ala
Val Val Ile Ala Phe Val Val Cys Trp Leu Pro Tyr 645 650 655 His Val
Arg Arg Leu Met Phe Cys Tyr Ile Ser Asp Glu Gln Trp Thr 660 665 670
Thr Phe Leu Phe Asp Phe Tyr His Tyr Phe Tyr Met Leu Thr Asn Ala 675
680 685 Leu Phe Tyr Val Ser Ser Ala Ile Asn Pro Ile Leu Tyr Asn Leu
Val 690 695 700 Ser Ala Asn Phe Arg Gln Val Phe Leu Ser Thr Leu Ala
Cys Leu Cys 705 710 715 720 Pro Gly Trp Arg His Arg Arg Lys Lys Arg
Pro Thr Phe Ser Arg Lys 725 730 735 Pro Asn Ser Met Ser Ser Asn His
Ala Phe Ser Thr Ser Ala Thr Arg 740 745 750 Glu Thr Leu Tyr Ala Ala
Ala Asp Tyr Lys Asp Asp Asp Asp Lys 755 760 765 <210> SEQ ID
NO 333 <211> LENGTH: 425 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 333 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser
Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys
Ile Ile Glu Ala Arg 20 25 30 Thr Ser Glu Ser Asp Thr Ala Gly Pro
Asn Ser Asp Leu Asp Val Asn 35 40 45 Thr Asp Ile Tyr Ser Lys Val
Leu Val Thr Ala Ile Tyr Leu Ala Leu 50 55 60 Phe Val Val Gly Thr
Val Gly Asn Ser Val Thr Ala Phe Thr Leu Ala 65 70 75 80 Arg Lys Lys
Ser Leu Gln Ser Leu Gln Ser Thr Val His Tyr His Leu 85 90 95 Gly
Ser Leu Ala Leu Ser Asp Leu Leu Ile Leu Leu Leu Ala Met Pro 100 105
110 Val Glu Leu Tyr Asn Phe Ile Trp Val His His Pro Trp Ala Phe Gly
115 120 125 Asp Ala Gly Cys Arg Gly Tyr Tyr Phe Leu Arg Asp Ala Cys
Thr Tyr 130 135 140 Ala Thr Ala Leu Asn Val Ala Ser Leu Ser Val Glu
Arg Tyr Leu Ala 145 150 155 160 Ile Cys His Pro Phe Lys Ala Lys Thr
Leu Met Ser Arg Ser Arg Thr 165 170 175 Lys Lys Phe Ile Ser Ala Ile
Trp Leu Ala Ser Ala Leu Leu Ala Ile 180 185 190 Pro Met Leu Phe Thr
Met Gly Leu Gln Asn Arg Ser Gly Asp Gly Thr 195 200 205 His Pro Gly
Gly Leu Val Cys Thr Pro Ile Val Asp Thr Ala Thr Val 210 215 220 Lys
Val Val Ile Gln Val Asn Thr Phe Met Ser Phe Leu Phe Pro Met 225 230
235 240 Leu Val Ile Ser Ile Leu Asn Thr Val Ile Ala Asn Lys Leu Thr
Val 245 250 255 Met Val His Gln Ala Ala Glu Gln Gly Arg Val Cys Thr
Val Gly Thr 260 265 270 His Asn Gly Leu Glu His Ser Thr Phe Asn Met
Thr Ile Glu Pro Gly 275 280 285 Arg Val Gln Ala Leu Arg His Gly Val
Leu Val Leu Arg Ala Val Val 290 295 300 Ile Ala Phe Val Val Cys Trp
Leu Pro Tyr His Val Arg Arg Leu Met 305 310 315 320 Phe Cys Tyr Ile
Ser Asp Glu Gln Trp Thr Thr Phe Leu Phe Asp Phe 325 330 335 Tyr His
Tyr Phe Tyr Met Leu Thr Asn Ala Leu Phe Tyr Val Ser Ser 340 345 350
Ala Ile Asn Pro Ile Leu Tyr Asn Leu Val Ser Ala Asn Phe Arg Gln 355
360 365 Val Phe Leu Ser Thr Leu Ala Cys Leu Cys Pro Gly Trp Arg His
Arg 370 375 380 Arg Lys Lys Arg Pro Thr Phe Ser Arg Lys Pro Asn Ser
Met Ser Ser 385 390 395 400 Asn His Ala Phe Ser Thr Ser Ala Thr Arg
Glu Thr Leu Tyr Ala Ala 405 410 415 Ala Asp Tyr Lys Asp Asp Asp Asp
Lys 420 425 <210> SEQ ID NO 334 <211> LENGTH: 878
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 334 Met Lys Ile Lys Thr Gly Ala
Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser
Ala Ser Ala Leu Ala Lys Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile
Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val
Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu 50 55
60 His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly
65 70 75 80 Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly
Gly Tyr 85 90 95 Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp
Lys Ala Phe Gln 100 105 110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala
Val Arg Tyr Asn Gly Lys 115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val
Glu Ala Leu Ser Leu Ile Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn
Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys
Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu
Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185
190 Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly
195 200 205 Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val
Asp Leu 210 215 220 Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr
Ser Ile Ala Glu 225 230 235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala
Met Thr Ile Asn Gly Pro Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr
Ser Lys Val Asn Tyr Gly Val Thr Val 260 265 270 Leu Pro Thr Phe Lys
Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu 275 280 285 Ser Ala Gly
Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe
Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310
315 320 Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu
Glu 325 330 335
Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Lys 340
345 350 Gly Glu Ile Met Pro Asn Ile Pro Gln Met Ser Ala Phe Trp Tyr
Ala 355 360 365 Val Leu Ile Glu Ala Arg Thr Ser Glu Ser Asp Thr Ala
Gly Pro Asn 370 375 380 Ser Asp Leu Asp Val Asn Thr Asp Ile Tyr Ser
Lys Val Leu Val Thr 385 390 395 400 Ala Ile Tyr Leu Ala Leu Phe Val
Val Gly Thr Val Gly Asn Ser Val 405 410 415 Thr Ala Phe Thr Leu Ala
Arg Lys Lys Ser Leu Gln Ser Leu Gln Ser 420 425 430 Thr Val His Tyr
His Leu Gly Ser Leu Ala Leu Ser Asp Leu Leu Ile 435 440 445 Leu Leu
Leu Ala Met Pro Val Glu Leu Tyr Asn Phe Ile Trp Val His 450 455 460
His Pro Trp Ala Phe Gly Asp Ala Gly Cys Arg Gly Tyr Tyr Phe Leu 465
470 475 480 Arg Asp Ala Cys Thr Tyr Ala Thr Ala Leu Asn Val Ala Ser
Leu Ser 485 490 495 Val Glu Arg Tyr Leu Ala Ile Cys His Pro Phe Lys
Ala Lys Thr Leu 500 505 510 Met Ser Arg Ser Arg Thr Lys Lys Phe Ile
Ser Ala Ile Trp Leu Ala 515 520 525 Ser Ala Leu Leu Ala Ile Pro Met
Leu Phe Thr Met Gly Leu Gln Asn 530 535 540 Arg Ser Gly Asp Gly Thr
His Pro Gly Gly Leu Val Cys Thr Pro Ile 545 550 555 560 Val Asp Thr
Ala Thr Val Lys Val Val Ile Gln Val Asn Thr Phe Met 565 570 575 Ser
Phe Leu Phe Pro Met Leu Val Ile Ser Ile Leu Asn Thr Val Ile 580 585
590 Ala Asn Lys Leu Thr Val Met Val His Gln Ala Ala Glu Gln Gly Arg
595 600 605 Val Cys Thr Val Gly Thr His Asn Gly Leu Glu His Ser Thr
Phe Asn 610 615 620 Met Thr Ile Glu Pro Gly Arg Val Gln Ala Leu Arg
His Gly Val Leu 625 630 635 640 Val Leu Arg Ala Val Val Ile Ala Phe
Val Val Cys Trp Leu Pro Tyr 645 650 655 His Val Arg Arg Leu Met Phe
Cys Tyr Ile Ser Asp Glu Gln Trp Thr 660 665 670 Thr Phe Leu Phe Asp
Phe Tyr His Tyr Phe Tyr Met Leu Thr Asn Ala 675 680 685 Leu Phe Tyr
Val Ser Ser Ala Ile Asn Pro Ile Leu Tyr Asn Leu Val 690 695 700 Ser
Ala Asn Phe Arg Gln Val Phe Leu Ser Thr Leu Ala Cys Leu Cys 705 710
715 720 Pro Gly Trp Arg His Arg Arg Lys Lys Arg Pro Thr Phe Ser Arg
Lys 725 730 735 Pro Asn Ser Met Ser Ser Asn His Ala Phe Ser Thr Ser
Ala Thr Arg 740 745 750 Glu Thr Leu Tyr Ala Ala Ala Ser Asp Lys Ile
Ile His Leu Thr Asp 755 760 765 Asp Ser Phe Asp Thr Asp Val Leu Lys
Ala Asp Gly Ala Ile Leu Val 770 775 780 Asp Phe Trp Ala Glu Trp Cys
Gly Pro Cys Lys Met Ile Ala Pro Ile 785 790 795 800 Leu Asp Glu Ile
Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys 805 810 815 Leu Asn
Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg 820 825 830
Gly Ile Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala Thr 835
840 845 Lys Val Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Phe Leu Asp
Ala 850 855 860 Asn Leu Ala Ala Ala Ala Asp Tyr Lys Asp Asp Asp Asp
Lys 865 870 875 <210> SEQ ID NO 335 <211> LENGTH: 536
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 335 Met Lys Ile Lys Thr Gly Ala
Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser
Ala Ser Ala Leu Ala Lys Ile Ile Glu Ala Arg 20 25 30 Thr Ser Glu
Ser Asp Thr Ala Gly Pro Asn Ser Asp Leu Asp Val Asn 35 40 45 Thr
Asp Ile Tyr Ser Lys Val Leu Val Thr Ala Ile Tyr Leu Ala Leu 50 55
60 Phe Val Val Gly Thr Val Gly Asn Ser Val Thr Ala Phe Thr Leu Ala
65 70 75 80 Arg Lys Lys Ser Leu Gln Ser Leu Gln Ser Thr Val His Tyr
His Leu 85 90 95 Gly Ser Leu Ala Leu Ser Asp Leu Leu Ile Leu Leu
Leu Ala Met Pro 100 105 110 Val Glu Leu Tyr Asn Phe Ile Trp Val His
His Pro Trp Ala Phe Gly 115 120 125 Asp Ala Gly Cys Arg Gly Tyr Tyr
Phe Leu Arg Asp Ala Cys Thr Tyr 130 135 140 Ala Thr Ala Leu Asn Val
Ala Ser Leu Ser Val Glu Arg Tyr Leu Ala 145 150 155 160 Ile Cys His
Pro Phe Lys Ala Lys Thr Leu Met Ser Arg Ser Arg Thr 165 170 175 Lys
Lys Phe Ile Ser Ala Ile Trp Leu Ala Ser Ala Leu Leu Ala Ile 180 185
190 Pro Met Leu Phe Thr Met Gly Leu Gln Asn Arg Ser Gly Asp Gly Thr
195 200 205 His Pro Gly Gly Leu Val Cys Thr Pro Ile Val Asp Thr Ala
Thr Val 210 215 220 Lys Val Val Ile Gln Val Asn Thr Phe Met Ser Phe
Leu Phe Pro Met 225 230 235 240 Leu Val Ile Ser Ile Leu Asn Thr Val
Ile Ala Asn Lys Leu Thr Val 245 250 255 Met Val His Gln Ala Ala Glu
Gln Gly Arg Val Cys Thr Val Gly Thr 260 265 270 His Asn Gly Leu Glu
His Ser Thr Phe Asn Met Thr Ile Glu Pro Gly 275 280 285 Arg Val Gln
Ala Leu Arg His Gly Val Leu Val Leu Arg Ala Val Val 290 295 300 Ile
Ala Phe Val Val Cys Trp Leu Pro Tyr His Val Arg Arg Leu Met 305 310
315 320 Phe Cys Tyr Ile Ser Asp Glu Gln Trp Thr Thr Phe Leu Phe Asp
Phe 325 330 335 Tyr His Tyr Phe Tyr Met Leu Thr Asn Ala Leu Phe Tyr
Val Ser Ser 340 345 350 Ala Ile Asn Pro Ile Leu Tyr Asn Leu Val Ser
Ala Asn Phe Arg Gln 355 360 365 Val Phe Leu Ser Thr Leu Ala Cys Leu
Cys Pro Gly Trp Arg His Arg 370 375 380 Arg Lys Lys Arg Pro Thr Phe
Ser Arg Lys Pro Asn Ser Met Ser Ser 385 390 395 400 Asn His Ala Phe
Ser Thr Ser Ala Thr Arg Glu Thr Leu Tyr Ala Ala 405 410 415 Ala Ser
Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp 420 425 430
Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp 435
440 445 Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala
Asp 450 455 460 Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile
Asp Gln Asn 465 470 475 480 Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg
Gly Ile Pro Thr Leu Leu 485 490 495 Leu Phe Lys Asn Gly Glu Val Ala
Ala Thr Lys Val Gly Ala Leu Ser 500 505 510 Lys Gly Gln Leu Lys Glu
Phe Leu Asp Ala Asn Leu Ala Ala Ala Ala 515 520 525 Asp Tyr Lys Asp
Asp Asp Asp Lys 530 535 <210> SEQ ID NO 336 <211>
LENGTH: 380 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic amino acid sequence <400> SEQUENCE: 336 Met Gly Cys
Leu Gly Asn Ser Lys Thr Glu Asp Gln Arg Asn Glu Glu 1 5 10 15 Lys
Ala Gln Arg Glu Ala Asn Lys Lys Ile Glu Lys Gln Leu Gln Lys 20 25
30 Asp Lys Gln Val Tyr Arg Ala Thr His Arg Leu Leu Leu Leu Gly Ala
35 40 45 Gly Glu Ser Gly Lys Ser Thr Ile Val Lys Gln Met Arg Ile
Leu His 50 55 60 Val Asn Gly Phe Asn Gly Asp Ser Glu Lys Ala Thr
Lys Val Gln Asp 65 70 75 80 Ile Lys Asn Asn Leu Lys Glu Ala Ile Glu
Thr Ile Val Ala Ala Met 85 90 95 Ser Asn Leu Val Pro Pro Val Glu
Leu Ala Asn Pro Glu Asn Gln Phe 100 105 110 Arg Val Asp Tyr Ile Leu
Ser Val Met Asn Val Pro Asp Phe Asp Phe 115 120 125 Pro Pro Glu Phe
Tyr Glu His Ala Lys Ala Leu Trp Glu Asp Glu Gly 130 135 140
Val Arg Ala Cys Tyr Glu Arg Ser Asn Glu Tyr Gln Leu Ile Asp Cys 145
150 155 160 Ala Gln Tyr Phe Leu Asp Lys Ile Asp Val Ile Lys Gln Ala
Asp Tyr 165 170 175 Val Pro Ser Asp Gln Asp Leu Leu Arg Cys Arg Val
Leu Thr Ser Gly 180 185 190 Ile Phe Glu Thr Lys Phe Gln Val Asp Lys
Val Asn Phe His Met Phe 195 200 205 Asp Val Gly Gly Gln Arg Asp Glu
Arg Arg Lys Trp Ile Gln Cys Phe 210 215 220 Asn Asp Val Thr Ala Ile
Ile Phe Val Val Ala Ser Ser Ser Tyr Asn 225 230 235 240 Met Val Ile
Arg Glu Asp Asn Gln Thr Asn Arg Leu Gln Glu Ala Leu 245 250 255 Asn
Leu Phe Lys Ser Ile Trp Asn Asn Arg Trp Leu Arg Thr Ile Ser 260 265
270 Val Ile Leu Phe Leu Asn Lys Gln Asp Leu Leu Ala Glu Lys Val Leu
275 280 285 Ala Gly Lys Ser Lys Ile Glu Asp Tyr Phe Pro Glu Phe Ala
Arg Tyr 290 295 300 Thr Thr Pro Glu Asp Ala Thr Pro Glu Pro Gly Glu
Asp Pro Arg Val 305 310 315 320 Thr Arg Ala Lys Tyr Phe Ile Arg Asp
Glu Phe Leu Arg Ile Ser Thr 325 330 335 Ala Ser Gly Asp Gly Arg His
Tyr Cys Tyr Pro His Phe Thr Cys Ala 340 345 350 Val Asp Thr Glu Asn
Ile Arg Arg Val Phe Asn Asp Cys Arg Asp Ile 355 360 365 Ile Gln Arg
Met His Leu Arg Gln Tyr Glu Leu Leu 370 375 380 <210> SEQ ID
NO 337 <211> LENGTH: 394 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 337 Met Gly Cys Leu Gly Asn Ser Lys Thr Glu Asp Gln Arg
Asn Glu Glu 1 5 10 15 Lys Ala Gln Arg Glu Ala Asn Lys Lys Ile Glu
Lys Gln Leu Gln Lys 20 25 30 Asp Lys Gln Val Tyr Arg Ala Thr His
Arg Leu Leu Leu Leu Gly Ala 35 40 45 Gly Glu Ser Gly Lys Ser Thr
Ile Val Lys Gln Met Arg Ile Leu His 50 55 60 Val Asn Gly Phe Asn
Gly Glu Gly Gly Glu Glu Asp Pro Gln Ala Ala 65 70 75 80 Arg Ser Asn
Ser Asp Gly Glu Lys Ala Thr Lys Val Gln Asp Ile Lys 85 90 95 Asn
Asn Leu Lys Glu Ala Ile Glu Thr Ile Val Ala Ala Met Ser Asn 100 105
110 Leu Val Pro Pro Val Glu Leu Ala Asn Pro Glu Asn Gln Phe Arg Val
115 120 125 Asp Tyr Ile Leu Ser Val Met Asn Val Pro Asp Phe Asp Phe
Pro Pro 130 135 140 Glu Phe Tyr Glu His Ala Lys Ala Leu Trp Glu Asp
Glu Gly Val Arg 145 150 155 160 Ala Cys Tyr Glu Arg Ser Asn Glu Tyr
Gln Leu Ile Asp Cys Ala Gln 165 170 175 Tyr Phe Leu Asp Lys Ile Asp
Val Ile Lys Gln Ala Asp Tyr Val Pro 180 185 190 Ser Asp Gln Asp Leu
Leu Arg Cys Arg Val Leu Thr Ser Gly Ile Phe 195 200 205 Glu Thr Lys
Phe Gln Val Asp Lys Val Asn Phe His Met Phe Asp Val 210 215 220 Gly
Gly Gln Arg Asp Glu Arg Arg Lys Trp Ile Gln Cys Phe Asn Asp 225 230
235 240 Val Thr Ala Ile Ile Phe Val Val Ala Ser Ser Ser Tyr Asn Met
Val 245 250 255 Ile Arg Glu Asp Asn Gln Thr Asn Arg Leu Gln Glu Ala
Leu Asn Leu 260 265 270 Phe Lys Ser Ile Trp Asn Asn Arg Trp Leu Arg
Thr Ile Ser Val Ile 275 280 285 Leu Phe Leu Asn Lys Gln Asp Leu Leu
Ala Glu Lys Val Leu Ala Gly 290 295 300 Lys Ser Lys Ile Glu Asp Tyr
Phe Pro Glu Phe Ala Arg Tyr Thr Thr 305 310 315 320 Pro Glu Asp Ala
Thr Pro Glu Pro Gly Glu Asp Pro Arg Val Thr Arg 325 330 335 Ala Lys
Tyr Phe Ile Arg Asp Glu Phe Leu Arg Ile Ser Thr Ala Ser 340 345 350
Gly Asp Gly Arg His Tyr Cys Tyr Pro His Phe Thr Cys Ala Val Asp 355
360 365 Thr Glu Asn Ile Arg Arg Val Phe Asn Asp Cys Arg Asp Ile Ile
Gln 370 375 380 Arg Met His Leu Arg Gln Tyr Glu Leu Leu 385 390
<210> SEQ ID NO 338 <211> LENGTH: 359 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 338 Met Thr Leu Glu Ser Ile Met Ala Cys Cys
Leu Ser Glu Glu Ala Lys 1 5 10 15 Glu Ala Arg Arg Ile Asn Asp Glu
Ile Glu Arg Gln Leu Arg Arg Asp 20 25 30 Lys Arg Asp Ala Arg Arg
Glu Leu Lys Leu Leu Leu Leu Gly Thr Gly 35 40 45 Glu Ser Gly Lys
Ser Thr Phe Ile Lys Gln Met Arg Ile Ile His Gly 50 55 60 Ser Gly
Tyr Ser Asp Glu Asp Lys Arg Gly Phe Thr Lys Leu Val Tyr 65 70 75 80
Gln Asn Ile Phe Thr Ala Met Gln Ala Met Ile Arg Ala Met Asp Thr 85
90 95 Leu Lys Ile Pro Tyr Lys Tyr Glu His Asn Lys Ala His Ala Gln
Leu 100 105 110 Val Arg Glu Val Asp Val Glu Lys Val Ser Ala Phe Glu
Asn Pro Tyr 115 120 125 Val Asp Ala Ile Lys Ser Leu Trp Asn Asp Pro
Gly Ile Gln Glu Cys 130 135 140 Tyr Asp Arg Arg Arg Glu Tyr Gln Leu
Ser Asp Ser Thr Lys Tyr Tyr 145 150 155 160 Leu Asn Asp Leu Asp Arg
Val Ala Asp Pro Ala Tyr Leu Pro Thr Gln 165 170 175 Gln Asp Val Leu
Arg Val Arg Val Pro Thr Thr Gly Ile Ile Glu Tyr 180 185 190 Pro Phe
Asp Leu Gln Ser Val Ile Phe Arg Met Val Asp Val Gly Gly 195 200 205
Gln Arg Ser Glu Arg Arg Lys Trp Ile His Cys Phe Glu Asn Val Thr 210
215 220 Ser Ile Met Phe Leu Val Ala Leu Ser Glu Tyr Asp Gln Val Leu
Val 225 230 235 240 Glu Ser Asp Asn Glu Asn Arg Met Glu Glu Ser Lys
Ala Leu Phe Arg 245 250 255 Thr Ile Ile Thr Tyr Pro Trp Phe Gln Asn
Ser Ser Val Ile Leu Phe 260 265 270 Leu Asn Lys Lys Asp Leu Leu Glu
Glu Lys Ile Met Tyr Ser His Leu 275 280 285 Val Asp Tyr Phe Pro Glu
Tyr Asp Gly Pro Gln Arg Asp Ala Gln Ala 290 295 300 Ala Arg Glu Phe
Ile Leu Lys Met Phe Val Asp Leu Asn Pro Asp Ser 305 310 315 320 Asp
Lys Ile Ile Tyr Ser His Phe Thr Cys Ala Thr Asp Thr Glu Asn 325 330
335 Ile Arg Phe Val Phe Ala Ala Val Lys Asp Thr Ile Leu Gln Leu Asn
340 345 350 Leu Lys Glu Tyr Asn Leu Val 355 <210> SEQ ID NO
339 <211> LENGTH: 355 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 339 Met Gly Cys Thr Val Ser Ala Glu Asp Lys Ala Ala Ala
Glu Arg Ser 1 5 10 15 Lys Met Ile Asp Lys Asn Leu Arg Glu Asp Gly
Glu Lys Ala Ala Arg 20 25 30 Glu Val Lys Leu Leu Leu Leu Gly Ala
Gly Glu Ser Gly Lys Ser Thr 35 40 45 Ile Val Lys Gln Met Lys Ile
Ile His Glu Asp Gly Tyr Ser Glu Glu 50 55 60 Glu Cys Arg Gln Tyr
Arg Ala Val Val Tyr Ser Asn Thr Ile Gln Ser 65 70 75 80 Ile Met Ala
Ile Val Lys Ala Met Gly Asn Leu Gln Ile Asp Phe Ala 85 90 95 Asp
Pro Ser Arg Ala Asp Asp Ala Arg Gln Leu Phe Ala Leu Ser Cys 100 105
110 Thr Ala Glu Glu Gln Gly Val Leu Pro Asp Asp Leu Ser Gly Val Ile
115 120 125 Arg Arg Leu Trp Ala Asp His Gly Val Gln Ala Cys Phe Gly
Arg Ser 130 135 140 Arg Glu Tyr Gln Leu Asn Asp Ser Ala Ala Tyr Tyr
Leu Asn Asp Leu 145 150 155 160 Glu Arg Ile Ala Gln Ser Asp Tyr Ile
Pro Thr Gln Gln Asp Val Leu 165 170 175
Arg Thr Arg Val Lys Thr Thr Gly Ile Val Glu Thr His Phe Thr Phe 180
185 190 Lys Asp Leu His Phe Lys Met Phe Asp Val Gly Gly Gln Arg Ser
Glu 195 200 205 Arg Lys Lys Trp Ile His Cys Phe Glu Gly Val Thr Ala
Ile Ile Phe 210 215 220 Cys Val Ala Leu Ser Ala Tyr Asp Leu Val Leu
Ala Glu Asp Glu Glu 225 230 235 240 Met Asn Arg Met His Glu Ser Met
Lys Leu Phe Asp Ser Ile Cys Asn 245 250 255 Asn Lys Trp Phe Thr Asp
Thr Ser Ile Ile Leu Phe Leu Asn Lys Lys 260 265 270 Asp Leu Phe Glu
Glu Lys Ile Thr His Ser Pro Leu Thr Ile Cys Phe 275 280 285 Pro Glu
Tyr Thr Gly Ala Asn Lys Tyr Asp Glu Ala Ala Ser Tyr Ile 290 295 300
Gln Ser Lys Phe Glu Asp Leu Asn Lys Arg Lys Asp Thr Lys Glu Ile 305
310 315 320 Tyr Thr His Phe Thr Cys Ala Thr Asp Thr Lys Asn Val Gln
Phe Val 325 330 335 Phe Asp Ala Val Thr Asp Val Ile Ile Lys Asn Asn
Leu Lys Asp Cys 340 345 350 Gly Leu Phe 355 <210> SEQ ID NO
340 <211> LENGTH: 381 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 340 Met Ser Gly Val Val Arg Thr Leu Ser Arg Cys Leu Leu
Pro Ala Glu 1 5 10 15 Ala Gly Gly Ala Arg Glu Arg Arg Ala Gly Ser
Gly Ala Arg Asp Ala 20 25 30 Glu Arg Glu Ala Arg Arg Arg Ser Arg
Asp Ile Asp Ala Leu Leu Ala 35 40 45 Arg Glu Arg Arg Ala Val Arg
Arg Leu Val Lys Ile Leu Leu Leu Gly 50 55 60 Ala Gly Glu Ser Gly
Lys Ser Thr Phe Leu Lys Gln Met Arg Ile Ile 65 70 75 80 His Gly Arg
Glu Phe Asp Gln Lys Ala Leu Leu Glu Phe Arg Asp Thr 85 90 95 Ile
Phe Asp Asn Ile Leu Lys Gly Ser Arg Val Leu Val Asp Ala Arg 100 105
110 Asp Lys Leu Gly Ile Pro Trp Gln Tyr Ser Glu Asn Glu Lys His Gly
115 120 125 Met Phe Leu Met Ala Phe Glu Asn Lys Ala Gly Leu Pro Val
Glu Pro 130 135 140 Ala Thr Phe Gln Leu Tyr Val Pro Ala Leu Ser Ala
Leu Trp Arg Asp 145 150 155 160 Ser Gly Ile Arg Glu Ala Phe Ser Arg
Arg Ser Glu Phe Gln Leu Gly 165 170 175 Glu Ser Val Lys Tyr Phe Leu
Asp Asn Leu Asp Arg Ile Gly Gln Leu 180 185 190 Asn Tyr Phe Pro Ser
Lys Gln Asp Ile Leu Leu Ala Arg Lys Ala Thr 195 200 205 Lys Gly Ile
Val Glu His Asp Phe Val Ile Lys Lys Ile Pro Phe Lys 210 215 220 Met
Val Asp Val Gly Gly Gln Arg Ser Gln Arg Gln Lys Trp Phe Gln 225 230
235 240 Cys Phe Asp Gly Ile Thr Ser Ile Leu Phe Met Val Ser Ser Ser
Glu 245 250 255 Tyr Asp Gln Val Leu Met Glu Asp Arg Arg Thr Asn Arg
Leu Val Glu 260 265 270 Ser Met Asn Ile Phe Glu Thr Ile Val Asn Asn
Lys Leu Phe Phe Asn 275 280 285 Val Ser Ile Ile Leu Phe Leu Asn Lys
Met Asp Leu Leu Val Glu Lys 290 295 300 Val Lys Thr Val Ser Ile Lys
Lys His Phe Pro Asp Phe Arg Gly Asp 305 310 315 320 Pro His Gln Leu
Glu Asp Val Gln Arg Tyr Leu Val Gln Cys Phe Asp 325 330 335 Arg Lys
Arg Arg Asn Arg Ser Lys Pro Leu Phe His His Phe Thr Thr 340 345 350
Ala Ile Asp Thr Glu Asn Val Arg Phe Val Phe His Ala Val Lys Asp 355
360 365 Thr Ile Leu Gln Glu Asn Leu Lys Asp Ile Met Leu Gln 370 375
380 <210> SEQ ID NO 341 <211> LENGTH: 50 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic amino acid
sequence <400> SEQUENCE: 341 Met Lys Ile Lys Thr Gly Ala Arg
Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala
Ser Ala Leu Ala Lys Ile Ile Glu Ala Arg 20 25 30 Leu Gln Ala Ser
Val Asp Ala Glu Ser Arg Asp Tyr Lys Asp Asp Asp 35 40 45 Asp Lys 50
<210> SEQ ID NO 342 <211> LENGTH: 27 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 342 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu
Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala
Leu Ala Lys 20 25 <210> SEQ ID NO 343 <211> LENGTH: 50
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 343 Met Lys Ile Lys Thr Gly Ala
Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser
Ala Ser Ala Leu Ala Lys Ile Ile Glu Ala Arg 20 25 30 Leu Gln Ala
Ser Val Asp Ala Glu Ser Arg Asp Tyr Lys Asp Asp Asp 35 40 45 Asp
Lys 50 <210> SEQ ID NO 344 <211> LENGTH: 50 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Synthetic amino acid
sequence <400> SEQUENCE: 344 Met Lys Ile Lys Thr Gly Ala Arg
Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala
Ser Ala Leu Ala Lys Ile Ile Glu Ala Arg 20 25 30 Leu Gln Ala Ser
Val Asp Ala Glu Ser Arg Asp Tyr Lys Asp Asp Asp 35 40 45 Asp Lys 50
<210> SEQ ID NO 345 <211> LENGTH: 343 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 345 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu
Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala
Leu Ala Lys Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile Trp Ile Asn
Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val Gly Lys Lys
Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu 50 55 60 His Pro
Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly 65 70 75 80
Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85
90 95 Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe
Gln 100 105 110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr
Asn Gly Lys 115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu
Ser Leu Ile Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys
Thr Trp Glu Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys Glu Leu Lys
Ala Lys Gly Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu Gln Glu Pro
Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185 190 Tyr Ala
Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly 195 200 205
Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu
210 215 220 Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile
Ala Glu 225 230 235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr
Ile Asn Gly Pro Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr Ser Lys
Val Asn Tyr Gly Val Thr Val 260 265 270 Leu Pro Thr Phe Lys Gly Gln
Pro Ser Lys Pro Phe Val Gly Val Leu 275 280 285 Ser Ala Gly Ile Asn
Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe Leu Glu
Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310 315 320
Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu 325
330 335 Leu Ala Lys Asp Pro Arg Ile 340 <210> SEQ ID NO 346
<211> LENGTH: 381 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic amino acid sequence <400> SEQUENCE:
346 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr
1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Glu Glu
Gly Lys 20 25 30 Leu Val Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn
Gly Leu Ala Glu 35 40 45 Val Gly Lys Lys Phe Glu Lys Asp Thr Gly
Ile Lys Val Thr Val Glu 50 55 60 His Pro Asp Lys Leu Glu Glu Lys
Phe Pro Gln Val Ala Ala Thr Gly 65 70 75 80 Asp Gly Pro Asp Ile Ile
Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85 90 95 Ala Gln Ser Gly
Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln 100 105 110 Asp Lys
Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys 115 120 125
Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn 130
135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro
Ala 145 150 155 160 Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala
Leu Met Phe Asn 165 170 175 Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu
Ile Ala Ala Asp Gly Gly 180 185 190 Tyr Ala Phe Lys Tyr Glu Asn Gly
Lys Tyr Asp Ile Lys Asp Val Gly 195 200 205 Val Asp Asn Ala Gly Ala
Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210 215 220 Ile Lys Asn Lys
His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu 225 230 235 240 Ala
Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp 245 250
255 Ala Trp Ser Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val
260 265 270 Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly
Val Leu 275 280 285 Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu
Leu Ala Lys Glu 290 295 300 Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu
Gly Leu Glu Ala Val Asn 305 310 315 320 Lys Asp Lys Pro Leu Gly Ala
Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 330 335 Leu Ala Lys Asp Pro
Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Ser 340 345 350 Ala Phe Trp
Tyr Ala Val Arg Ile Glu Ala Arg Leu Gln Ala Ser Val 355 360 365 Asp
Ala Glu Ser Arg Asp Tyr Lys Asp Asp Asp Asp Lys 370 375 380
<210> SEQ ID NO 347 <211> LENGTH: 381 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 347 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu
Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala
Leu Ala Lys Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile Trp Ile Asn
Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val Gly Lys Lys
Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu 50 55 60 His Pro
Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly 65 70 75 80
Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85
90 95 Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe
Gln 100 105 110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr
Asn Gly Lys 115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu
Ser Leu Ile Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys
Thr Trp Glu Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys Glu Leu Lys
Ala Lys Gly Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu Gln Glu Pro
Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185 190 Tyr Ala
Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly 195 200 205
Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210
215 220 Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala
Glu 225 230 235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile
Asn Gly Pro Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr Ser Lys Val
Asn Tyr Gly Val Thr Val 260 265 270 Leu Pro Thr Phe Lys Gly Gln Pro
Ser Lys Pro Phe Val Gly Val Leu 275 280 285 Ser Ala Gly Ile Asn Ala
Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe Leu Glu Asn
Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310 315 320 Lys
Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 330
335 Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Ser
340 345 350 Ala Phe Trp Tyr Ala Val Arg Ile Glu Ala Arg Leu Gln Ala
Ser Val 355 360 365 Asp Ala Glu Ser Arg Asp Tyr Lys Asp Asp Asp Asp
Lys 370 375 380 <210> SEQ ID NO 348 <211> LENGTH: 381
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 348 Met Lys Ile Lys Thr Gly Ala
Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser
Ala Ser Ala Leu Ala Lys Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile
Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val
Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu 50 55
60 His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly
65 70 75 80 Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly
Gly Tyr 85 90 95 Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp
Lys Ala Phe Gln 100 105 110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala
Val Arg Tyr Asn Gly Lys 115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val
Glu Ala Leu Ser Leu Ile Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn
Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys
Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu
Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185
190 Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly
195 200 205 Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val
Asp Leu 210 215 220 Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr
Ser Ile Ala Glu 225 230 235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala
Met Thr Ile Asn Gly Pro Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr
Ser Lys Val Asn Tyr Gly Val Thr Val 260 265 270
Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu 275
280 285 Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys
Glu 290 295 300 Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu
Ala Val Asn 305 310 315 320 Lys Asp Lys Pro Leu Gly Ala Val Ala Leu
Lys Ser Tyr Glu Glu Glu 325 330 335 Leu Ala Lys Asp Pro Arg Ile Ala
Ala Thr Met Glu Asn Ala Gln Ser 340 345 350 Ala Phe Trp Tyr Ala Val
Arg Ile Glu Ala Arg Leu Gln Ala Ser Val 355 360 365 Asp Ala Glu Ser
Arg Asp Tyr Lys Asp Asp Asp Asp Lys 370 375 380 <210> SEQ ID
NO 349 <211> LENGTH: 120 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 349 Ala Ser Val Asp Ala Glu Ser Arg Ser Asp Lys Ile Ile
His Leu Thr 1 5 10 15 Asp Asp Ser Phe Asp Thr Asp Val Leu Lys Ala
Asp Gly Ala Ile Leu 20 25 30 Val Asp Phe Trp Ala Glu Trp Cys Gly
Pro Cys Lys Met Ile Ala Pro 35 40 45 Ile Leu Asp Glu Ile Ala Asp
Glu Tyr Gln Gly Lys Leu Thr Val Ala 50 55 60 Lys Leu Asn Ile Asp
Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile 65 70 75 80 Arg Gly Ile
Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala 85 90 95 Thr
Lys Val Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Asn Leu Ala 100 105
110 Asp Tyr Lys Asp Asp Asp Asp Lys 115 120 <210> SEQ ID NO
350 <211> LENGTH: 120 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 350 Ala Ser Val Asp Ala Glu Ser Arg Ser Asp Lys Ile Ile
His Leu Thr 1 5 10 15 Asp Asp Ser Phe Asp Thr Asp Val Leu Lys Ala
Asp Gly Ala Ile Leu 20 25 30 Val Asp Phe Trp Ala Glu Trp Cys Gly
Pro Cys Lys Met Ile Ala Pro 35 40 45 Ile Leu Asp Glu Ile Ala Asp
Glu Tyr Gln Gly Lys Leu Thr Val Ala 50 55 60 Lys Leu Asn Ile Asp
Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile 65 70 75 80 Arg Gly Ile
Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala 85 90 95 Thr
Lys Val Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Asn Leu Ala 100 105
110 Asp Tyr Lys Asp Asp Asp Asp Lys 115 120 <210> SEQ ID NO
351 <211> LENGTH: 120 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 351 Ala Ser Val Asp Ala Glu Ser Arg Ser Asp Lys Ile Ile
His Leu Thr 1 5 10 15 Asp Asp Ser Phe Asp Thr Asp Val Leu Lys Ala
Asp Gly Ala Ile Leu 20 25 30 Val Asp Phe Trp Ala Glu Trp Cys Gly
Pro Cys Lys Met Ile Ala Pro 35 40 45 Ile Leu Asp Glu Ile Ala Asp
Glu Tyr Gln Gly Lys Leu Thr Val Ala 50 55 60 Lys Leu Asn Ile Asp
Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile 65 70 75 80 Arg Gly Ile
Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala 85 90 95 Thr
Lys Val Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Asn Leu Ala 100 105
110 Asp Tyr Lys Asp Asp Asp Asp Lys 115 120 <210> SEQ ID NO
352 <211> LENGTH: 120 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 352 Ala Ser Val Asp Ala Glu Ser Arg Ser Asp Lys Ile Ile
His Leu Thr 1 5 10 15 Asp Asp Ser Phe Asp Thr Asp Val Leu Lys Ala
Asp Gly Ala Ile Leu 20 25 30 Val Asp Phe Trp Ala Glu Trp Cys Gly
Pro Cys Lys Met Ile Ala Pro 35 40 45 Ile Leu Asp Glu Ile Ala Asp
Glu Tyr Gln Gly Lys Leu Thr Val Ala 50 55 60 Lys Leu Asn Ile Asp
Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile 65 70 75 80 Arg Gly Ile
Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala 85 90 95 Thr
Lys Val Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Asn Leu Ala 100 105
110 Asp Tyr Lys Asp Asp Asp Asp Lys 115 120 <210> SEQ ID NO
353 <211> LENGTH: 154 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 353 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser
Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys
Ile Ile Glu Ala Arg 20 25 30 Leu Gln Ala Ser Val Asp Ala Glu Ser
Arg Ser Asp Lys Ile Ile His 35 40 45 Leu Thr Asp Asp Ser Phe Asp
Thr Asp Val Leu Lys Ala Asp Gly Ala 50 55 60 Ile Leu Val Asp Phe
Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile 65 70 75 80 Ala Pro Ile
Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr 85 90 95 Val
Ala Lys Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr 100 105
110 Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val
115 120 125 Ala Ala Thr Lys Val Gly Ala Leu Ser Lys Gly Gln Leu Lys
Glu Asn 130 135 140 Leu Ala Asp Tyr Lys Asp Asp Asp Asp Lys 145 150
<210> SEQ ID NO 354 <211> LENGTH: 154 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 354 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu
Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala
Leu Ala Lys Ile Ile Glu Ala Arg 20 25 30 Leu Gln Ala Ser Val Asp
Ala Glu Ser Arg Ser Asp Lys Ile Ile His 35 40 45 Leu Thr Asp Asp
Ser Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala 50 55 60 Ile Leu
Val Asp Phe Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile 65 70 75 80
Ala Pro Ile Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr 85
90 95 Val Ala Lys Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys
Tyr 100 105 110 Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys Asn
Gly Glu Val 115 120 125 Ala Ala Thr Lys Val Gly Ala Leu Ser Lys Gly
Gln Leu Lys Glu Asn 130 135 140 Leu Ala Asp Tyr Lys Asp Asp Asp Asp
Lys 145 150 <210> SEQ ID NO 355 <211> LENGTH: 154
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 355 Met Lys Ile Lys Thr Gly Ala
Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser
Ala Ser Ala Leu Ala Lys Ile Ile Glu Ala Arg
20 25 30 Leu Gln Ala Ser Val Asp Ala Glu Ser Arg Ser Asp Lys Ile
Ile His 35 40 45 Leu Thr Asp Asp Ser Phe Asp Thr Asp Val Leu Lys
Ala Asp Gly Ala 50 55 60 Ile Leu Val Asp Phe Trp Ala Glu Trp Cys
Gly Pro Cys Lys Met Ile 65 70 75 80 Ala Pro Ile Leu Asp Glu Ile Ala
Asp Glu Tyr Gln Gly Lys Leu Thr 85 90 95 Val Ala Lys Leu Asn Ile
Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr 100 105 110 Gly Ile Arg Gly
Ile Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val 115 120 125 Ala Ala
Thr Lys Val Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Asn 130 135 140
Leu Ala Asp Tyr Lys Asp Asp Asp Asp Lys 145 150 <210> SEQ ID
NO 356 <211> LENGTH: 154 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 356 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser
Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys
Ile Ile Glu Ala Arg 20 25 30 Leu Gln Ala Ser Val Asp Ala Glu Ser
Arg Ser Asp Lys Ile Ile His 35 40 45 Leu Thr Asp Asp Ser Phe Asp
Thr Asp Val Leu Lys Ala Asp Gly Ala 50 55 60 Ile Leu Val Asp Phe
Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile 65 70 75 80 Ala Pro Ile
Leu Asp Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr 85 90 95 Val
Ala Lys Leu Asn Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr 100 105
110 Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val
115 120 125 Ala Ala Thr Lys Val Gly Ala Leu Ser Lys Gly Gln Leu Lys
Glu Asn 130 135 140 Leu Ala Asp Tyr Lys Asp Asp Asp Asp Lys 145 150
<210> SEQ ID NO 357 <211> LENGTH: 485 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic amino acid sequence
<400> SEQUENCE: 357 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu
Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala
Leu Ala Lys Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile Trp Ile Asn
Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val Gly Lys Lys
Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu 50 55 60 His Pro
Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly 65 70 75 80
Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85
90 95 Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe
Gln 100 105 110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr
Asn Gly Lys 115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu
Ser Leu Ile Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys
Thr Trp Glu Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys Glu Leu Lys
Ala Lys Gly Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu Gln Glu Pro
Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185 190 Tyr Ala
Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly 195 200 205
Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210
215 220 Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala
Glu 225 230 235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile
Asn Gly Pro Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr Ser Lys Val
Asn Tyr Gly Val Thr Val 260 265 270 Leu Pro Thr Phe Lys Gly Gln Pro
Ser Lys Pro Phe Val Gly Val Leu 275 280 285 Ser Ala Gly Ile Asn Ala
Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe Leu Glu Asn
Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310 315 320 Lys
Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 330
335 Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Ser
340 345 350 Ala Phe Trp Tyr Ala Val Arg Ile Glu Ala Arg Leu Gln Ala
Ser Val 355 360 365 Asp Ala Glu Ser Arg Ser Asp Lys Ile Ile His Leu
Thr Asp Asp Ser 370 375 380 Phe Asp Thr Asp Val Leu Lys Ala Asp Gly
Ala Ile Leu Val Asp Phe 385 390 395 400 Trp Ala Glu Trp Cys Gly Pro
Cys Lys Met Ile Ala Pro Ile Leu Asp 405 410 415 Glu Ile Ala Asp Glu
Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn 420 425 430 Ile Asp Gln
Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile 435 440 445 Pro
Thr Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val 450 455
460 Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Asn Leu Ala Asp Tyr Lys
465 470 475 480 Asp Asp Asp Asp Lys 485 <210> SEQ ID NO 358
<211> LENGTH: 485 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic amino acid sequence <400> SEQUENCE:
358 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser Ala Leu Thr
1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys Ile Glu Glu
Gly Lys 20 25 30 Leu Val Ile Trp Ile Asn Gly Asp Lys Gly Tyr Asn
Gly Leu Ala Glu 35 40 45 Val Gly Lys Lys Phe Glu Lys Asp Thr Gly
Ile Lys Val Thr Val Glu 50 55 60 His Pro Asp Lys Leu Glu Glu Lys
Phe Pro Gln Val Ala Ala Thr Gly 65 70 75 80 Asp Gly Pro Asp Ile Ile
Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85 90 95 Ala Gln Ser Gly
Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln 100 105 110 Asp Lys
Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys 115 120 125
Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile Tyr Asn 130
135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro
Ala 145 150 155 160 Leu Asp Lys Glu Leu Lys Ala Lys Gly Lys Ser Ala
Leu Met Phe Asn 165 170 175 Leu Gln Glu Pro Tyr Phe Thr Trp Pro Leu
Ile Ala Ala Asp Gly Gly 180 185 190 Tyr Ala Phe Lys Tyr Glu Asn Gly
Lys Tyr Asp Ile Lys Asp Val Gly 195 200 205 Val Asp Asn Ala Gly Ala
Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210 215 220 Ile Lys Asn Lys
His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu 225 230 235 240 Ala
Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile Asn Gly Pro Trp 245 250
255 Ala Trp Ser Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly Val Thr Val
260 265 270 Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro Phe Val Gly
Val Leu 275 280 285 Ser Ala Gly Ile Asn Ala Ala Ser Pro Asn Lys Glu
Leu Ala Lys Glu 290 295 300 Phe Leu Glu Asn Tyr Leu Leu Thr Asp Glu
Gly Leu Glu Ala Val Asn 305 310 315 320 Lys Asp Lys Pro Leu Gly Ala
Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 330 335 Leu Ala Lys Asp Pro
Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Ser 340 345 350 Ala Phe Trp
Tyr Ala Val Arg Ile Glu Ala Arg Leu Gln Ala Ser Val 355 360 365 Asp
Ala Glu Ser Arg Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser 370 375
380
Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe 385
390 395 400 Trp Ala Glu Trp Cys Gly Pro Cys Lys Met Ile Ala Pro Ile
Leu Asp 405 410 415 Glu Ile Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val
Ala Lys Leu Asn 420 425 430 Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys
Tyr Gly Ile Arg Gly Ile 435 440 445 Pro Thr Leu Leu Leu Phe Lys Asn
Gly Glu Val Ala Ala Thr Lys Val 450 455 460 Gly Ala Leu Ser Lys Gly
Gln Leu Lys Glu Asn Leu Ala Asp Tyr Lys 465 470 475 480 Asp Asp Asp
Asp Lys 485 <210> SEQ ID NO 359 <211> LENGTH: 485
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 359 Met Lys Ile Lys Thr Gly Ala
Arg Ile Leu Ala Leu Ser Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser
Ala Ser Ala Leu Ala Lys Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile
Trp Ile Asn Gly Asp Lys Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val
Gly Lys Lys Phe Glu Lys Asp Thr Gly Ile Lys Val Thr Val Glu 50 55
60 His Pro Asp Lys Leu Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly
65 70 75 80 Asp Gly Pro Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly
Gly Tyr 85 90 95 Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp
Lys Ala Phe Gln 100 105 110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala
Val Arg Tyr Asn Gly Lys 115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val
Glu Ala Leu Ser Leu Ile Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn
Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys
Glu Leu Lys Ala Lys Gly Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu
Gln Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185
190 Tyr Ala Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly
195 200 205 Val Asp Asn Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val
Asp Leu 210 215 220 Ile Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr
Ser Ile Ala Glu 225 230 235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala
Met Thr Ile Asn Gly Pro Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr
Ser Lys Val Asn Tyr Gly Val Thr Val 260 265 270 Leu Pro Thr Phe Lys
Gly Gln Pro Ser Lys Pro Phe Val Gly Val Leu 275 280 285 Ser Ala Gly
Ile Asn Ala Ala Ser Pro Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe
Leu Glu Asn Tyr Leu Leu Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310
315 320 Lys Asp Lys Pro Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu
Glu 325 330 335 Leu Ala Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn
Ala Gln Ser 340 345 350 Ala Phe Trp Tyr Ala Val Arg Ile Glu Ala Arg
Leu Gln Ala Ser Val 355 360 365 Asp Ala Glu Ser Arg Ser Asp Lys Ile
Ile His Leu Thr Asp Asp Ser 370 375 380 Phe Asp Thr Asp Val Leu Lys
Ala Asp Gly Ala Ile Leu Val Asp Phe 385 390 395 400 Trp Ala Glu Trp
Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp 405 410 415 Glu Ile
Ala Asp Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn 420 425 430
Ile Asp Gln Asn Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile 435
440 445 Pro Thr Leu Leu Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys
Val 450 455 460 Gly Ala Leu Ser Lys Gly Gln Leu Lys Glu Asn Leu Ala
Asp Tyr Lys 465 470 475 480 Asp Asp Asp Asp Lys 485 <210> SEQ
ID NO 360 <211> LENGTH: 485 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 360 Met Lys Ile Lys Thr Gly Ala Arg Ile Leu Ala Leu Ser
Ala Leu Thr 1 5 10 15 Thr Met Met Phe Ser Ala Ser Ala Leu Ala Lys
Ile Glu Glu Gly Lys 20 25 30 Leu Val Ile Trp Ile Asn Gly Asp Lys
Gly Tyr Asn Gly Leu Ala Glu 35 40 45 Val Gly Lys Lys Phe Glu Lys
Asp Thr Gly Ile Lys Val Thr Val Glu 50 55 60 His Pro Asp Lys Leu
Glu Glu Lys Phe Pro Gln Val Ala Ala Thr Gly 65 70 75 80 Asp Gly Pro
Asp Ile Ile Phe Trp Ala His Asp Arg Phe Gly Gly Tyr 85 90 95 Ala
Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro Asp Lys Ala Phe Gln 100 105
110 Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val Arg Tyr Asn Gly Lys
115 120 125 Leu Ile Ala Tyr Pro Ile Ala Val Glu Ala Leu Ser Leu Ile
Tyr Asn 130 135 140 Lys Asp Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu
Glu Ile Pro Ala 145 150 155 160 Leu Asp Lys Glu Leu Lys Ala Lys Gly
Lys Ser Ala Leu Met Phe Asn 165 170 175 Leu Gln Glu Pro Tyr Phe Thr
Trp Pro Leu Ile Ala Ala Asp Gly Gly 180 185 190 Tyr Ala Phe Lys Tyr
Glu Asn Gly Lys Tyr Asp Ile Lys Asp Val Gly 195 200 205 Val Asp Asn
Ala Gly Ala Lys Ala Gly Leu Thr Phe Leu Val Asp Leu 210 215 220 Ile
Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Glu 225 230
235 240 Ala Ala Phe Asn Lys Gly Glu Thr Ala Met Thr Ile Asn Gly Pro
Trp 245 250 255 Ala Trp Ser Asn Ile Asp Thr Ser Lys Val Asn Tyr Gly
Val Thr Val 260 265 270 Leu Pro Thr Phe Lys Gly Gln Pro Ser Lys Pro
Phe Val Gly Val Leu 275 280 285 Ser Ala Gly Ile Asn Ala Ala Ser Pro
Asn Lys Glu Leu Ala Lys Glu 290 295 300 Phe Leu Glu Asn Tyr Leu Leu
Thr Asp Glu Gly Leu Glu Ala Val Asn 305 310 315 320 Lys Asp Lys Pro
Leu Gly Ala Val Ala Leu Lys Ser Tyr Glu Glu Glu 325 330 335 Leu Ala
Lys Asp Pro Arg Ile Ala Ala Thr Met Glu Asn Ala Gln Ser 340 345 350
Ala Phe Trp Tyr Ala Val Arg Ile Glu Ala Arg Leu Gln Ala Ser Val 355
360 365 Asp Ala Glu Ser Arg Ser Asp Lys Ile Ile His Leu Thr Asp Asp
Ser 370 375 380 Phe Asp Thr Asp Val Leu Lys Ala Asp Gly Ala Ile Leu
Val Asp Phe 385 390 395 400 Trp Ala Glu Trp Cys Gly Pro Cys Lys Met
Ile Ala Pro Ile Leu Asp 405 410 415 Glu Ile Ala Asp Glu Tyr Gln Gly
Lys Leu Thr Val Ala Lys Leu Asn 420 425 430 Ile Asp Gln Asn Pro Gly
Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile 435 440 445 Pro Thr Leu Leu
Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val 450 455 460 Gly Ala
Leu Ser Lys Gly Gln Leu Lys Glu Asn Leu Ala Asp Tyr Lys 465 470 475
480 Asp Asp Asp Asp Lys 485 <210> SEQ ID NO 361 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleic acid sequence <400> SEQUENCE: 361
atgcctaaag ccgctccctc a 21 <210> SEQ ID NO 362 <211>
LENGTH: 15 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleic acid sequence <400> SEQUENCE: 362
atgaagccgc tccct 15
<210> SEQ ID NO 363 <211> LENGTH: 15 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleic acid sequence
<400> SEQUENCE: 363 augaagccgc ucccu 15 <210> SEQ ID NO
364 <211> LENGTH: 5 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic amino acid sequence <400>
SEQUENCE: 364 Met Pro Lys Ala Ala 1 5 <210> SEQ ID NO 365
<211> LENGTH: 15 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic nucleic acid sequence <400> SEQUENCE:
365 tgggttactc actca 15 <210> SEQ ID NO 366 <211>
LENGTH: 15 <212> TYPE: RNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic nucleic acid sequence <400> SEQUENCE: 366
uggguuacuc acuca 15 <210> SEQ ID NO 367 <211> LENGTH: 5
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic amino
acid sequence <400> SEQUENCE: 367 Trp Val Thr His Ser 1 5
<210> SEQ ID NO 368 <211> LENGTH: 15 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic nucleic acid sequence
<400> SEQUENCE: 368 atgaagccgc actca 15 <210> SEQ ID NO
369 <211> LENGTH: 15 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic nucleic acid sequence <400>
SEQUENCE: 369 augaagccgc acuca 15 <210> SEQ ID NO 370
<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic amino acid sequence <400> SEQUENCE:
370 Met Pro Lys His Ser 1 5
* * * * *
References