U.S. patent application number 10/369845 was filed with the patent office on 2003-10-02 for methods for isolating novel antimicrobial agents from hypermutable mammalian cells.
Invention is credited to Grasso, Luigi, Nicolaides, Nicholas C., Sass, Philip M..
Application Number | 20030186441 10/369845 |
Document ID | / |
Family ID | 24844790 |
Filed Date | 2003-10-02 |
United States Patent
Application |
20030186441 |
Kind Code |
A1 |
Nicolaides, Nicholas C. ; et
al. |
October 2, 2003 |
Methods for isolating novel antimicrobial agents from hypermutable
mammalian cells
Abstract
Dominant-negative alleles of human mismatch repair genes can be
used to generate hypermutable cells and organisms. By introducing
these genes into mammalian cells new cell lines with novel and
useful properties can be prepared more efficiently than by relying
on the natural rate of mutation or introduction of mutations by
chemical mutagens. These methods are useful for generating novel
and highly active antimicrobial molecules as well as superior
antimicrobial agents from pre-existing chemicals. These methods are
also useful for generating cell lines expressing novel
antimicrobials that are useful for pharmaceutical
manufacturing.
Inventors: |
Nicolaides, Nicholas C.;
(Boothwyn, PA) ; Grasso, Luigi; (Philadelphia,
PA) ; Sass, Philip M.; (Audubon, PA) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
ONE LIBERTY PLACE, 46TH FLOOR
1650 MARKET STREET
PHILADELPHIA
PA
19103
US
|
Family ID: |
24844790 |
Appl. No.: |
10/369845 |
Filed: |
February 19, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10369845 |
Feb 19, 2003 |
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09708200 |
Nov 7, 2000 |
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6576468 |
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Current U.S.
Class: |
435/455 ;
435/366 |
Current CPC
Class: |
C12N 15/102 20130101;
C07K 14/47 20130101; C12N 15/01 20130101 |
Class at
Publication: |
435/455 ;
435/366 |
International
Class: |
C12N 015/85; C12N
005/08 |
Claims
What is claimed is:
1. A method for making a mammalian cell hypermutable, comprising:
introducing into said mammalian cell a polynucleotide comprising a
dominant-negative allele of a mismatch repair gene, whereby said
cell becomes hypermutable.
2. The method of claim 1 wherein said polynucleotide is introduced
into said cell by transfection in vitro.
3. The method of claim 1, wherein said polynucleotide is introduced
into said cell by transfection of an adherent cell in vitro.
4. The method of claim 1 wherein said mismatch repair gene is
PMS2.
5. The method of claim 1 wherein said mismatch repair gene is human
PMS2.
6. The method of claim 1 wherein said mismatch repair gene is human
MLH1.
7. The method of claim 1 wherein said mismatch repair gene is human
PMS1.
8. The method of claim 1 wherein said mismatch repair gene is human
MSH2.
9. The method of claim 4 wherein said allele comprises a truncation
mutation.
10. The method of claim 4 wherein said allele comprises a
truncation mutation at codon 134.
11. The method of claim 9 wherein said truncation mutation is a
thymidine at nucleotide 424 of wild-type PMS2.
12. A homogeneous composition comprising a cultured, hypermutable,
mammalian cell comprising a dominant negative allele of a mismatch
repair gene.
13. The composition of claim 12 wherein said mismatch repair gene
is PMS2.
14. The composition of claim 12 wherein said mismatch repair gene
is human PMS2.
15. The composition of claim 12 wherein said mismatch repair gene
is human MLH1.
16. The composition of claim 12 wherein said mismatch repair gene
is human PMS1.
17. The composition of claim 12 wherein said mismatch repair gene
is human MSH2.
18. The composition of claim 12 wherein said cell expresses a
protein consisting of the first 133 amino acids of hPMS2.
19. A method for obtaining a mammalian cell that is resistant to a
selected microbe comprising: growing a culture of mammalian cells
wherein said cells have a dominant-negative allele of a mismatch
repair gene; exposing said cells to said selected microbe; and
selecting said mammalian cell that is resistant to said selected
microbe.
20. The method of claim 19 wherein said hypermutable cell is
selected for resistance to a gram-negative microbe.
21. The method of claim 19 wherein said hypermutable cell is
selected for resistance to a gram-positive microbe.
22. The method of claim 19 wherein said hypermutable cell is
selected for resistance to a protozoan.
23. The method of claim 19 wherein said hypermutable cell is
selected for resistance to a bacteria.
24. The method of claim 19 wherein said hypermutable cell is
selected for resistance to a fungi.
25. The method of claim 19 wherein said step of selecting for
microbial resistance comprises isolating and testing conditioned
medium from said hypermutable cell.
26. A method for obtaining a cell comprising a mutation in a gene
encoding an antimicrobial activity comprising: growing a culture of
mammalian cells having said gene encoding said antimicrobial
activity, and a dominant negative allele of a mismatch repair gene;
selecting a cell comprising said antimicrobial activity; and
determining whether said gene comprises a mutation.
27. The method of claim 26 wherein said hypermutable cell is
selected for resistance to a gram-negative microbe.
28. The method of claim 26 wherein said hypermutable cell is
selected for resistance to a gram-positive microbe.
29. The method of claim 26 wherein said hypermutable cell is
selected for resistance to a protozoan.
30. The method of claim 26 wherein said hypermutable cell is
selected for resistance to a bacteria.
31. The method of claim 26 wherein said hypermutable cell is
selected for resistance to a fungi.
32. The method of claim 26 wherein said step of selecting a cell
for antimicrobial activity comprises isolating and testing
conditioned medium from said hypermutable cell.
33. The method of claim 26 wherein said step of examining said cell
to determine whether said gene comprises a mutation comprises
analyzing a nucleotide sequence of said gene.
34. The method of claim 26 wherein said step of examining said cell
to determine whether said gene comprises a mutation comprises
analyzing mRNA transcribed from said gene.
35. The method of claim 26 wherein said step of examining said cell
to determine whether said gene comprises a mutation comprises
analyzing a protein encoded by said gene.
36. The method of claim 26 wherein said step of examining said cell
to determine whether said gene comprises a mutation comprises
analyzing the phenotype of said gene.
Description
FIELD OF THE INVENTION
[0001] The present invention is related to the area of
antimicrobial agents and cellular production of those agents. In
particular, it is related to the field of identification of novel
antimicrobial agents by placing mammalian cells under selection in
the presence of the microbe.
BACKGROUND OF THE INVENTION
[0002] For as long as man has shared the planet with microorganisms
there have been widespread outbreaks of infectious disease and
subsequent widespread mortality associated with it. Although
microorganisms and man frequently share a symbiotic relationship,
microorganisms can, under some conditions, lead to sickness and
death. The discovery, wide use and dissemination of antibiotics to
treat microbial infection in both human and animal populations over
the last one hundred or so years has done much to control, and in
some instances, eradicate some microbes and associated infectious
disease. However, microbes have a strong propensity to evolve and
alter their genetic makeup when confronted with toxic substances
that place them under life and death selective pressures.
Therefore, emerging infectious diseases currently pose an important
public health problem in both developed as well as developing
countries. Not only have microbes evolved to evade and defeat
current antibiotic therapeutics, but also there are novel and
previously unrecognized and/or characterized bacterial, fungal,
viral, and parasitic diseases that have emerged within the past two
decades. Sass, Curr. Opin. in Drug Discov. & Develop. 2000,
3(5):646-654.
[0003] Since the accidental discovery of a penicillin-producing
mold by Fleming there has been steady progress in synthesizing,
isolating and characterizing new and more effective beta-lactam
antibiotics. In addition to the great success of the beta-lactam
family of antibiotics, the newer fluoroquinolones have a
broad-spectrum of bactericidal activity as well as excellent oral
bio-availability, tissue penetration and favorable safety and
tolerability profiles. King et al., Am. Fam. Physician, 2000, 61,
2741-2748. A newly devised four-generation classification of the
quinolone drugs accounts for the expanded antimicrobial spectrum of
the more recently introduced fluoroquinolones and their clinical
indications. The so-called first generation drugs, which include
nalidixic acid, are capable of achieving minimal serum levels. The
second-generation quinolones, such as ciprofloxacin, have an
increased gram-negative and systemic activity. The third-generation
drugs comprise pharmaceuticals such as levofloxacin and are have
significant and expanded action against gram-positive bacteria and
a typical pathogens. Finally, the fourth-generation quinolone
drugs, which, to date, only includes trovofloxacin, are highly
active against anaerobes in addition to the activity described for
the third-generation drugs. Furthermore, the quinolone class of
anti-microbial drugs can be divided based on their pharmacokinetic
properties and bioavailability.
[0004] Mammalian epithelial surfaces are remarkable for their
ability to provide critical physiologic functions in the face of
frequent microbial challenges. The fact that these mucosal surfaces
remain infection-free in the normal host suggests that highly
effective mechanisms of host defense have evolved to protect these
environmentally exposed tissues. Throughout the animal and plant
kingdoms, endogenous genetically encoded antimicrobial peptides
have been shown to be key elements in the response to epithelial
compromise and microbial invasion. Zasloff, Curr. Opin. Immunol.,
1992, 4, 3-7; and Bevins, Ciba Found Symp., 1994, 186, 250-69. In
mammals, a variety of such peptides have been identified, including
the well-characterized defensins and cathelicidins and others
(andropin, magainin, tracheal antimicrobial peptide, and PR-39; see
Bevins, Ciba Found. Symp., 1994, 186, 250-69 and references
therein). A major source of these host defense molecules is
circulating phagocytic leukocytes. However, more recently, it has
been shown that resident epithelial cells of the skin and
respiratory, alimentary, and genitourinary tracts also synthesize
and release antimicrobial peptides. Both in vitro and in vivo data
support the hypothesis that these molecules are important
contributors to intrinsic mucosal immunity. Alterations in their
level of expression or biologic activity can predispose the
organism to microbial infection. Huttner et al., Pediatr. Res.,
1999, 45, 785-94.
[0005] Across the evolutionary scale species from insects to
mammals to plants defend themselves against invading pathogenic
microorganisms by utilizing cationic antimicrobial peptides that
rapidly kill microbes without exerting toxicity to the host.
Physicochemical peptide-lipid interactions provide attractive
mechanisms for innate immunity as discussed below. Many of these
peptides form cationic amphipathic secondary structures, typically
alpha-helices and beta-sheets, which can selectively interact with
anionic bacterial membranes via electrostatic interactions. Rapid,
peptide-induced membrane permeabilization and subsequent cellular
lysis is the result. Matsuzaki, Biochim. Biophys. Acta, 1999, 1462,
1-10.
[0006] The primary structures of a large number of these
host-defense peptides have been determined. While there is no
primary structure homology, the peptides are characterized by a
preponderance of cationic and hydrophobic amino acids. The
secondary structures of many of the host-defense peptides have been
determined by a variety of techniques. Sitaram et al., Biochim,
Biophys. Acta, 1999, 1462, 29-54. The acyclic peptides tend to
adopt helical conformation, especially in media of low dielectric
constant, whereas peptides with more than one disulfide bridge
adopt beta-structures.
[0007] As described above, one reason for the rise in microbial
drug resistance to the first line antimicrobial therapies in
standard use today is the inappropriate and over-use of
prescription antibiotics. Although bacteria are the most common
organisms to develop drug-resistance, there are numerous examples
of demonstrated resistance in fungi, viruses, and parasites. The
development of a resistant phenotype is a complex phenomenon that
involves an interaction of the microorganism, the environment, and
the patient, separately as well as in combination. Sitaram et al.,
Biochim. Biophys. Acta, 1999, 1462, 29-54. The microorganism in
question may develop resistance while under antibiotic selection or
it may be a characteristic of the microbe prior to exposure to a
given agent. There are a number of mechanisms of resistance to
antibiotics that have been described, including genes that encode
antibiotic resistance enzymes that are harbored on extrachromosomal
plasmids as well as DNA elements (e.g. transposable elements) that
can reside either extra-chromosomally or within the host
genome.
[0008] Due to the ability of microorganisms to acquire the ability
to develop resistance to antibiotics there is a need to continually
develop novel antibiotics. Traditional methods to develop novel
antibiotics have included medicinal chemistry approaches to modify
existing antibiotics (Kang et al., Bioorg. Med. Chem. Lett., 2000,
10, 95-99) as well as isolation of antibiotics from new organisms
(Alderson et al., Res. Microbiol., 1993, 144, 665-72). Each of
these methods, however, has limitations. The traditional medicinal
chemistry approach entails modification of an existing molecule to
impart a more effective activity. The chemist makes a "best guess"
as to which parts of the molecule to alter, must then devise a
synthetic strategy, synthesize the molecule, and then have it
tested. This approach is laborious, requires large numbers of
medicinal chemists and frequently results in a molecule that is
lower in activity than the original antibiotic. The second
approach, isolation of novel antimicrobial agents, requires
screening large numbers of diverse organisms for novel
antimicrobial activity. Then, the activity must be isolated from
the microorganism. This is not a small task, and frequently takes
many years of hard work to isolate the active molecule. Even after
the molecule is identified, it may not be possible for medicinal
chemists to effectively devise a synthetic strategy due to the
complexity of the molecule. Furthermore, the synthetic strategy
must allow for a cost-effective synthesis. Therefore, a method that
would allow for creation of more effective antibiotics from
existing molecules or allow rapid isolation of novel antimicrobial
agents is needed to combat the ever-growing list of antibiotic
resistant organisms. The present invention described herein is
directed to the use of random genetic mutation of a cell to produce
novel antibiotics by blocking the endogenous mismatch repair
activity of a host cell. The cell can be a mammalian cell that
produces an antimicrobial agent naturally, or a cell that is placed
under selective pressure to obtain a novel antimicrobial molecule
that attacks a specific microbe. Moreover, the invention describes
methods for obtaining enhanced antimicrobial activity of a cell
line that produces an antimicrobial activity due to recombinant
expression or as part of the innate capacity of the cell to harbor
such activity.
[0009] In addition, the generation of genetically altered host
cells that are capable of secreting an antimicrobial activity,
which can be protein or non-protein based, will be valuable
reagents for manufacturing the entity for clinical studies. An
embodiment of the invention described herein is directed to the
creation of genetically altered host cells with novel and/or
increased antimicrobial production that are generated by a method
that interferes with the highly ubiquitous and phylogenetically
conserved process of mismatch repair.
[0010] The present invention facilitates the generation of novel
antimicrobial agents and the production of cell lines that express
elevated levels of antimicrobial activity. Advantages of the
present invention are further described in the examples and figures
described herein.
SUMMARY OF THE INVENTION
[0011] One embodiment of the present invention provides a method
for generating genetically altered mammalian cells and placing the
cells under direct microbial selection as a means to isolate novel
antimicrobial agents. Another embodiment provides a method for
identifying novel microbe-specific toxic molecules by altering the
ability of the cell to correct natural defects that occur in the
DNA during the process of DNA replication. Interference with this
process, called mismatch repair, leads to genetically dissimilar
sibling cells. These genetically dissimilar cells contain
mutations, ranging from one mutation/genome to two or more
mutations/genome, offer a rich population of cells from which to
select for specific output traits, such as the novel ability to
resist microbial insult. The genetically altered cell generated by
manipulation of the mismatch repair process is then incubated with
a microbe that is normally toxic to cells. Most of the cells will
rapidly lose viability and die; however, a subset of resistant
cells will have the capacity to resist the microbial insult. These
cells express a molecule, protein or non-protein in structure, that
imbues an antimicrobial activity to the newly selected mammalian
clones. These newly created cells can be expanded in vitro and the
new molecule isolated and characterized by standard methods that
are well described it the art. The novel molecule(s) are then
tested for their ability to kill or inhibit the growth of the
microbe by standard microbial assays that are well described in the
art. Finally, the novel cell line generated serves as an additional
resource for large-scale production of the novel antimicrobial
agent for use in clinical studies. The processes described herein
are applicable to any mammalian cell and any microbe for which an
antibiotic agent is sought.
[0012] The invention provides methods for rendering mammalian cells
hypermutable as a means to generate antimicrobial agents.
[0013] The invention also provides methods for generating
genetically altered cell lines that secrete enhanced amounts of a
known or novel antimicrobial polypeptide.
[0014] The invention also provides methods for generating
genetically altered cell lines that secrete enhanced amounts of a
known or novel antimicrobial non-polypeptide based molecule.
[0015] The invention also provides methods for generating
genetically altered cell lines that do not secrete enhanced amounts
of an antimicrobial peptide or non-peptide molecule but rather have
a cell-surface active molecule that detoxifies the microbe under
test.
[0016] The invention also provides methods for producing an
enhanced rate of genetic hypermutation in a mammalian cell and use
of this as the basis to select for microbial-resistant cell
lines.
[0017] The invention also provides methods of mutating a known
antimicrobial encoding gene of interest in a mammalian cell as a
means to obtain a molecule with enhanced bactericidal activity.
[0018] The invention also provides methods for creating genetically
altered antimicrobial molecules in vivo.
[0019] The invention also provides methods for creating novel
antimicrobial molecules from preexisting antimicrobial molecules by
altering the innate enzymatic or binding ability of the molecules
by altering the mismatch repair system within the host mammalian
cell.
[0020] The invention also provides methods for creating a novel
anti-microbial polypeptide or non-polypeptide based molecule that
has the capacity to bind in an irreversible manner to a microbe and
thereby block binding of the pathogenic microbe to a host target
organism and result in loss of viability of the microbe.
[0021] The invention also provides methods for creating a novel
antimicrobial polypeptide or non-polypeptide based small molecule
that can block microbial cell growth and/or survival.
[0022] The invention also provides methods for creating a novel
antimicrobial polypeptide or non-polypeptide based biochemical that
are able to irreversibly bind to toxic chemicals produced by
pathogenic microbes.
[0023] The invention also provides methods for creating genetically
altered antimicrobial molecules, either peptide of non-peptide
based, that have enhanced pharmacokinetic properties in host
organisms.
[0024] The invention also provides methods for creating genetically
altered cell lines that manufacture an antimicrobial molecules,
either peptide of non-peptide based, for use in large-scale
production of the antimicrobial agent for clinical studies.
[0025] These and other aspects of the invention are described in
the embodiments below. In one embodiment of the invention
described, a method for making a microbial-sensitive mammalian cell
microbe resistant by rendering the cell line hypermutable is
provided. A polynucleotide encoding a dominant negative allele of a
mismatch repair gene is introduced into an mammalian cell. The cell
becomes hypermutable as a result of the introduction of the
gene.
[0026] In another embodiment of the invention, an isolated
hypermutable cell is provided. The cell comprises a dominant
negative allele of a mismatch repair gene. The cell exhibits an
enhanced rate of hypermutation.
[0027] In another embodiment of the invention, an isolated
hypermutable cell is provided. The cell comprises a dominant
negative allele of a mismatch repair gene. The cell exhibits an
enhanced rate of hypermutation. The populations of cells generated
by introduction of the mismatch repair gene are grown in the
presence of microbes that are toxic to the wild type non-mutant
cells. Cells are selected that are resistant to the microbe and the
novel molecule(s) isolated and characterized for antimicrobial
activity by standard methods well described in the art.
[0028] In another embodiment of the invention, an isolated
hypermutable cell is described to create a novel antimicrobial
molecule from a pre-existing antimicrobial molecule by altering the
innate enzymatic or binding ability of the molecule.
[0029] In another embodiment of the invention, a method of creating
a novel antimicrobial polypeptide or non-polypeptide based molecule
that has the capacity to bind in an irreversible manner to a
microbe and thereby block binding of the pathogenic microbe to a
host target organism and result in loss of viability of the
microbe.
[0030] In another embodiment of the invention, a method of creating
a novel antimicrobial polypeptide or non-polypeptide based small
molecule that can block microbial cell growth and/or survival is
described.
[0031] In another embodiment of the invention, a method of creating
a novel antimicrobial polypeptide or non-polypeptide based
biochemical that are able to irreversibly bind to toxic chemicals
produced by pathogenic microbes is described.
[0032] In another embodiment of the invention, a method is provided
for introducing a mutation into a known endogenous gene encoding
for an antimicrobial polypeptide or a non-protein based
antimicrobial molecule as a means to create a more efficacious
antimicrobial. A polynucleotide encoding a dominant negative allele
of a mismatch repair gene is introduced into a cell. The cell
becomes hypermutable as a result of the introduction of the gene.
The cell further comprises an antimicrobial gene(s) of interest.
The cell is grown and tested to determine whether the gene encoding
for an antimicrobial is altered and whether the novel molecule is
more active by standard microbiology assays well known in the
art.
[0033] In another embodiment of the invention, a gene or genes
encoding for an antimicrobial molecule is introduced into a
mammalian cell host that is mismatch repair defective. The cell is
grown, and then clones are analyzed for enhanced antimicrobial
characteristics.
[0034] In another embodiment of the invention, a method is provided
for producing new phenotypes of a cell. A polynucleotide encoding a
dominant negative allele of a mismatch repair gene is introduced
into a cell. The cell becomes hypermutable as a result of the
introduction of the gene. The cell is grown and tested for the
expression of new phenotypes where the phenotype is enhanced
secretion of a novel or known antimicrobial polypeptide.
[0035] In another embodiment of the invention, a method is provided
for producing new phenotypes of a cell. A polynucleotide encoding a
dominant negative allele of a mismatch repair gene is introduced
into a cell. The cell becomes hypermutable as a result of the
introduction of the gene. The cell is grown and tested for the
expression of new phenotypes where the phenotype is enhanced
secretion of a novel or known antimicrobial non-polypeptide based
molecule.
[0036] In another embodiment of the invention, a method is provided
for producing new phenotypes of a cell. A polynucleotide encoding a
dominant negative allele of a mismatch repair gene is introduced
into a cell. The cell becomes hypermutable as a result of the
introduction of the gene. The cell is grown and tested for the
expression of new phenotypes where the phenotype is enhanced
antimicrobial activity of a novel or known antimicrobial
polypeptide that is not secreted.
[0037] In another embodiment of the invention, a method is provided
for producing new phenotypes of a cell. A polynucleotide encoding a
dominant negative allele of a mismatch repair gene is introduced
into a cell. The cell becomes hypermutable as a result of the
introduction of the gene. The cell is grown and tested for the
expression of new phenotypes where the phenotype is enhanced
antimicrobial activity of a novel or known antimicrobial
non-polypeptide based molecule that is not secreted.
[0038] In another embodiment of the invention, a method is provided
for restoring genetic stability in a cell containing a
polynucleotide encoding a dominant negative allele of a mismatch
repair gene. The expression of the dominant negative mismatch
repair gene is suppressed and the cell is restored to its former
genetic stability.
[0039] In another embodiment of the invention, a method is provided
for restoring genetic stability in a cell containing a
polynucleotide encoding a dominant negative allele of a mismatch
repair gene and a newly selected phenotype. The expression of the
dominant negative mismatch repair gene is suppressed and the cell
restores its genetic stability and the new phenotype is stable.
[0040] These and other embodiments of the invention provide the art
with methods that generate enhanced mutability in cells and animals
as well as providing cells and animals harboring potentially useful
mutations and novel protein and non-protein based molecules.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1 is a representative in situ .beta.-galactosidase
staining of TK-hPMS2-134 or TKvect cells to measure for cells
containing genetically altered .beta.-galactosidase genes; arrows
indicate Blue (.beta.-galactosidase positive) cells.
[0042] FIG. 2 is a schematic representation of sequence of
alterations of the .beta.-galactosidase gene produced by expression
of TK-hPMS2-134 host cells in TK cells.
[0043] FIGS. 3A, 3B and 3C show a representative
immunoprecipitation of in vitro translated hPMS2 and hMLH1
proteins.
[0044] FIG. 4 shows representative complementation of MMR activity
in transduced SH cells.
[0045] FIG. 5 is a representative photograph of Syrian hamster
TK-ts 13 cells transfected with a eukaryotic expression vector that
produces a novel anti-microbial polypeptide.
[0046] FIG. 6 is a representative graph showing TK-HPMS-134
transfected TK cells can suppress the growth of bacteria in
vitro.
[0047] The presented invention is directed to, in part, methods for
developing hypermutable mammalian cells by taking advantage of the
conserved mismatch repair process of host cells. Mismatched repair
process is described in several references. Baker et al., Cell,
1995, 82, 309 319; Bronner et al., Nature, 1994, 368, 258 261; de
Wind et al., Cell, 1995, 82, 321 330; and Drummond et al., Science,
1995, 268, 1909 1912. Dominant negative alleles of such genes, when
introduced into cells or transgenic animals, increase the rate of
spontaneous mutations by reducing the effectiveness of DNA repair
and thereby render the cells or animals hypermutable. Hypermutable
cells or animals can then be utilized to develop new mutations in a
gene of interest or in a gene whose function has not been
previously described. Blocking mismatch repair in cells such as,
for example, mammalian cells or mammalian cells transfected with
genes encoding for specific antimicrobial peptides or non-peptide
based antimicrobials, can enhance the rate of mutation within these
cells leading to clones that have novel or enhanced antimicrobial
activity or production and/or cells that contain genetically
altered antimicrobials with enhanced biochemical activity against a
range of opportunistic microbes.
[0048] The process of mismatch repair, also called mismatch
proofreading, is carried out by protein complexes in cells ranging
from bacteria to mammalian cells. Modrich, Science, 1994, 266, 1959
1960. A mismatch repair gene is a gene that encodes for one of the
proteins of such a mismatch repair complex. Baker et. al., Cell,
1995, 82, 309 319; Bronner et al., Nature, 1994, 368, 258 261; de
Wind et al., Cell, 1995, 82, 321 330; Drummond et al., Science,
1995, 268, 1909 1912; and Modrich, Science, 1994, 266, 1959 1960.
Although not wanting to be bound by any particular theory of
mechanism of action, a mismatch repair complex is believed to
detect distortions of the DNA helix resulting from
non-complementary pairing of nucleotide bases. The
non-complementary base on the newer DNA strand is excised, and the
excised base is replaced with the appropriate base that is
complementary to the older DNA strand. In this way, cells eliminate
many mutations, which occur as a result of mistakes in DNA
replication.
[0049] Dominant negative alleles cause a mismatch repair defective
phenotype even in the presence of a wild-type allele in the same
cell. An example of a dominant negative allele of a mismatch repair
gene is the human gene hPMS2-134, which carries a truncation
mutation at codon 134. Nicolaides et al., Mol. Cell. Biol., 1998,
18, 1635-1641. The mutation causes the product of this gene to
abnormally terminate at the position of the 134th amino acid,
resulting in a shortened polypeptide containing the N-terminal 133
amino acids. Such a mutation causes an increase in the rate of
mutations that accumulate in cells after DNA replication.
Expression of a dominant negative allele of a mismatch repair gene
results in impairment of mismatch repair activity, even in the
presence of the wild-type allele. Any allele, which produces such
effect, can be used in this invention.
[0050] Dominant negative alleles of a mismatch repair gene can be
obtained from the cells of humans, animals, yeast, bacteria, or
other organisms. Prolla et al., Science, 1994, 264, 1091 1093;
Strand et al., Nature, 1993, 365, 274 276; and Su et al., J. Biol.
Chem., 1988, 263, 6829 6835. Screening cells for defective mismatch
repair activity can identify such alleles. Cells from animals or
humans with cancer can be screened for defective mismatch repair.
Cells from colon cancer patients may be particularly useful.
Parsons et al., Cell, 1993, 75, 1227 1236; and Papadopoulos et al.,
Science, 1993, 263, 1625 1629. Genomic DNA, cDNA, or mRNA from any
cell encoding a mismatch repair protein can be analyzed for
variations from the wild type sequence. Perucho, Biol. Chem., 1996,
377, 675 684. Dominant negative alleles of a mismatch repair gene
can also be created artificially, for example, by producing
variants of the hPMS2-134 allele or other mismatch repair genes.
Various techniques of site-directed mutagenesis can be used. The
suitability of such alleles, whether natural or artificial, for use
in generating hypermutable cells or animals can be evaluated by
testing the mismatch repair activity caused by the allele in the
presence of one or more wild-type alleles, to determine if it is a
dominant negative allele.
[0051] A cell or an animal into which a dominant negative allele of
a mismatch repair gene has been introduced will become
hypermutable. This means that the spontaneous mutation rate of such
cells or animals is elevated compared to cells or animals without
such alleles. The degree of elevation of the spontaneous mutation
rate can be at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold,
100-fold, 200-fold, 500-fold, or 1000-fold that of the normal cell
or animal.
[0052] According to one aspect of the invention, a polynucleotide
encoding a dominant negative form of a mismatch repair protein is
introduced into a cell. The gene can be any dominant negative
allele encoding a protein that is part of a mismatch repair
complex, for example, PMS2, PMS1, MLH1, or MSH2. The dominant
negative allele can be naturally occurring or made in the
laboratory. The polynucleotide can be in the form of genomic DNA,
cDNA, RNA, or a chemically synthesized polynucleotide.
[0053] The polynucleotide can be cloned into an expression vector
containing a constitutively active promoter segment (such as but
not limited to CMV, SV40, Elongation Factor or LTR sequences) or to
inducible promoter sequences such as the steroid inducible pIND
vector (InVitrogen), where the expression of the dominant negative
mismatch repair gene can be regulated. The polynucleotide can be
introduced into the cell by transfection.
[0054] Transfection is any process whereby a polynucleotide is
introduced into a cell. The process of transfection can be carried
out in a living animal, e.g., using a vector for gene therapy, or
it can be carried out in vitro, e.g., using a suspension of one or
more isolated cells in culture. The cell can be any type of
eukaryotic cell, including, for example, cells isolated from humans
or other primates, mammals or other vertebrates, invertebrates, and
single celled organisms such as protozoa, yeast, or bacteria.
[0055] In general, transfection will be carried out using a
suspension of cells, or a single cell, but other methods can also
be applied as long as a sufficient fraction of the treated cells or
tissue incorporates the polynucleotide so as to allow transfected
cells to be grown and utilized. The protein product of the
polynucleotide may be transiently or stably expressed in the cell.
Techniques for transfection are well known and available techniques
for introducing polynucleotides include but are not limited to
electroporation, transduction, cell fusion, the use of calcium
chloride, and packaging of the polynucleotide together with lipid
for fusion with the cells of interest. Once a cell has been
transfected with the mismatch repair gene, the cell can be grown
and reproduced in culture. If the transfection is stable, such that
the gene is expressed at a consistent level for many cell
generations, then a cell line results.
[0056] An isolated cell is a cell obtained from a tissue of humans
or animals by mechanically separating out individual cells and
transferring them to a suitable cell culture medium, either with or
without pretreatment of the tissue with enzymes, e.g., collagenase
or trypsin. Such isolated cells are typically cultured in the
absence of other types of cells. Cells selected for the
introduction of a dominant negative allele of a mismatch repair
gene may be derived from a eukaryotic organism in the form of a
primary cell culture or an immortalized cell line, or may be
derived from suspensions of single-celled organisms.
[0057] The invention described herein is useful for creating
microbial-resistant mammalian cells that secrete new antimicrobial
biochemical agents, either protein or non-protein in nature.
Furthermore, the invention can be applied to cell lines that
express known antimicrobial agents as a means to enhance the
biochemical activity of the antimicrobial agent.
[0058] Once a transfected cell line has been produced, it can be
used to generate new mutations in one or more gene(s) of interest
or in genes that have not been previously described. A gene of
interest can be any gene naturally possessed by the cell line or
introduced into the cell line by standard methods known in the art.
An advantage of using transfected cells or to induce, mutation(s)
in a gene or genes of interest that encode antimicrobial activity
is that the cell need not be exposed to mutagenic chemicals or
radiation, which may have secondary harmful effects, both on the
object of the exposure and on the workers. Furthermore, it has been
demonstrated that chemical and physical mutagens are base pair
specific in the way they alter the structure of DNA; the invention
described herein results in mutations that are not dependent upon
the specific nucleotide or a specific string of nucleotides and is
a truly random genetic approach. Therefore, use of the present
invention to obtain mutations in novel or known antimicrobial genes
will be much more efficient and have a higher likelihood of success
in contrast to conventional mutagenesis with chemical or
irradiation. Once a new antimicrobial trait is identified in a
sibling cell, the dominant negative allele can be removed from the
cell by a variety of standard methods known in the art. For
example, the gene can be directly knocked out the allele by
technologies used by those skilled in the art or use of a inducible
expression system; the dominant-negative allele is driven by a
standard promoter that is regulated by inclusion of an inducer,
withdrawal of the inducer results in attenuation of the expression
of the dominant negative mismatch repair mutant and a normal DNA
repair process will ensue.
[0059] New antimicrobial agents are selected from cells that have
been exposed to the dominant negative mismatch repair process
followed by incubating the mutant cells in the presence of the
microbe for which an novel antimicrobial agent is sought. The novel
antimicrobial agent is purified by standard methods known to those
skilled in the art and characterized. The antimicrobial agents are
re-screened to determine the specific activity of the novel
antimicrobial as well as tested against a broad range of microbes
to determine spectrum of activity. The gene(s) that encode the
novel antimicrobial are isolated by standard well known methods to
those in the art. The mutations can be detected by analyzing for
alterations in the genotype of the cells by examining the sequence
of genomic DNA, cDNA, messenger RNA, or amino acids associated with
the gene of interest. A mutant polypeptide can be detected by
identifying alterations in electrophoretic mobility, spectroscopic
properties, or other physical or structural characteristics of a
protein encoded by a mutant gene when the cell that has undergone
alteration encodes a known antimicrobial that is altered by the
means described in the current invention to obtain a more
efficacious antimicrobial. Examples of mismatch repair proteins and
nucleic acid sequences include the following:
1 PMS2 (mouse) (SEQ ID NO:7) MEQTEGVSTE CAKAIKPIDG KSVHQICSGQ
VILSLSTAVK ELIENSVDAG ATTIDLRLKD 60 YGVDLTEVSD NGCGVEEENF
EGLALKHHTS KIQEFADLTQ VETFGERGEA LSSLCALSDV 120 TISTCHGSAS
VGTRLVFDHN GKITQKTPYP RPKGTTVSVQ HLFYTLPVRY KEFQRNIKKE 180
YSKMVQVLQA YCIISAGVRV SCTNQLGQGK RHAVVCTSGT SGMKENIGSV FGQKQLQSLI
240 PFVQLPPSDA VCEEYGLSTS GRHKTFSTFR ASFESARTAP GGVQQTGSFS
SSIRGPVTQQ 300 RSLSLSMRFY HMYNRHQYPF VVLNVSVDSE CVDINVTPDK
RQILLQEEKL LLAVLKTSLI 360 GMFDSDANKL NVNQQPLLDV EGNLVKLHTA
ELEKPVPGKQ DNSPSLKSTA DEKRVASISR 420 LREAFSLHPT KEIKSRGPET
AELTRSFPSE KRGVLSSYPS DVISYRGLRG SQDKLVSPTD 480 SPGDCMDREK
IEKDSGLSST SAGSEEEFST PEVASSFSSD YNVSSLEDRP SQETINCGDL 540
DCRPPGTGQS LKPEDHGYQC KALPLARLSP TNAKRFKTEE RPSNVNISQR LPGPQSTSAA
600 EVDVAIKMNK RIVLLEFSLS SLAKRMKQLQ HLKAQNKHEL SYRKFRAKIC
PGENQAAEDE 660 LRKEISKSMF AEMEILGQFN LGFTVTKLKE DLFLVDQHAA
DEKYNFEMLQ QHTVLQAQRL 720 ITPQTLNLTA VNEAVLIENL EIFRKNGFDF
VTDEDAPVTE RAKLISLPTS KNWTFGPQDI 780 DELIFMLSDS PGVMCRPSRV
RQMFASRACR KSVMIGTALN ASEMKKLITH MGEMDHPWNC 840 PHGRPTMRHV
ANLDVISQN 859 PMS2 (mouse cDNA) (SEQ ID NO:8) gaattccggt gaaggtcctg
aagaatttcc agattcctga gtatcattgg aggagacaga 60 taacctgtcg
tcaggtaacg atggtgtata tgcaacagaa atgggtgttc ctggagacgc 120
gtcttttccc gagagcggca ccgcaactct cccgcggtga ctgtgactgg aggagtcctg
180 catccatgga gcaaaccgaa ggcgtgagta cagaatgtgc taaggccatc
aagcctattg 240 atgggaagtc agtccatcaa atttgttctg ggcaggtgat
actcagttta agcaccgctg 300 tgaaggagtt gatagaaaat agtgtagatg
ctggtgctac tactattgat ctaaggctta 360 aagactatgg ggtggacctc
attgaagttt cagacaatgg atgtggggta gaagaagaaa 420 actttgaagg
tctagctctg aaacatcaca catctaagat tcaagagttt gccgacctca 480
cgcaggttga aactttcggc tttcgggggg aagctctgag ctctctgtgt gcactaagtg
540 atgtcactat atctacctgc cacgggtctg caagcgttgg gactcgactg
gtgtttgacc 600 ataatgggaa aatcacccag aaaactccct acccccgacc
taaaggaacc acagtcagtg 660 tgcagcactt attttataca ctacccgtgc
gttacaaaga gtttcagagg aacattaaaa 720 aggagtattc caaaatggtg
caggtcttac aggcgtactg tatcatctca gcaggcgtcc 780 gtgtaagctg
cactaatcag ctcggacagg ggaagcggca cgctgtggtg tgcacaagcg 840
gcacgtctgg catgaaggaa aatatcgggt ctgtgtttgg ccagaagcag ttgcaaagcc
900 tcattccttt tgttcagctg ccccctagtg acgctgtgtg tgaagagtac
ggcctgagca 960 cttcaggacg ccacaaaacc ttttctacgt ttcgggcttc
atttcacagt gcacgcacgg 1020 cgccgggagg agtgcaacag acaggcagtt
tttcttcatc aatcagaggc cctgtgaccc 1080 agcaaaggtc tctaagcttg
tcaatgaggt tttatcacat gtataaccgg catcagtacc 1140 catttgtcgt
ccttaacgtt tccgttgact cagaatgtgt ggatattaat gtaactccag 1200
ataaaaggca aattctacta caagaagaga agctattgct ggccgtttta aagacctcct
1260 tgataggaat gtttgacagt gatgcaaaca agcttaatgt caaccagcag
ccactgctag 1320 atgttgaagg taacttagta aagctgcata ctgcagaact
agaaaagcct gtgccaggaa 1380 agcaagataa ctctccttca ctgaagagca
cagcagacga gaaaagggta gcatccatct 1440 ccaggctgag agaggccttt
tctcttcatc ctactaaaga gatcaagtct aggggtccag 1500 agactgctga
actgacacgg agttttccaa gtgagaaaag gggcgtgtta tcctcttatc 1560
cttcagacgt catctcttac agaggcctcc gtggctcgca ggacaaattg gtgagtccca
1620 cggacagccc tggtgactgt atggacagag agaaaataga aaaagactca
gggctcagca 1680 gcacctcagc tggctctgag gaagagttca gcaccccaga
agtggccagt agctttagca 1740 gtgactataa cgtgagctcc ctagaagaca
gaccttctca ggaaaccata aactgtggtg 1800 acctggactg ccgtcctcca
ggtacaggac agtccttgaa gccagaagac catggatatc 1860 aatgcaaagc
tctacctcta gctcgtctgt cacccacaaa tgccaagcgc ttcaagacag 1920
aggaaagacc ctcaaatgtc aacatttctc aaagattqcc tggtcctcag agcacctcag
1980 cagctgaggt cgatgtagcc ataaaaatga ataagagaat cgtgctcctc
gagttctctc 2040 tgagttctct agctaagcga atgaagcagt tacagcacct
aaaggcgcag aacaaacatg 2100 aactgagtta cagaaaattt agggccaaga
tttgccctgg agaaaaccaa gcagcagaag 2160 atgaactcag aaaagagatt
agtaaatcga tgtttgcaga gatggagatc ttgggtcagt 2220 ttaacctggg
atttatagta accaaactga aagaggacct cttcctggtg gaccagcatg 2280
ctgcggatga gaagtacaac tttgagatgc tgcagcagca cacggtgctc caggcgcaga
2340 ggctcatcac accccagact ctgaacttaa ctgctgtcaa tgaaqctgta
ctgatagaaa 2400 atctggaaat attcagaaag aatggctttg actttqtcat
tgatgaggat gctccagtca 2460 ctgaaagggc taaattgatt tccttaccaa
ctagtaaaaa ctggaccttt ggaccccaag 2520 atatagatga actgatcttt
atgttaagtg acagccctgg ggtcatgtgc cggccctcac 2580 gagtcagaca
gatgtttgct tccagagcct gtcggaagtc agtgatgatt ggaacggcgc 2640
tcaatgcgag cgagatgaag aagctcatca cccacatggg tgagatggac cacccctgga
2700 actgccccca cggcaggcca accatgaggc acgttgccaa tctggatgtc
atctctcaga 2760 actgacacac cccttgtagc atagagttta ttacagattg
ttcggtttgc aaagagaagg 2820 ttttaagtaa tctgattatc gttgtacaaa
aattagcatg ctgctttaat gtactggatc 2880 catttaaaag cagtgttaag
gcaggcatga tggagtgttc ctctagctca gctacttggg 2940 tgatccggtg
ggagctcatg tgagcccagg actttgagac cactccgagc cacattcatg 3000
agactcaatt caaggacaaa aaaaaaaaga tatttttgaa gccttttaaa aaaaaa 3056
PMS2 (human) (SEQ ID NO:9) MERAESSSTE PAKAIKPIDR KSVHQICSGQ
VVLSLSTAVK ELVENSLDAG ATNIDLKLKD 60 YGVDLIEVSD NGCGVEEENF
EGLTLKHHTS KIQEFADLTQ VETFGFRGEA LSSLCALSDV 120 TISTCHASAK
VGTRLMFDHN GKIIQKTPYP RPRGTTVSVQ QLFSTLPVRH KEFQRNIKKE 180
YAKMVQVLHA YCIISAGIRV SCTNQLGQGK RQPVVCTGGS PSIKENIGSV FGQKQLQSLI
240 PFVQLPPSDS VCEEYGLSCS DALHNLFYIS GFISQCTHGV GRSSTDRQFF
FINRRPCDPA 300 KVCRLVNEVY HMYNRHQYPF VVLNISVDSE CVDINVTPDK
RQILLQEEKL LLAVLKTSLI 360 GMFDSDVNKL NVSQQPLLDV EGNLIKMHAA
DLEKPMVEKQ DQSPSLRTGE EKKDVSISRL 420 REAFSLRHTT ENKPHSPKTP
EPRRSPLGQK RGMLSSSTSG AISDKGVLRP QKEAVSSSHG 480 PSDPTDRAEV
EKDSGHGSTS VDSEGFSIPD TGSHCSSEYA ASSPGDRGSQ EHVDSQEKAP 540
ETDDSFSDVD CHSNQEDTGC KFRVLPQPTN LATPNTKRFK KEEILSSSDI CQKLVNTQDM
600 SASQVDVAVK INKKVVPLDF SMSSLAKRIK QLHHEAQQSE GEQNYRKFRA
KICPGENQAA 660 EDELRKEISK TMFAEMEIIG QFNLGFIITK LNEDIFIVDQ
HATDEKYNFE MLQQHTVLQG 720 QRLIAPQTLN LTAVNEAVLI ENLETFRKNG
FDFVIDENAP VTERAKLISL PTSKNWTFGP 780 QDVDELIFML SDSPGVMCRP
SRVKQMFASR ACRKSVMIGT ALNTSEMKKL ITHMGEMDHP 840 WNCPHGRPTM
RHIANLGVIS QN 862 PMS2 (human cDNA) (SEQ ID NO:10) cgaggcggat
cgggtgttgc atccatggag cgagctgaga gctcgagtac agaacctgct 60
aaggccatca aacctattga tcggaagtca gtccatcaga tttgctctgg gcaggtggta
120 ctgagtctaa gcactgcggt aaaggagtta gtagaaaaca gtctggatgc
tggtgccact 180 aatattgatc taaagcttaa ggactatgga gtggatctta
ttgaagtttc agacaatgga 240 tgtggggtag aagaagaaaa cttcgaaggc
ttaactctga aacatcacac atctaagatt 300 caagagtttg ccgacctaac
tcaggttgaa acttttggct ttcgggggga agctctgagc 360 tcactttgtg
cactgagcga tgtcaccatt tctacctgcc acgcatcggc gaaggttgga 420
actcgactga tgtttgatca caatgggaaa attatccaga aaacccccta cccccgcccc
480 agagggacca cagtcagcgt gcagcagtta ttttccacac tacctgtgcg
ccataaggaa 540 tttcaaagga atattaagaa ggagtatgcc aaaatggtcc
aggtcttaca tgcatactgt 600 atcatttcag caggcatccg tgtaagttgc
accaatcagc ttggacaagg aaaacgacag 660 cctgtggtat gcacaggtgg
aagccccagc ataaaggaaa atatcggctc tgtgtttggg 720 cagaagcagt
tgcaaagcct cattcctttt gttcagctgc cccctagtga ctccgtgtgt 780
gaagagtacg gtttgagctg ttcggatgct ctgcataatc ttttttacat ctcaggtttc
840 atttcacaat gcacgcatgg agttggaagg agttcaacag acagacagtt
tttctttatc 900 aaccggcggc cttgtgaccc agcaaaggtc tgcagactcg
tgaatgaggt ctaccacatg 960 tataatcgac accagtatcc atttgttgtt
cttaacattt ctgttgattc agaatgcgtt 1020 gatatcaatg ttactccaga
taaaaggcaa attttgctac aagaggaaaa gcttttgttg 1080 gcagttttaa
agacctcttt gataggaatg tttgatagtg atgtcaacaa gctaaatgtc 1140
agtcagcagc cactgctgga tgttgaaggt aacttaataa aaatgcatgc agcggatttg
1200 gaaaagccca tggtagaaaa gcaggatcaa tccccttcat taaggactgg
agaagaaaaa 1260 aaagacgtgt ccatttccag actgcgagag gccttttctc
ttcgtcacac aacagagaac 1320 aagcctcaca gcccaaagac tccagaacca
agaaggagcc ctctaggaca gaaaaggggt 1380 atgctgtctt ctagcacttc
aggtgccatc tctgacaaag gcgtcctgag acctcagaaa 1440 gaggcagtga
gttccagtca cggacccagt gaccctacgg acagagcgga ggtqgagaag 1500
gactcggggc acggcagcac ttccgtggat tctgaggggt tcagcatccc agacacgggc
1560 agtcactgca gcagcgagta tgcggccagc tccccagggg acaggggctc
gcaggaacat 1620 gtggactctc aggagaaagc gcctgaaact gacgactctt
tttcagatgt ggactgccat 1680 tcaaaccagg aagataccgg atgtaaattt
cgagttttgc ctcagccaac taatctcgca 1740 accccaaaca caaagcgttt
taaaaaagaa gaaattcttt ccagttctga catttgtcaa 1800 aagttagtaa
atactcagga catgtcagcc tctcaggttg atgtagctgt gaaaattaat 1860
aagaaagttg tgcccctgga cttttctatg agttctttag ctaaacgaat aaagcagtta
1920 catcatgaag cacagcaaag tgaaggggaa cagaattaca ggaagtttag
ggcaaagatt 1980 tgtcctggag aaaatcaagc agccgaagat gaactaagaa
aagagataag taaaacgatg 2040 tttgcagaaa tqgaaatcat tggtcagttt
aacctgggat ttataataac caaactgaat 2100 gaggatatct tcatagtgga
ccagcatgcc acggacgaga agtataactt cgagatgctg 2160 cagcagcaca
ccgtgctcca ggggcagagg ctcatagcac ctcagactct caacttaact 2220
gctgttaatg aagctgttct gatagaaaat ctggaaatat ttagaaagaa tggctttgat
2280 tttgttatcg atgaaaatgc tccagtcact gaaagggcta aactgatttc
cttgccaact 2340 agtaaaaact ggaccttcgg acaccaggac gtcgatgaac
tgatcttcat gctgagcgac 2400 agccctgggg tcatgtgccg gccttcccga
gtcaagcaga tgtttgcctc cagagcctgc 2460 cggaagtcgg tgatgattgg
gactgctctt aacacaagcg agatgaagaa actgatcacc 2520 cacatggggg
agatggacca cccctggaac tgtccccatg gaaggccaac catgagacac 2580
atcgccaacc tgggtgtcat ttctcagaac tgaccgtagt cactgtatgg aataattggt
2640 tttatcgcag atttttatgt tttgaaagac agagtcttca ctaacctttt
ttgttttaaa 2700 atgaaacctg ctacttaaaa aaaatacaca tcacacccat
ttaaaagtga tcttgagaac 2760 cttttcaaac c 2771 PMS 1 (human) (SEQ ID
NO:11) MKQLPAATVR LLSSSQIITS VVSVVKELIE NSLDAGATSV DVKLENYGFD
KIEVRDNGEG 60 IKAVDAPVMA MKYYTSKINS HEDLENLTTY GERGEALGSI
CCTAEVLITT RTAADNFSTQ 120 YVLDGSGHIL SQKPSHLGQG TTVTALRLFK
NLPVRKQFYS TAKKCKDEIK KIQDLLMSFG 180 ILKPDLRIVF VHNKAVIWQK
SRVSDHKMAL MSVLGTAVMN NMESFQYHSE ESQIYLSGFL 240 PKCDADHSFT
SLSTPERSFI FINSRPVHQK DILKLIRHHY NLKCLKESTR LYPVFFLKID 300
VPTADVDVNL TPDKSQVLLQ NKESVLIALE NLMTTCYGPL PSTNSYENNK TDVSAADIVL
360 SKTAETDVLF NKVESSGKNY SNVDTSVIPF QNDMHNDESG KNTDDCLNHQ
ISIGDFGYGH 420 CSSEISNIDK NTKNAFQDIS MSNVSWENSQ TEYSKTCFIS
SVKHTQSENG NKDHIDESGE 480 NEEEAGLENS SEISADEWSR GNILKNSVGE
NIEPVKILVP EKSLPCKVSN NNYPIPEQMN 540 LNEDSCNKKS NVIDNKSGKV
TAYDLLSNRV IKKPMSASAL FVQDHRPQFL IENPKTSLED 600 ATLQIEELWK
TLSEEEKLKY EEKATKDLER YNSQMKRAIE QESQMSLKDG RKKIKPTSAW 660
NLAQKHKLKT SLSNQPKLDE LLQSQIEKRR SQNIKMVQIP FSMKNLKINF KKQNKVDLEE
720 KDEPCLIHNL RFPDAWLMTS KTEVMLLNPY RVEEALLFKR LLENHKLPAE
PLEKPIMLTE 780 SLFNGSHYLD VLYKMTADDQ RYSGSTYLSD PRLTANGFKI
KLIPGVSITE NYLEIEGMAN 840 CLPFYGVADL KEILNAILNR NAKEVYECRP
RKVISYLEGE AVRLSRQLPM YLSKEDIQDI 900 IYRMKHQFGN EIKECVHGRP
FFHHLTYLPE TT 932 PMS 1 (human) (SEQ ID NO:12) ggcacgagtg
gctgcttgcg gctagtggat qgtaattgcc tgcctcgcgc tagcagcaag 60
ctgctctgtt aaaagcgaaa atgaaacaat tgcctgcggc aacagttcga ctcctttcaa
120 gttctcagat catcacttcg gtggtcagtg ttgtaaaaga gcttattgaa
aactccttgg 180 atgctggtgc cacaagcgta gatgttaaac tggagaacta
tggatttgat aaaattgagg 240 tgcgagataa cggggagggt atcaaqgctg
ttgatgcacc tgtaatggca atgaagtact 300 acacctcaaa aataaatagt
catgaagatc ttgaaaattt gacaacttac ggttttcgtg 360 gagaagcctt
ggggtcaatt tgttgtatag ctgaggtttt aattacaaca agaacggctg 420
ctgataattt tagcacccag tatgttttag atggcagtgg ccacatactt tctcagaaac
480 cttcacatct tggtcaaggt acaactgtaa ctgctttaag attatttaag
aatctacctg 540 taagaaagca gttttactca actgcaaaaa aatgtaaaga
tgaaataaaa aagatccaag 600 atctcctcat gagctttggt atccttaaac
ctgacttaag gattgtcttt gtacataaca 660 aggcagttat ttggcagaaa
agcagagtat cagatcacaa gatggctctc atgtcagttc 720 tggggactgc
tgttatgaac aatatggaat cctttcagta ccactctgaa gaatctcaga 780
tttatctcaq tggatttctt ccaaagtgtg atgcagacca ctctttcact agtctttcaa
840 caccagaaag aagtttcatc ttcataaaca gtcgaccagt acatcaaaaa
gatatcttaa 900 agttaatccg acatcattac aatctgaaat gcctaaagga
atctactcgt ttgtatcctg 960 ttttctttct gaaaatcgat gttcctacag
ctgatgttga tgtaaattta acaccagata 1020 aaagccaagt attattacaa
aataaggaat ctgttttaat tgctcttgaa aatctgatga 1080 cgacttgtta
tggaccatta cctagtacaa attcttatga aaataataaa acagatgttt 1140
ccgcagctga catcgttctt agtaaaacag cagaaacaga tgtgcttttt aataaagtgg
1200 aatcatctgq aaagaattat tcaaatqttg atacttcagt cattccattc
caaaatgata 1260 tgcataatga tgaatctgga aaaaacactg atgattgttt
aaatcaccag ataagtattg 1320 gtgactttgg ttatggtcat tgtagtagtg
aaatttctaa cattgataaa aacactaaga 1380 atgcatttca ggacatttca
atgagtaatg tatcatggga gaactctcag acggaatata 1440 gtaaaacttg
ttttataagt tccgttaagc acacccagtc agaaaatggc aataaagacc 1500
atatagatga gagtggggaa aatgaggaag aagcaggtct tgaaaactct tcggaaattt
1560 ctgcagatga gtggagcagg ggaaatatac ttaaaaattc agtgggagag
aatattgaac 1620 ctgtgaaaat tttagtgcct gaaaaaagtt taccatgtaa
agtaagtaat aataattatc 1680 caatccctga acaaatgaat cttaatgaag
attcatgtaa caaaaaatca aatgtaatag 1740 ataataaatc tggaaaagtt
acagcttatg atttacttag caatcgagta atcaagaaac 1800 ccatgtcagc
aagtgctctt tttgttcaag atcatcgtcc tcagtttctc atagaaaatc 1860
ctaagactag tttagaggat gcaacactac aaattgaaga actgtggaag acattgagtg
1920 aagaggaaaa actgaaatat gaagagaagg ctactaaaga cttggaacga
tacaatagtc 1980 aaatgaagag agccattgaa caggagtcac aaatgtcact
aaaagatggc agaaaaaaga 2040 taaaacccac cagcgcatgg aatttggccc
agaagcacaa gttaaaaacc tcattatcta 2100 atcaaccaaa acttgatgaa
ctccttcagt cccaaattga aaaaagaagg agtcaaaata 2160 ttaaaatggt
acagatcccc ttttctatga aaaacttaaa aataaatttt aagaaacaaa 2220
acaaagttga cttagaagag aaggatgaac cttgcttgat ccacaatctc aggtttcctg
2280 atgcatggct aatgacatcc aaaacagagg taatgttatt aaatccatat
agagtagaag 2340 aagccctgct atttaaaaga cttcttgaga atcataaact
tcctgcagag ccactggaaa 2400 agccaattat gttaacagag agtcttttta
atggatctca ttatttagac gttttatata 2460 aaatgacagc agatgaccaa
agatacagtg gatcaactta cctgtctgat cctcgtctta 2520 cagcgaatgg
tttcaagata aaattgatac caggagtttc aattactgaa aattacttgg 2580
aaatagaagg aatggctaat tgtctcccat tctatggagt agcagattta aaagaaattc
2640 ttaatgctat attaaacaga aatgcaaagg aagtttatga atgtagacct
cgcaaagtga 2700 taagttattt agagggagaa gcagtgcgtc tatccagaca
attacccatg tacttatcaa 2760 aagaggacat ccaagacatt atctacagaa
tgaagcacca gtttggaaat gaaattaaag 2820 agtgtgttca tggtcgccca
ttttttcatc atttaaccta tcttccagaa actacatgat 2880 taaatatgtt
taagaagatt agttaccatt gaaattggtt ctgtcataaa acagcatgag 2940
tctggtttta aattatcttt gtattatgtg tcacatggtt attttttaaa tgaggattca
3000 ctgacttgtt tttatattga aaaaagttcc acgtattgta gaaaacgtaa
ataaactaat 3060 3063 aac 3063 MSH2 (human) (SEQ ID NO:13)
MAVQPKETLQ LESAAEVGFV RFFQGMPEKP TTTVRLFDRG DFYTAHGEDA LLAAREVFKT
60 QGVIKYMGPA GAKNLQSVVL SKMNFESFVK DLLLVRQYRV EVYKNRAGNK
ASKENDWYLA 120 YKASPGNLSQ FEDILFGNND MSASIGVVGV KMSAVDGQRQ
VGVGYVDSIQ RKLGLGEFPD 180 NDQFSNLEAL LIQIGPKECV LPGGETAGDM
GKLRQIIQRG GILITERKKA DFSTKDIYQD 240
LNRLLKGKKG EQMNSAVLPE MENQVAVSSL SAVIKFLELL SDDSNFGQFE LTTFDFSQYM
300 KLDIAAVRAL NLFQGSVEDT TGSQSLAALL NKCKTPQGQR LVNQWIKQPL
MDKNRIEERL 360 NLVEAFVEDA ELRQTLQEDL LRRFPDLNRL AKKFQRQAAN
LQDGYRLYQG INQLPNVIQA 420 LEKHEGKHQK LLLAVFVTPL TDLRSDFSKF
QEMIETTLDM DQVENHEFLV KPSFDPNLSE 480 LREIMNDLEK KMQSTLTSAA
RDLGLDPGKQ IKLDSSAQFG YYFRVTCKEE KVLRNNKNFS 540 TVDIQKNGVK
FTNSKLTSLN EEYTKNKTEY EEAQDAIVKE IVNISSGYVE PMQTLNDVLA 600
QLDAVVSFAH VSNGAPVPYV RPAILEKGQG RIILKASRHA CVEVQDEIAF IPNDVYFEKD
660 KQMFHIITGP NMGGKSTYTR QTGVIVLMAQ IGCFVPCESA EVSIVDCILA
RVGAGDSQLK 720 GVSTFMAEML ETASILRSAT KDSLITIDEL GRGTSTYDGF
GLAWAISEYI ATKIGAFCMF 780 ATHFHELTAL ANQIPTVNNL HVTALTTEET
LTMLYQVKKG VCDQSFGIHV AELANFPKHV 840 IECAKQKALE LEEFQYIGES
QGYDIMEPAA KKCYLEREQG EKIIQEFLSK VKQMPFTEMS 900 EENITIKLKQ
LKAEVIAKNN SFVNEIISRI KVTT 934 MSH2 (human cDNA) (SEQ ID NO:14)
ggcgggaaac agcttagtgg gtgtggggtc gcgcattttc ttcaaccagg aggtgaggag
60 gtttcgacat ggcggtgcag ccgaaggaga cgctgcagtt ggagagcgcg
gccgaggtcg 120 gcttcgtgcg cttctttcag ggcatgccgg agaagccgac
caccacagtg cgccttttcg 180 accggggcga cttctatacg gcgcacggcg
aggacgcgct gctggccgcc cgggaggtgt 240 tcaagaccca gggggtgatc
aagtacatgg ggccggcagg agcaaagaat ctgcagagtg 300 ttgtgcttag
taaaatgaat tttgaatctt ttgtaaaaga tcttcttctg gttcgtcagt 360
atagagttga agtttataag aatagagctg gaaataaggc atccaaggag aatgattggt
420 atttggcata taaggcttct cctggcaatc tctctcagtt tgaagacatt
ctctttggta 480 acaatgatat gtcagcttcc attggtgttg tgggtgttaa
aatgtccgca gttgatggcc 540 agagacaggt tggagttggg tatgtggatt
ccatacagag gaaactagga ctgtgtgaat 600 tccctgataa tgatcagttc
tccaatcttg aggctctcct catccagatt ggaccaaagg 660 aatgtgtttt
acccggagga gagactgctg gagacatggg gaaactgaga cagataattc 720
aaagaggagg aattctgatc acagaaagaa aaaaagctga cttttccaca aaagacattt
780 atcaggacct caaccggttg ttgaaaggca aaaagggaga gcagatgaat
agtgctgtat 840 tgccagaaat ggagaatcag gttgcagttt catcactgtc
tgcggtaatc aagtttttag 900 aactcttatc agatgattcc aactttggac
agtttgaact gactactttt gacttcagcc 960 agtatatgaa attggatatt
gcagcagtca gagcccttaa cctttttcag ggttctgttg 1020 aagataccac
tggctctcag tctctggctg ccttgctgaa taagtgtaaa acccctcaag 1080
gacaaagact tgttaaccag tggattaagc agcctctcat ggataagaac agaatagagg
1140 agagattgaa tttagtggaa gcttttgtag aagatgcaga attgaggcag
actttacaag 1200 aagatttact tcgtcgattc ccagatctta accgacttgc
caagaagttt caaagacaag 1260 cagcaaactt acaagattgt taccgactct
atcagggtat aaatcaacta cctaatgtta 1320 tacaggctct ggaaaaacat
gaaggaaaac accagaaatt attgttggca gtttttgtga 1380 ctcctcttac
tgatcttcgt tctgacttct ccaagtttca ggaaatgata gaaacaactt 1440
tagatatgga tcaggtggaa aaccatgaat tccttgtaaa accttcattt gatcctaatc
1500 tcagtgaatt aagagaaata atgaatgact tggaaaagaa gatgcagtca
acattaataa 1560 gtgcagccag agatcttggc ttggaccctg gcaaacagat
taaactggat tccagtgcac 1620 agtttggata ttactttcgt gtaacctgta
aggaagaaaa agtccttcgt aacaataaaa 1680 actttagtac tgtagatatc
cagaagaatg gtgttaaatt taccaacagc aaattgactt 1740 ctttaaatga
agagtatacc aaaaataaaa cagaatatga agaagcccag gatgccattg 1800
ttaaagaaat tgtcaatatt tcttcaggct atgtagaacc aatgcagaca ctcaatgatg
1860 tgttagctca gctagatgct gttgtcagct ttgctcacgt gtcaaatgga
gcacctgttc 1920 catatgtacg accagccatt ttggagaaag gacaaggaag
aattatatta aaagcatcca 1980 ggcatgcttg tgttgaagtt caagatgaaa
ttgcatttat tcctaatgac gtatactttg 2040 aaaaagataa acagatgttc
cacatcatta ctggccccaa tatgggaggt aaatcaacat 2100 atattcgaca
aactggggtg atagtactca tggcccaaat tgggtgtttt gtgccatgtg 2160
agtcagcaga agtgtccatt gtggactgca tcttagcccg agtaggggct ggtgacagtc
2220 aattgaaagg agtctccacg ttcatggctg aaatgttgga aactgcttct
atcctcaggt 2280 ctgcaaccaa agattcatta ataatcatag atgaattggg
aagaggaact tctacctacg 2340 atggatttgg gttagcatgg gctatatcag
aatacattgc aacaaagatt ggtgcttttt 2400 gcatgtttgc aacccatttt
catgaactta ctgccttggc caatcagata ccaactgtta 2460 ataatctaca
tgtcacagca ctcaccactg aagagacctt aactatgctt tatcaggtga 2520
agaaaggtgt ctgtgatcaa agttttqgga ttcatgttgc agagcttgct aatttcccta
2580 agcatgtaat agagtgtgct aaacagaaag ccctggaact tgaggagttt
cagtatattg 2640 gagaatcgca aggatatgat atcatggaac cagcagcaaa
gaagtgctat ctggaaagag 2700 agcaaggtga aaaaattatt caggagttcc
tgtccaaggt gaaacaaatg ccctttactg 2760 aaatgtcaga agaaaacatc
acaataaagt taaaacagct aaaagctgaa gtaatagcaa 2820 agaataatag
ctttgtaaat gaaatcattt cacgaataaa agttactacg tgaaaaatcc 2880
cagtaatgga atgaaggtaa tattgataag ctattgtctg taatagtttt atattgtttt
2940 atattaaccc tttttccata gtgttaactg tcagtgccca tgggctatca
acttaataag 3000 atatttagta atattttact ttgaggacat tttcaaagat
ttttattttg aaaaatgaga 3060 gctgtaactg aqgactgttt gcaattgaca
taggcaataa taagtgatgt gctgaatttt 3120 ataaataaaa tcatgtagtt tgtgg
3145 MLH1 (human) (SEQ ID NO:15) MSFVAGVIRR LDETVVNRIA AGEVIQRPAN
AIKEMIENCL DAKSTSTQVI VKEGGLKLIQ 60 IQDNGTGIRK EDLDIVCERF
TTSKLQSFED LASISTYGFR GEALASISHV AHVTITTKTA 120 DGKCAYRASY
SDGKLKAPPK PCAGNQGTQI TVEDLFYNIA TRRKALKNPS EEYGKILEVV 180
GRYSVHNAGI SFSVKKQGET VADVRTLPNA STVDNIRSIF GNAVSRELIE TGCEDKTLAF
240 KMNGYISNAN YSVKKCIFLL FINHRLVEST SLRKATETVY AAYLPKNTHP
FLYLSLEISP 300 QNVDVNVHPT KHEVHFLHEE SILERVQQHI ESKLLGSNSS
RMYFTQTLLP GLAGPSGEMV 360 KSTTSLTSSS TSGSSDKVYA HQMVRTDSRE
QKLDAFLQPL SKPLSSQPQA IVTEDKTDIS 420 SGRARQQDEE MLELPAPAEV
AAKNQSLEGD TTKGTSEMSE KRGPTSSNPR KRHREDSDVE 480 MVEDDSRKEM
TAACTPRRRI INLTSVLSLQ EEINEQGHEV LREMLHNHSF VGCVNPQWAL 540
AQHQTKLYLL NTTKLSEELF YQILTYDFAN FGVLRLSEPA PLFDLAMLAL DSPESGWTEE
600 DGPKEGLAEY IVEFLKKKAE MLADYFSLEI DEEGNLIGLP LLIDNYVPPL
EGLPIFILRL 660 ATEVNWDEEK ECFESLSKEC ANFYSIRKQY ISEESTLSGQ
QSEVPGSIPN SWKWTVEHIV 720 YKALRSHILP PKHFTEDGNI LQLANLPDLY KVFERC
756 MLH1 (human) (SEQ ID NO:16) cttggctctt ctggcgccaa aatgtcgttc
gtggcagggg ttattcggcg gctggacgag 60 acagtggtga accgcatcgc
ggcgggggaa gttatccagc ggccagctaa tgctatcaaa 120 gagatgattg
agaactgttt agatgcaaaa tccacaagta ttcaagtgat tgttaaagag 180
ggaggcctga agttgattca gatccaagac aatggcaccg ggatcaqgaa agaagatctg
240 gatattgtat gtgaaaggtt cactactagt aaactgcagt cctttgagga
tttagccagt 300 atttctacct atggctttcg aggtgaggct ttggccagca
taagccatgt ggctcatgtt 360 actattacaa cgaaaacagc tgatggaaag
tgtgcataca gagcaagtta ctcagatgga 420 aaactgaaag cccctcctaa
accatgtgct ggcaatcaag ggacccagat cacggtggag 480 gacctttttt
acaacatagc cacgaggaga aaagctttaa aaaatccaag tgaagaatat 540
gggaaaattt tggaagttgt tggcaggtat tcagtacaca atgcaggcat tagtttctca
600 gttaaaaaac aaggagagac agtagctgat gttaggacac tacccaatgc
ctcaaccgtg 660 gacaatattc gctccatctt tggaaatgct gttagtcgag
aactgataga aattggatgt 720 gaggataaaa ccctagcctt caaaatgaat
ggttacatat ccaatgcaaa ctactcagtg 780 aagaagtgca tcttcttact
cttcatcaac catcgtctgg tagaatcaac ttccttgaga 840 aaagccatag
aaacagtgta tgcagcctat ttgcccaaaa acacacaccc attcctgtac 900
ctcagtttag aaatcagtcc ccagaatgtg gatgttaatg tgcaccccac aaagcatgaa
960 gttcacttcc tgcacgagga gagcatcctg gagcgggtgc agcagcacat
cgagagcaag 1020 ctcctgggct ccaattcctc caggatgtac ttcacccaga
ctttgctacc aggacttgct 1080 ggcccctctg gggagatggt taaatccaca
acaagtctga cctcgtcttc tacttctgga 1140 agtagtgata aggtctatgc
ccaccagatg gttcgtacag attcccggga acagaagctt 1200 gatgcatttc
tgcagcctct gagcaaaccc ctgtccagtc agccccaggc cattgtcaca 1260
gaggataaga cagatatttc tagtggcagg gctaggcagc aagatgagga gatgcttgaa
1320 ctcccagccc ctgctgaagt ggctgccaaa aatcagagct tggaggggga
tacaacaaag 1380 gggacttcag aaatgtcaga gaagagagga cctacttcca
gcaaccccag aaagagacat 1440 cgggaagatt ctgatgtgga aatggtggaa
gatgattccc gaaaggaaat gactgcagct 1500 tgtacccccc ggagaaggat
cattaacctc actagtgttt tgagtctcca ggaagaaatt 1560 aatgagcagg
gacatgaggt tctccgggag atgttgcata accactcctt cgtgggctgt 1620
gtgaatcctc agtgggcctt ggcacagcat caaaccaagt tataccttct caacaccacc
1680 aagcttagtg aagaactgtt ctaccagata ctcatttatg attttgccaa
ttttggtgtt 1740 ctcaggttat cggagccagc accgctcttt gaccttgcca
tgcttgcctt agatagtcca 1800 gagagtggct ggacagagga agatggtccc
aaagaaggac ttgctgaata cattgttgag 1860 tttctgaaga agaaggctga
gatgcttgca gactatttct ctttggaaat tgatgaggaa 1920 gggaacctga
ttggattacc ccttctgatt gacaactatg tgcccccttt ggagggactg 1980
cctatcttca ttcttcgact agccactgag gtgaattggg acgaagaaaa ggaatgtttt
2040 gaaagcctca gtaaagaatg cgctatgttc tattccatcc ggaagcagta
catatctgag 2100 gagtcgaccc tctcaggcca gcagagtgaa gtgcctggct
ccattccaaa ctcctggaag 2160 tggactgtgg aacacattgt ctataaagcc
ttgcgctcac acattctgcc tcctaaacat 2220 ttcacagaag atggaaatat
cctgcagctt gctaacctgc ctgatctata caaagtcttt 2280 gagaggtgtt
aaatatggtt atttatgcac tgtgggatgt gttcttcttt ctctgtattc 2340
cgatacaaag tgttgtatca aagtgtgata tacaaagtgt accaacataa gtgttggtag
2400 cacttaagac ttatacttgc cttctgatag tattccttta tacacagtgg
attgattata 2460 aataaataga tgtgtcttaa cata 2484 hPMS2-134 (human)
(SEQ ID NO:17) MERAESSSTE PAKAIKPIDR KSVHQICSGQ VVLSLSTAVK
ELVENSLDAG ATNIDLKLKD 60 YGVDLIEVSD NGCGVEEENF EGLTLKHHTS
KIQEFADLTQ VETFGFRGEA LSSLCALSDV 120 TISTCHASAK VGT 133 hPMS2-134
(human cDNA) (SEQ ID NO:18) cgaggcggat cgggtgttgc atccatggag
cgagctgaga gctcgagtac agaacctgct 60 aaggccatca aacctattga
tcggaagtca gtccatcaga tttgctctgg gcaggtggta 120 ctgagtctaa
gcactgcggt aaaggagtta gtagaaaaca gtctggatgc tggtgccact 180
aatattgatc taaagcttaa ggactatgga gtggatctta ttgaagtttc agacaatgga
240 tgtggggtag aagaagaaaa cttcgaaggc ttaactctga aacatcacac
atctaagatt 300 caagagtttg ccgacctaac tcaggttgaa acttttggct
ttcgggggga agctctgagc 360 tcactttgtg cactgagcga tgtcaccatt
tctacctgcc acgcatcggc gaaggttgga 420 acttga 426
[0060] The above disclosure generally describes the present
invention. A more complete understanding can be obtained by
reference to the following specific examples, which are provided
herein for purposes of illustration only, and are not intended to
limit the scope of the invention.
EXAMPLES
Example 1
hPMS2-134 Encodes a Dominant Negative Mismatch Repair Protein
[0061] A profound defect in MMR was found in the normal cells of
two HNPCC patients. That this defect was operative in vivo was
demonstrated by the widespread presence of microsatellite
instability in non neoplastic cells of such patients. One of the
two patients had a germ line truncating mutation of the hPMS2 gene
at codon 134 (the hPMS2 134 mutation), while the other patient had
a small germ line deletion within the hMLH1 gene. Leach et al.,
Cell, 1993, 75, 1215 1225. These data contradicted the two hit
model generally believed to explain the biochemical and biological
features of HNPCC patients. The basis for this MMR deficiency in
the normal cells of these patients was unclear, and several
potential explanations were offered. For example, it was possible
that the second allele of the relevant MMR gene was inactivated in
the germ line of these patients through an undiscovered mechanism,
or that unknown mutations of other genes involved in the MMR
process were present that cooperated with the known germ line
mutation. It is clear from knock out experiments in mice that MMR
deficiency is compatible with normal growth and development,
supporting these possibilities. Edelmann et al., Cell, 1996, 85,
1125 1134. Alternatively, it was possible that the mutant alleles
exerted a dominant-negative effect, resulting in MMR deficiency
even in the presence of the wild type allele of the corresponding
MMR gene and all other genes involved in the MMR process. To
distinguish between these possibilities, the truncated polypeptide
encoded by the hPMS2 134 mutation was expressed in an MMR
proficient cell line its affect on MMR activity was analyzed. The
results showed that this mutant could indeed exert a
dominant-negative effect, resulting in biochemical and genetic
manifestations of MMR deficiency. One embodiment of the present
invention is demonstrated in Table 1, where a Syrian hamster
fibroblast cell line (TK) was transfected with an expression vector
containing the hPMS2-134 (TK-PMS2-134) or the empty expression
vector (TKvect), which also contains the NEO gene as a selectable
marker. TK-PMS2-134 cells were determined to be stably expressing
the gene via western blot analysis (data not shown). Nuclear
lysates from hPMS2-134 and control cells were measured for the
ability to correct mismatched DNA substrates. As shown in Table 1,
TK-PMS2-134 cells had a dramatic decrease in repair activity while
TKvect control cells were able to repair mismatched DNA duplexes at
a rate of .about.4.0 fmol/15 minutes (p<0.01).
2TABLE 1 Relative endogenous MMR activity of MMR-proficient cells
expressing an ectopically expressed morphogene or an empty
expression vector 5' DNA Repair activity of G/T mismatch Cell Lines
(fmol/15 minutes) TKvect 1 3.5 2 2.9 3 5.5 TK-PMS2-134 1 0 2 0 3
0.5
[0062] These data show that the expression of the TK-PMS2-134
results in suppressed MMR of a host organism and allows for an
enhanced mutation rate of genetic loci with each mitosis.
Example 2
hPMS2-134 Can Alter Genes In Vivo
[0063] An example of the ability to alter mismatch repair comes
from experiments using manipulation of mismatch repair TK cells
(described above) that expressed the TK-hPMS2-134 mutant were used
by transfection of the mammalian expression construct containing a
defective .beta.-galactosidase gene (referred to as pCAR-OF) which
was transfected into TK-hPMS2-134 or TKvect cells as described
above. The pCAR OF vector consists of a .beta.-galactosidase gene
containing a 29-basepair poly-CA tract inserted at the 5' end of
its coding region, which causes the wild-type reading frame to
shift out-of-frame. This chimeric gene is cloned into the pCEP4,
which contains the constitutively active cytomegalovirus (CMV)
promoter upstream of the cloning site and also contains the
hygromycin-resistance gene that allows for selection of cells
containing this vector. The pCAR-OF reporter cannot generate
.beta.-galactosidase activity unless a frame-restoring mutation
(i.e., insertion or deletion) arises following transfection into a
host. Another reporter vector called pCAR-IF contains a
.beta.-galactosidase in which a 27-bp poly-CA repeat was cloned
into the same site as the pCAR-OF gene, but it is biologically
active because the removal of a single repeat restores the open
reading frame and produces a functional chimeric
.beta.-galactosidase polypeptide (not shown). In these experiments,
TK-hPMS2-134 and TKvect cells were transfected with the pCAR-OF
reporter vector and selected for 17 days in neomycin plus
hygromycin selection medium. After the 17 days, resistant colonies
were stained for .beta.-galactosidase production to determine the
number of clones containing a genetically altered
.beta.-galactosidase gene. All conditions produced a relatively
equal number of neomycin/hygromycin resistant cells, however, only
the cells expressing the TK-hPMS2-134 contained a subset of clones
that were positive for .beta.-galactosidase activity.
Representative results are shown in Table 2, which shows the data
from these experiments where cell colonies were stained in situ for
.beta.-galactosidase activity and scored for activity. Cells were
scored positive if the colonies turned blue in the presence of
X-gal substrate and scored negative if colonies remained white.
Analysis of triplicate experiments showed that a significant
increase in the number of functional .beta.-galactosidase positive
cells was found in the TK-hPMS2-134 cultures, while no
.beta.-galactosidase positive cells were seen in the control TKvect
cells.
3TABLE 2 Number of TKmorph and TKvect cells containing functional
.beta.-galactosidase activity % Clones with Cells White Colonies
Blue Colonies altered B-gal Tkvect 65 +/- 9 0 0/65 = 0% TK-PMS2-134
40 +/- 12 28 +/- 4 28/68 = 41%
[0064] TK-PMS2-134 40+/-12 28+/-4 28/68=41% TK-PMS2-134/pCAR-OF
clones that were pooled and expanded also showed a number of cells
that contained a functional .beta.-galactosidase gene. No
.beta.-galactosidase positive cells were observed in TKvect cells
transfected with the pCAR-OF vector. These data are shown in FIG. 1
where the dark staining in panel B represent .beta.-galactosidase
positive cells present in the TK-PMS2-134/pCAR-OF cultures while
none are found in the TKvect cells grown under similar conditions
(panel A). These data demonstrate the ability of the mutant
mismatch repair gene, hPMS2-134, to generate gene alterations in
vivo, which allows for the rapid screening of clones with altered
polypeptides exhibiting new biochemical features.
[0065] To confirm that alterations within the nucleotide sequences
of the .beta.-galactosidase gene was indeed responsible for the in
vivo .beta.-galactosidase activity present in TK-hPMS2-134 clones,
RNA was isolated from TK-hPMS2-134/pCAR-OF and TKvect/pCAR-OF and
the .beta.-galactosidase mRNA primary structure was examined by
reverse transcriptase polymerase chain reaction (RT-PCR)
amplification and sequencing. Sequence analysis of
.beta.-galactosidase message from TKvect cells found no structural
alterations in the input gene sequence. Analysis of the
.beta.-galactosidase message from TK-hPMS-134 cells found several
changes within the coding sequences of the gene. These sequence
alterations included insertion and deletions of the poly CA tract
in the amino terminus as expected. Other alterations included
insertions of sequences outside of the polyCA repeat as well as a
series of single base alterations contained throughout the length
of the gene.
[0066] A summary of the genetic alterations are given in FIG. 2
where a schematic representation of the .beta.-galactosidase gene
is shown with the regions and types of genetic alterations depicted
below.
[0067] Plasmids. The full length wild type hPMS2 cDNA was obtained
from a human Hela cDNA library as described in Strand et al.,
Nature, 1993, 365, 274 276, which is incorporated herein by
reference in its entirety. An hPMS2 cDNA containing a termination
codon at amino acid 134 was obtained via RT PCR from the patient in
which the mutation was discovered. Nicolaides et al., Mol. Cell.
Biol., 1998, 18, 1635-1641, which is incorporated herein by
reference in its entirety. The cDNA fragments were cloned into the
BamHI site into the pSG5 vector, which contains an SV40 promoter
followed by an SV40 polyadenylation signal. Nicolaides et al.,
Genomics, 1995, 29, 329 334, which is incorporated herein by
reference in its entirety. The pCAR reporter vectors described in
FIG. 1 were constructed as described in Palombo et al., Nature,
1994, 36, 417, which is incorporated herein by reference in its
entirety.
[0068] .beta.-galactosidase assay. Seventeen days following
transfection with pCAR, .beta.-galactosidase assays were performed
using 20 .mu.g of protein in 45 mM 2 mercaptoethanol, 1 mM
MgCl.sub.2, 0.1 M NaPO.sub.4 and 0.6 mg/ml Chlorophenol red
.beta.-D galatopyranoside (CPRG, Boehringer Mannheim). Reactions
were incubated for 1 hour, terminated by the addition of 0.5 M
Na.sub.2CO.sub.3, and analyzed by spectrophotometry at 576 nm.
Nicolaides et al., Mol. Cell. Biol., 1998, 18, 1635-1641. For in
situ .beta.-galactosidase staining, cells were fixed in 1%
glutaraldehyde in PBS and incubated in 0.15 M NaCl, 1 mM
MgCl.sub.2, 3.3 mM K.sub.4Fe(CN).sub.6, 3.3 mM K.sub.3Fe(CN).sub.6,
0.2%.times.Gal for 2 hours at 37.degree. C.
Example 3
hPMS2-134 Causes a Defect in MMR Activity
[0069] The differences in .beta.-galactosidase activity between
PMS2 WT and PMS2 134 transfected cells can be due to the PMS2 134
protein disturbing MMR activity resulting in a higher frequency of
mutation within the pCAR OF reporter and re establishing the ORF.
To directly test whether MMR was altered, a biochemical assay for
MMR with the individual clones described in Example 1 was employed.
Nuclear extracts were prepared from the clones and incubated with
heteroduplex substrates containing either a /CA.backslash.
insertion deletion or a G/T mismatch under conditions described
previously. The /CA.backslash. and G/T heteroduplexes were used to
test repair from the 3' and 5' directions, respectively. There was
a dramatic difference between the PMS2-134 expressing clones and
the other clones in these assays (Table 3).
4TABLE 3 MMR activity of nuclear extracts from SH clones or pooled
culturesa Cell Amt of repaired substrate (fmol/15 min) line
3'/CA.backslash. 3' G/T 5' G/T 3'/CTG.backslash. 5'/CTG.backslash.
SH clonesb PMS2- NOT Clone A 10.2 3.5 Clone B 12.7 2.9 Clone C 13.5
5.5 PMS2- WT Clone A 2.8 2.2 Clone B 5.7 4.8 Clone c 4.7 2.9 PMS2-
134 Clone A 2.5 0.0 Clone B 2.4 0.0 Clone C 5.0 0.5 Pooled cultures
PMS2- 2.07 .+-. 2.37 .+-. 0.37 3.45 .+-. 1.35 2.77 .+-. 1.37 NOT
0.09 PMS2- 1.65 .+-. 1.86 .+-. 0.57 1.13 .+-. 0.23 1.23 .+-. 0.65
WT 0.94 PMS2- 0.14 .+-. 0.0 .+-. 0.0 1.31 .+-. 0.66 0.0 .+-. 0.0
134 0.2 aThe extracts were tested for MMR activity with 24 fmol of
heteroduplex. bThese data represent similar results derived from
more than five independent experiments.
[0070] While all clones repaired substrates from the 3' direction
(/CA.backslash. heteroduplex), cells expressing the PMS2 134
polypeptide had very little 5' repair activity. A similar
directional defect in mismatch repair was evident with pooled
clones resulting from PMS2 134 transfection, or when the
heteroduplex contained a 2 4 base pair loop, examples of which are
shown in Table 3. A small decrease in MMR activity was observed in
the 3'/CA.backslash. PMS2-WT repair assays, perhaps a result of
interference in the biochemical assays by over-expression of the
PMS2 protein. No significant activity was caused by PMS2-WT in the
in situ .beta.-galactosidase assays, a result more likely to
reflect the in vivo condition.
[0071] Biochemical assays for mismatch repair. MMR activity in
nuclear extracts was performed as described, using 24 fmol of
substrate, in Bronner et al., Nature, 1994, 368, 258 261 and
Nicolaides et al., Mol. Cell. Biol., 1998, 18, 1635-1641, each of
which is incorporated herein by reference in its entirety.
Complementation assays were done by adding .about.100 ng of
purified MutL" or MutS" components to 100 .mu.g of nuclear extract,
adjusting the final KCl concentration to 100 mM. Bevins, Ciba
Found. Symp., 1994, 186, 250-69 and Alderson et al., Res.
Microbiol., 1993, 144, 665-72. The substrates used in these
experiments contain a strand break 181 nucleotides 5' or 125
nucleotides 3' to the mismatch. Values represent experiments
performed at least in duplicate.
Example 4
C-Terminus of hPMS2 Mediates Interaction Between hPMS2 and
hMLH1
[0072] To elucidate the mechanism by which hPMS2 134 affected MMR,
the interaction between hPMS2 and hMLH1 was analyzed. Previous
studies have shown that these two proteins dimerize to form a
functionally active complex. Bronner et al., Nature, 1994, 368, 258
261. Proteins were synthesized in vitro using reticulocyte lysates,
employing RNA generated from cloned templates. The full length
hMLH1 and hPMS2 proteins bound to each other and were co
precipitated with antibodies to either protein, as expected (data
not shown). To determine the domain of hPMS2 that bound to hMLH1,
the amino terminus (codons 1-134), containing the most highly
conserved domain among mutL proteins (Su et al., J. Biol. Chem.,
1988, 263, 6829 6835 and Edelmann et al., Cell, 1996, 85, 1125
1134), and the carboxyl terminus (codons 135-862) were separately
cloned and proteins produced in vitro in coupled transcription
translation reactions. FIGS. 3A, 3B, and 3C show a representative
immunoprecipitation of in vitro-translated hPMS2 and hMLH1
proteins. FIG. 3A shows labeled (indicated by an asterisk) or
unlabelled proteins incubated with an antibody to the C-terminus of
hPMS2 in lanes 1 to 3 and to hMLH1 in lanes 4 to 6. Lane 7 contains
a nonprogrammed reticulocyte lysate. PMS2-135 contains codons 135
to 862 of hPMS2. The major translation products of hPMS2 and hMLH1
are indicated. FIG. 3B shows labeled hPMS2-134 (containing codons
1-134 of hPMS2) incubated in the presence or absence of unlabelled
hMLH1 plus an antibody to hMLH1 (lanes 1 and 2, respectively). Lane
3 contains lysate from a nonprogrammed reticulolysate. FIG. 3C
shows labeled proteins incubated with an antibody to the N terminus
of hPMS2. Lane 6 contains a nonprogrammed reticulocyte lysate. In
both panels A and B, autoradiographs of immunoprecipitated products
are shown. When a 35S labelled, full-length hMLH1 protein (FIG. 3A,
lane 5) was mixed with the unlabelled carboxyl terminal hPMS2
polypeptide, a monoclonal antibody (mAb) to the carboxyl terminus
of hPMS2 efficiently immunoprecipitated the labeled hMLH1 protein
(lane 1). No hMLH1 protein was precipitated in the absence of hPMS2
(lane 2). Conversely, when the 35S labelled carboxyl terminus of
hPMS2 (lane 3) was incubated with unlabelled, full length hMLH1
protein, an anti hMLH1 mAb precipitated the hPMS2 polypeptide (lane
4). In the absence of the unlabelled hMLH1 protein, no hPMS2
protein was precipitated by this mAb (lane 6). The same antibody
failed to immunoprecipitate the amino terminus of hPMS2 (amino
acids 1 134) when mixed with unlabelled MLH1 protein (FIG. 3B, lane
1). This finding was corroborated by the converse experiment in
which radiolabelled hPMS2-134 (FIG. 3C, lane 1) was unable to
coprecipitate radiolabelled MLH1 when precipitations were done
using an N terminal hPMS2 antibody (FIG. 3C, lane 2) while this
antibody was shown to be able to coprecipitate MLH1 when mixed with
wild type hPMS2 (FIG. 3C, lane 4).
[0073] The initial steps of MMR are dependent on two protein
complexes, called MutS" and MutL". Drummond et al., Science, 1995,
268, 1909 1912. As the amino terminus of hPMS2 did not mediate
binding of hPMS2 to hMLH1, it was of interest to determine whether
it might instead mediate the interaction between the MutL" complex
(comprised of hMLH1 and hPMS2) and the MutS" complex (comprised of
MSH2 and GTBP). Because previous studies have demonstrated that
MSH2 and the MutL" components do not associate in solution, direct
hPMS2-134:MutS" interaction was unable to be assayed. A different
approach was used to address this issue, and attempted to
complement nuclear extracts from the various SH cell lines with
MutS" or MutL". If the truncated protein present in the PMS2-134
expressing SH cells was binding to MutS" and lowering its effective
concentration in the extract, then adding intact MutS" should
rescue the MMR defect in such extracts. FIG. 4 shows
complementation of MMR activity in transduced SH cells. Lysates
from pooled clones stably transduced with PMS2-NOT, PMS2-WT, or
PMS2-134 were complemented with purified MutS" or MutL" MMR
components by using the 5' G/T heteroduplex substrate. The values
are presented as the percentage of repair activity in each case
compared to that in lysates complemented with both purified MutL"
and MutS" components to normalize for repair efficiency in the
different lysate backgrounds. The values shown represent the
average of at least three different determinations. Purified MutS"
added to such extracts had no effect (FIG. 4). In contrast,
addition of intact MutL" to the extract completely restored
directional repair to the extracts from PMS2-134 cells (FIG.
4).
[0074] The results described above lead to several conclusions.
First, expression of the amino terminus of hPMS2 results in an
increase in microsatellite instability, consistent with a
replication error (RER) phenotype. That this elevated
microsatellite instability is due to MMR deficiency was proven by
evaluation of extracts from stably transduced cells. Interestingly,
the expression of PMS2-134 resulted in a polar defect in MMR, which
was only observed using heteroduplexes designed to test repair from
the 5' direction (no significant defect in repair from the 3'
direction was observed in the same extracts). Interestingly, cells
deficient in hMLH1 also have a polar defect in MMR, but in this
case preferentially affecting repair from the 3' direction. Huttner
et al., Pediatr. Res., 1999, 45, 785-94. It is known from previous
studies in both prokaryotes and eukaryotes that the separate
enzymatic components mediate repair from the two different
directions. Our results indicate a model in which 5' repair is
primarily dependent on hPMS2 while 3' repair is primarily dependent
on hMLH1. It is easy to envision how the dimeric complex between
PMS2 and MLH1 might set up this directionality. The combined
results also demonstrate that a defect in directional MMR is
sufficient to produce a RER+phenotype.
[0075] The dominant-negative function of the PMS2-134 polypeptide
can result from its binding to MLH1 and consequent inhibition of
MutL" function. This is based in part on the fact that the most
highly conserved domain of the PMS2 gene is located in its amino
terminus, and the only known biochemical partner for PMS2 is MLH1.
The binding studies revealed, however, that the carboxyl terminus
of PMS2, rather than the highly conserved amino terminus, actually
mediated binding to MLH1. This result is consistent with those
recently obtained in S. cerevisciae, in which the MLH 1 interacting
domain of PMS1 (the yeast homolog of human PMS2) was localized to
its carboxyl terminus. Leach et al., Cell, 1993, 75, 1215 1225. The
add back experiments additionally showed that the hPMS2-134 mutant
was not likely to mediate an interaction with the MutS" complex
(FIG. 4). The hPMS2-134 polypeptide does not inhibit the initial
steps in MMR, but rather interacts with and inhibits a downstream
component of the pathway, perhaps a nuclease required for repair
from the 5' direction.
[0076] The demonstration that the hPMS2-134 mutation can confer a
dominant-negative MMR defect to transfected cells helps to explain
the phenotype of the kindred in which this mutant was discovered.
Three individuals from this kindred were found to carry the
mutation, a father and his two children. Both children exhibited
microsatellite instability in their normal tissues and both
developed tumors at an early age, while the father had no evidence
of microsatellite instability in his normal cells and was
completely healthy at age 35. The only difference known to us with
respect to the MMR genes in this family is that the father's mutant
allele was expressed at lower levels than the wild type allele as
assessed by sequencing of RT PCR products of RNA from lymphocytes.
The children expressed both alleles at approximately equal levels.
The dominant negative attribute of the hPMS2-134 mutant may only be
manifest when it is present at sufficient concentrations (at least
equimolar) thus, explaining the absence of MMR deficiency in the
father. The reason for the differential expression of the hPMS2-134
allele in this kindred is not clear, though imprinting is a
possibility. Ascertainment of additional, larger kindreds with such
mutations will facilitate the investigation of this issue.
[0077] Western blots. Equal number of cells were lysed directly in
lysis buffer (60 mM Tris, pH 6.8, 2% SDS, 10% glycerol, 0.1 M 2
mercaptoethanol, 0.001% bromophenol blue) and boiled for 5 minutes.
Lysate proteins were separated by electrophoresis on 4 12% Tris
glycine gels (for analysis of full length hPMS2) or 4 20% Tris
glycine gels (for analysis of hPMS2-134). Gels were electroblotted
onto Immobilon P (Millipore) in 48 mM Tris base, 40 mM glycine,
0.0375% SDS, 20% methanol and blocked overnight at 4.degree. C. in
Tris buffered saline plus 0.05% Tween 20 and 5% condensed milk.
Filters were probed with a polyclonal antibody generated against
residues 2-20 of hPMS2 (Santa Cruz Biotechnology, Inc.) and a
horseradish peroxidase conjugated goat anti rabbit secondary
antibody, using chemiluminescence for detection (Pierce).
[0078] In vitro translation. Linear DNA fragments containing hPMS2
and hMLH1 cDNA sequences were prepared by PCR, incorporating
sequences for in vitro transcription and translation in the sense
primer. A full length hMLH1 fragment was prepared using the sense
primer 5' ggatcctaatacgactcactatagggaga ccaccatgtcgttcgtggcaggg 3'
(SEQ ID NO:1) (codons 1-6) and the antisense primer 5'
taagtcttaagtgctaccaac 3' (SEQ ID NO:2) (located in the 3'
untranslated region, nt 2411 2433), using a wild type HMLH1 cDNA
clone as template. A full length hPMS2 fragment was prepared with
the sense primer 5' ggatcctaatacgactcactatagggag
accaccatggaacaattgcctgcgg 3' (SEQ ID NO:3) (codons 1-6) and the
antisense primer 5' aggttagtgaagactctgtc 3' (SEQ ID NO:4) (located
in 3' untranslated region, nt 2670 2690) using a cloned hPMS2 cDNA
as template. A fragment encoding the amino terminal 134 amino acids
of hPMS2 was prepared using the same sense primer and the antisense
primer 5' agtcgagttccaaccttcg 3' (SEQ ID NO:5). A fragment
containing codons 135-862 of hPMS135 was generated using the sense
primer 5' ggatcctaatacgactcactatagggagaccaccatgatgtttgatcacaatgg 3'
(SEQ ID NO:6) (codons 135-141) and the same antisense primer as
that used for the full length hPMS2 protein. These fragments were
used to produce proteins via the coupled transcription translation
system (Promega). The reactions were supplemented with 35S labelled
methionine or unlabelled methionine, as indicated in the text. The
PMS135 and hMLH1 proteins could not be simultaneously radiolabelled
and immunoprecipitated because of their similar molecular weights
precluded resolution. Lower molecular weight bands are presumed to
be degradation products and/or polypeptides translated from
alternative internal methionines.
[0079] Immunoprecipitation. Immunoprecipitations were performed on
in vitro translated proteins by mixing the translation reactions
with 1 .mu.g of the MLH1 specific monoclonal antibody (mAB) MLH14
(Oncogene Science, Inc.), a polyclonal antibody generated to codons
2-20 of hPMS2 described above, or a polyclonal antibody generated
to codons 843-862 of hPMS2 (Santa Cruz Biotechnology, Inc.) in
400:1 of EBC buffer (50 mM Tris, pH 7.5, 0.1 M NaCl, 0.5% NP40).
After incubation for 1 hour at 4.degree. C., protein A sepharose
(Sigma) was added to a final concentration of 10% and reactions
were incubated at 4.degree. C. for 1 hour. Proteins bound to
protein A were washed five times in EBC and separated by
electrophoresis on 4 20% Tris glycine gels, which were then dried
and autoradiographed.
Example 5
Syrian Hamster Tk-ts-13 Cells Produce a Novel Anti-Microbial
Polypeptide can Suppress the Growth of Bacillus subtilis
[0080] The feasibility of creating microbial-resistant mammalian
cells is demonstrated as follows. Syrian Hamster TK fibroblasts
were transfected with a mammalian expression vector containing a
novel anti-microbial polypeptide called mlg1 or the empty
expression vector called psg. When cells expressing the mlg
polypeptide (referred to as TK-mlg1) were grown in the presence of
Bacillus subtilis, these cells were able to suppress the growth of
the microbes and allow the TK host to remain viable in contrast to
TK cells transfected with the empty vector (TK=psg), which all died
from the toxic effects that Bacillus subtilis has on mammalian
cells. FIG. 5 shows a photograph of TK-mlg1 and TK=psg cultures
grown in the presence of Bacillus for 4 days. Syrian hamster Tk-ts
13 cells were transfected with a eukaryotic expression vector that
produces a novel antimicrobial polypeptide referred to as mlg1
(Panel A) or the expression vector lacking an inserted cDNA for
expression (TK=psg, Panel B). Cells were plated at a density of
5.times.10.sup.5 cells/ml in a 10 cm falcon pyrogenic-free petri
dish in growth medium for 24 hours and then inoculated with 10:1 of
an exponentially growing culture of Bacillus subtilis. Cultures
were then incubated for 4 days at which time Bacilli grow and begin
to lyse the Tk-ts13 parental culture as shown in panel B (indicated
by arrows), while cells expressing the anti-microbial
mlglpolypeptide (Panel A) remain viable in the presence of Bacillus
(small granular structures present in panels A and B). These data
demonstrate the feasibility of cells to survive in the presence of
Bacillus contamination when they produce an anti-microbial agent.
These data show that antimicrobial producing mammalian cells are
capable of growing and surviving in the presence of toxic
microbes.
[0081] Cell lines and transfection. Syrian Hamster fibroblast Tk
ts13 cells were obtained from ATCC and cultured as described.
Modrich, Science, 1994,266, 1959 1960. Stably transfected cell
lines expressing hPMS2 were created by cotransfection of the PMS2
expression vectors and the pLHL4 plasmid encoding the hygromycin
resistance gene at a ratio of 3:1 (pCAR:pLHL4) and selected with
hygromycin. Stably transfected cell lines containing pCAR reporters
were generated by co transfection of pCAR vectors together with
either pNTK plasmid encoding the neomycin resistance plasmid or
with pLHL4. All transfections were performed using calcium
phosphate as previously described in Modrich, Science, 1994, 266,
1959 1960, which is incorporated herein by reference in its
entirety.
Example 6
TK-hPMS2-134 Cells can Suppress the Growth of Escherichia coli In
Vitro
[0082] While TK-hPMS2-134 TK-ts13 cells have been previously shown
to be capable of altering genes in vivo (refer to Table 2 and FIG.
1), the ability to generate "naturally microbial-resistant" clones
has not been reported in the literature. To generate
microbial-resistant TK cells, TK-ts13 cells constitutively
expressing the a dominant-negative mismatch repair gene,
TK-hPMS2-134 or the empty vector (TKvect) that have been in culture
for >3 months (.about.60 passages) were seeded at
5.times.10.sup.5 cells/ml in Dulbelcco's Modified Eagles Medium
(DMEM) plus 10% fetal bovine serum (FBS) and plated into 10 cm
dishes (Falcon) in duplicate. These cells were grown overnight at
37.degree. C. in 5%CO2 to allow cells to adhere to the plastic. The
next day, TK cultures were inoculated with 10:1 of an exponentially
growing culture of Escherichia coli. Cultures were then grown at
37.degree. C. in 5%CO2 and observed on day 7 and 14 for
microbial-resistant cell clones; these cells appear as clones of
cells surrounded by "cleared" areas on the plate. At day 7, all
cells in the control transfected TKvect culture were dead, while a
subset of cells were viable in the TK-hPMS2-134 transfected
cultures. At day 14, there were no clones in the control
transfected TKvect cultures, while there were 34 and 40 Escherichia
coli-resistant colonies formed in the TK-hPMS2-134 transfected
cultures. Growing clones from each dish were then pooled as
individual cultures and grown to confluence. These cultures were
named TK-hPMS2-134 (R1) and TK-hPMS2-134 (R2). Cultures were cured
of Escherichia coli by the addition of 1 mg/ml G418, in which the
TK-hPMS2-134 cells are resistant due to expression of the
neomycin-resistance gene contained on the mammalian expression
vector used to generate the cells.
[0083] TKvect, TK-hPMS2-134 (R1) and TK-hPMS2-134 (R2) cells were
plated at 5.times.10.sup.5 cell/ml in 10 mls and plated into 10 cm
dishes in duplicate. The next day, 10:1 of a logarithmic stage
Escherichia coli culture was added to each TK culture and cultures
were grown for 48 hours at 37.degree. C. in 5% CO.sub.2. An aliquot
of supernatant from each culture was harvested immediately after
inoculation to establish a baseline density of bacteria for each
culture. After 48 hours, 2 ml of supernatant were harvested from
each culture as well as from uninfected TK cultures. One ml of each
supernatant was then analyzed by a spectrophotometer at an OD6% to
measure for bacterial density. Supernatants from uninfected
cultures were used as a blank to correct for background. As shown
in FIG. 6, bacterial growth was significantly suppressed in
TK-hPMS2-134 (R1) and TK-hPMS2-134 (R2) cultures in contrast to
TKvect control cells. These data demonstrate the feasibility of
using a dominant-negative mismatch repair mutant hPMS2-134 on
mammalian cells to produce genetically altered clones capable of
producing a molecule(s) that can suppress microbial growth.
[0084] As those skilled in the art will appreciate, numerous
changes and modifications may be made to the preferred embodiments
of the invention without departing from the spirit of the
invention. It is intended that all such variations fall within the
scope of the invention. In addition, the entire disclosure of each
publication cited herein is hereby incorporated herein by
reference.
Sequence CWU 1
1
18 1 52 DNA Artificial Sequence Description of Artificial
Sequenceoligonu cleotide primer 1 ggatcctaat acgactcact atagggagac
caccatgtcg ttcgtggcag gg 52 2 21 DNA Artificial Sequence
Description of Artificial Sequenceoligonu cleotide primer 2
taagtcttaa gtgctaccaa c 21 3 53 DNA Artificial Sequence Description
of Artificial Sequenceoligonu cleotide primer 3 ggatcctaat
acgactcact atagggagac caccatggaa caattgcctg cgg 53 4 20 DNA
Artificial Sequence Description of Artificial Sequenceoligonu
cleotide primer 4 aggttagtga agactctgtc 20 5 19 DNA Artificial
Sequence Description of Artificial Sequenceprimer 5 agtcgagttc
caaccttcg 19 6 54 DNA Artificial Sequence Description of Artificial
Sequenceprimer 6 ggatcctaat acgactcact atagggagac caccatgatg
tttgatcaca atgg 54 7 859 PRT Mus musculus 7 Met Glu Gln Thr Glu Gly
Val Ser Thr Glu Cys Ala Lys Ala Ile Lys 1 5 10 15 Pro Ile Asp Gly
Lys Ser Val His Gln Ile Cys Ser Gly Gln Val Ile 20 25 30 Leu Ser
Leu Ser Thr Ala Val Lys Glu Leu Ile Glu Asn Ser Val Asp 35 40 45
Ala Gly Ala Thr Thr Ile Asp Leu Arg Leu Lys Asp Tyr Gly Val Asp 50
55 60 Leu Ile Glu Val Ser Asp Asn Gly Cys Gly Val Glu Glu Glu Asn
Phe 65 70 75 80 Glu Gly Leu Ala Leu Lys His His Thr Ser Lys Ile Gln
Glu Phe Ala 85 90 95 Asp Leu Thr Gln Val Glu Thr Phe Gly Phe Arg
Gly Glu Ala Leu Ser 100 105 110 Ser Leu Cys Ala Leu Ser Asp Val Thr
Ile Ser Thr Cys His Gly Ser 115 120 125 Ala Ser Val Gly Thr Arg Leu
Val Phe Asp His Asn Gly Lys Ile Thr 130 135 140 Gln Lys Thr Pro Tyr
Pro Arg Pro Lys Gly Thr Thr Val Ser Val Gln 145 150 155 160 His Leu
Phe Tyr Thr Leu Pro Val Arg Tyr Lys Glu Phe Gln Arg Asn 165 170 175
Ile Lys Lys Glu Tyr Ser Lys Met Val Gln Val Leu Gln Ala Tyr Cys 180
185 190 Ile Ile Ser Ala Gly Val Arg Val Ser Cys Thr Asn Gln Leu Gly
Gln 195 200 205 Gly Lys Arg His Ala Val Val Cys Thr Ser Gly Thr Ser
Gly Met Lys 210 215 220 Glu Asn Ile Gly Ser Val Phe Gly Gln Lys Gln
Leu Gln Ser Leu Ile 225 230 235 240 Pro Phe Val Gln Leu Pro Pro Ser
Asp Ala Val Cys Glu Glu Tyr Gly 245 250 255 Leu Ser Thr Ser Gly Arg
His Lys Thr Phe Ser Thr Phe Arg Ala Ser 260 265 270 Phe His Ser Ala
Arg Thr Ala Pro Gly Gly Val Gln Gln Thr Gly Ser 275 280 285 Phe Ser
Ser Ser Ile Arg Gly Pro Val Thr Gln Gln Arg Ser Leu Ser 290 295 300
Leu Ser Met Arg Phe Tyr His Met Tyr Asn Arg His Gln Tyr Pro Phe 305
310 315 320 Val Val Leu Asn Val Ser Val Asp Ser Glu Cys Val Asp Ile
Asn Val 325 330 335 Thr Pro Asp Lys Arg Gln Ile Leu Leu Gln Glu Glu
Lys Leu Leu Leu 340 345 350 Ala Val Leu Lys Thr Ser Leu Ile Gly Met
Phe Asp Ser Asp Ala Asn 355 360 365 Lys Leu Asn Val Asn Gln Gln Pro
Leu Leu Asp Val Glu Gly Asn Leu 370 375 380 Val Lys Leu His Thr Ala
Glu Leu Glu Lys Pro Val Pro Gly Lys Gln 385 390 395 400 Asp Asn Ser
Pro Ser Leu Lys Ser Thr Ala Asp Glu Lys Arg Val Ala 405 410 415 Ser
Ile Ser Arg Leu Arg Glu Ala Phe Ser Leu His Pro Thr Lys Glu 420 425
430 Ile Lys Ser Arg Gly Pro Glu Thr Ala Glu Leu Thr Arg Ser Phe Pro
435 440 445 Ser Glu Lys Arg Gly Val Leu Ser Ser Tyr Pro Ser Asp Val
Ile Ser 450 455 460 Tyr Arg Gly Leu Arg Gly Ser Gln Asp Lys Leu Val
Ser Pro Thr Asp 465 470 475 480 Ser Pro Gly Asp Cys Met Asp Arg Glu
Lys Ile Glu Lys Asp Ser Gly 485 490 495 Leu Ser Ser Thr Ser Ala Gly
Ser Glu Glu Glu Phe Ser Thr Pro Glu 500 505 510 Val Ala Ser Ser Phe
Ser Ser Asp Tyr Asn Val Ser Ser Leu Glu Asp 515 520 525 Arg Pro Ser
Gln Glu Thr Ile Asn Cys Gly Asp Leu Asp Cys Arg Pro 530 535 540 Pro
Gly Thr Gly Gln Ser Leu Lys Pro Glu Asp His Gly Tyr Gln Cys 545 550
555 560 Lys Ala Leu Pro Leu Ala Arg Leu Ser Pro Thr Asn Ala Lys Arg
Phe 565 570 575 Lys Thr Glu Glu Arg Pro Ser Asn Val Asn Ile Ser Gln
Arg Leu Pro 580 585 590 Gly Pro Gln Ser Thr Ser Ala Ala Glu Val Asp
Val Ala Ile Lys Met 595 600 605 Asn Lys Arg Ile Val Leu Leu Glu Phe
Ser Leu Ser Ser Leu Ala Lys 610 615 620 Arg Met Lys Gln Leu Gln His
Leu Lys Ala Gln Asn Lys His Glu Leu 625 630 635 640 Ser Tyr Arg Lys
Phe Arg Ala Lys Ile Cys Pro Gly Glu Asn Gln Ala 645 650 655 Ala Glu
Asp Glu Leu Arg Lys Glu Ile Ser Lys Ser Met Phe Ala Glu 660 665 670
Met Glu Ile Leu Gly Gln Phe Asn Leu Gly Phe Ile Val Thr Lys Leu 675
680 685 Lys Glu Asp Leu Phe Leu Val Asp Gln His Ala Ala Asp Glu Lys
Tyr 690 695 700 Asn Phe Glu Met Leu Gln Gln His Thr Val Leu Gln Ala
Gln Arg Leu 705 710 715 720 Ile Thr Pro Gln Thr Leu Asn Leu Thr Ala
Val Asn Glu Ala Val Leu 725 730 735 Ile Glu Asn Leu Glu Ile Phe Arg
Lys Asn Gly Phe Asp Phe Val Ile 740 745 750 Asp Glu Asp Ala Pro Val
Thr Glu Arg Ala Lys Leu Ile Ser Leu Pro 755 760 765 Thr Ser Lys Asn
Trp Thr Phe Gly Pro Gln Asp Ile Asp Glu Leu Ile 770 775 780 Phe Met
Leu Ser Asp Ser Pro Gly Val Met Cys Arg Pro Ser Arg Val 785 790 795
800 Arg Gln Met Phe Ala Ser Arg Ala Cys Arg Lys Ser Val Met Ile Gly
805 810 815 Thr Ala Leu Asn Ala Ser Glu Met Lys Lys Leu Ile Thr His
Met Gly 820 825 830 Glu Met Asp His Pro Trp Asn Cys Pro His Gly Arg
Pro Thr Met Arg 835 840 845 His Val Ala Asn Leu Asp Val Ile Ser Gln
Asn 850 855 8 3056 DNA Mus musculus 8 gaattccggt gaaggtcctg
aagaatttcc agattcctga gtatcattgg aggagacaga 60 taacctgtcg
tcaggtaacg atggtgtata tgcaacagaa atgggtgttc ctggagacgc 120
gtcttttccc gagagcggca ccgcaactct cccgcggtga ctgtgactgg aggagtcctg
180 catccatgga gcaaaccgaa ggcgtgagta cagaatgtgc taaggccatc
aagcctattg 240 atgggaagtc agtccatcaa atttgttctg ggcaggtgat
actcagttta agcaccgctg 300 tgaaggagtt gatagaaaat agtgtagatg
ctggtgctac tactattgat ctaaggctta 360 aagactatgg ggtggacctc
attgaagttt cagacaatgg atgtggggta gaagaagaaa 420 actttgaagg
tctagctctg aaacatcaca catctaagat tcaagagttt gccgacctca 480
cgcaggttga aactttcggc tttcgggggg aagctctgag ctctctgtgt gcactaagtg
540 atgtcactat atctacctgc cacgggtctg caagcgttgg gactcgactg
gtgtttgacc 600 ataatgggaa aatcacccag aaaactccct acccccgacc
taaaggaacc acagtcagtg 660 tgcagcactt attttataca ctacccgtgc
gttacaaaga gtttcagagg aacattaaaa 720 aggagtattc caaaatggtg
caggtcttac aggcgtactg tatcatctca gcaggcgtcc 780 gtgtaagctg
cactaatcag ctcggacagg ggaagcggca cgctgtggtg tgcacaagcg 840
gcacgtctgg catgaaggaa aatatcgggt ctgtgtttgg ccagaagcag ttgcaaagcc
900 tcattccttt tgttcagctg ccccctagtg acgctgtgtg tgaagagtac
ggcctgagca 960 cttcaggacg ccacaaaacc ttttctacgt ttcgggcttc
atttcacagt gcacgcacgg 1020 cgccgggagg agtgcaacag acaggcagtt
tttcttcatc aatcagaggc cctgtgaccc 1080 agcaaaggtc tctaagcttg
tcaatgaggt tttatcacat gtataaccgg catcagtacc 1140 catttgtcgt
ccttaacgtt tccgttgact cagaatgtgt ggatattaat gtaactccag 1200
ataaaaggca aattctacta caagaagaga agctattgct ggccgtttta aagacctcct
1260 tgataggaat gtttgacagt gatgcaaaca agcttaatgt caaccagcag
ccactgctag 1320 atgttgaagg taacttagta aagctgcata ctgcagaact
agaaaagcct gtgccaggaa 1380 agcaagataa ctctccttca ctgaagagca
cagcagacga gaaaagggta gcatccatct 1440 ccaggctgag agaggccttt
tctcttcatc ctactaaaga gatcaagtct aggggtccag 1500 agactgctga
actgacacgg agttttccaa gtgagaaaag gggcgtgtta tcctcttatc 1560
cttcagacgt catctcttac agaggcctcc gtggctcgca ggacaaattg gtgagtccca
1620 cggacagccc tggtgactgt atggacagag agaaaataga aaaagactca
gggctcagca 1680 gcacctcagc tggctctgag gaagagttca gcaccccaga
agtggccagt agctttagca 1740 gtgactataa cgtgagctcc ctagaagaca
gaccttctca ggaaaccata aactgtggtg 1800 acctggactg ccgtcctcca
ggtacaggac agtccttgaa gccagaagac catggatatc 1860 aatgcaaagc
tctacctcta gctcgtctgt cacccacaaa tgccaagcgc ttcaagacag 1920
aggaaagacc ctcaaatgtc aacatttctc aaagattgcc tggtcctcag agcacctcag
1980 cagctgaggt cgatgtagcc ataaaaatga ataagagaat cgtgctcctc
gagttctctc 2040 tgagttctct agctaagcga atgaagcagt tacagcacct
aaaggcgcag aacaaacatg 2100 aactgagtta cagaaaattt agggccaaga
tttgccctgg agaaaaccaa gcagcagaag 2160 atgaactcag aaaagagatt
agtaaatcga tgtttgcaga gatggagatc ttgggtcagt 2220 ttaacctggg
atttatagta accaaactga aagaggacct cttcctggtg gaccagcatg 2280
ctgcggatga gaagtacaac tttgagatgc tgcagcagca cacggtgctc caggcgcaga
2340 ggctcatcac accccagact ctgaacttaa ctgctgtcaa tgaagctgta
ctgatagaaa 2400 atctggaaat attcagaaag aatggctttg actttgtcat
tgatgaggat gctccagtca 2460 ctgaaagggc taaattgatt tccttaccaa
ctagtaaaaa ctggaccttt ggaccccaag 2520 atatagatga actgatcttt
atgttaagtg acagccctgg ggtcatgtgc cggccctcac 2580 gagtcagaca
gatgtttgct tccagagcct gtcggaagtc agtgatgatt ggaacggcgc 2640
tcaatgcgag cgagatgaag aagctcatca cccacatggg tgagatggac cacccctgga
2700 actgccccca cggcaggcca accatgaggc acgttgccaa tctggatgtc
atctctcaga 2760 actgacacac cccttgtagc atagagttta ttacagattg
ttcggtttgc aaagagaagg 2820 ttttaagtaa tctgattatc gttgtacaaa
aattagcatg ctgctttaat gtactggatc 2880 catttaaaag cagtgttaag
gcaggcatga tggagtgttc ctctagctca gctacttggg 2940 tgatccggtg
ggagctcatg tgagcccagg actttgagac cactccgagc cacattcatg 3000
agactcaatt caaggacaaa aaaaaaaaga tatttttgaa gccttttaaa aaaaaa 3056
9 862 PRT Homo sapiens 9 Met Glu Arg Ala Glu Ser Ser Ser Thr Glu
Pro Ala Lys Ala Ile Lys 1 5 10 15 Pro Ile Asp Arg Lys Ser Val His
Gln Ile Cys Ser Gly Gln Val Val 20 25 30 Leu Ser Leu Ser Thr Ala
Val Lys Glu Leu Val Glu Asn Ser Leu Asp 35 40 45 Ala Gly Ala Thr
Asn Ile Asp Leu Lys Leu Lys Asp Tyr Gly Val Asp 50 55 60 Leu Ile
Glu Val Ser Asp Asn Gly Cys Gly Val Glu Glu Glu Asn Phe 65 70 75 80
Glu Gly Leu Thr Leu Lys His His Thr Ser Lys Ile Gln Glu Phe Ala 85
90 95 Asp Leu Thr Gln Val Glu Thr Phe Gly Phe Arg Gly Glu Ala Leu
Ser 100 105 110 Ser Leu Cys Ala Leu Ser Asp Val Thr Ile Ser Thr Cys
His Ala Ser 115 120 125 Ala Lys Val Gly Thr Arg Leu Met Phe Asp His
Asn Gly Lys Ile Ile 130 135 140 Gln Lys Thr Pro Tyr Pro Arg Pro Arg
Gly Thr Thr Val Ser Val Gln 145 150 155 160 Gln Leu Phe Ser Thr Leu
Pro Val Arg His Lys Glu Phe Gln Arg Asn 165 170 175 Ile Lys Lys Glu
Tyr Ala Lys Met Val Gln Val Leu His Ala Tyr Cys 180 185 190 Ile Ile
Ser Ala Gly Ile Arg Val Ser Cys Thr Asn Gln Leu Gly Gln 195 200 205
Gly Lys Arg Gln Pro Val Val Cys Thr Gly Gly Ser Pro Ser Ile Lys 210
215 220 Glu Asn Ile Gly Ser Val Phe Gly Gln Lys Gln Leu Gln Ser Leu
Ile 225 230 235 240 Pro Phe Val Gln Leu Pro Pro Ser Asp Ser Val Cys
Glu Glu Tyr Gly 245 250 255 Leu Ser Cys Ser Asp Ala Leu His Asn Leu
Phe Tyr Ile Ser Gly Phe 260 265 270 Ile Ser Gln Cys Thr His Gly Val
Gly Arg Ser Ser Thr Asp Arg Gln 275 280 285 Phe Phe Phe Ile Asn Arg
Arg Pro Cys Asp Pro Ala Lys Val Cys Arg 290 295 300 Leu Val Asn Glu
Val Tyr His Met Tyr Asn Arg His Gln Tyr Pro Phe 305 310 315 320 Val
Val Leu Asn Ile Ser Val Asp Ser Glu Cys Val Asp Ile Asn Val 325 330
335 Thr Pro Asp Lys Arg Gln Ile Leu Leu Gln Glu Glu Lys Leu Leu Leu
340 345 350 Ala Val Leu Lys Thr Ser Leu Ile Gly Met Phe Asp Ser Asp
Val Asn 355 360 365 Lys Leu Asn Val Ser Gln Gln Pro Leu Leu Asp Val
Glu Gly Asn Leu 370 375 380 Ile Lys Met His Ala Ala Asp Leu Glu Lys
Pro Met Val Glu Lys Gln 385 390 395 400 Asp Gln Ser Pro Ser Leu Arg
Thr Gly Glu Glu Lys Lys Asp Val Ser 405 410 415 Ile Ser Arg Leu Arg
Glu Ala Phe Ser Leu Arg His Thr Thr Glu Asn 420 425 430 Lys Pro His
Ser Pro Lys Thr Pro Glu Pro Arg Arg Ser Pro Leu Gly 435 440 445 Gln
Lys Arg Gly Met Leu Ser Ser Ser Thr Ser Gly Ala Ile Ser Asp 450 455
460 Lys Gly Val Leu Arg Pro Gln Lys Glu Ala Val Ser Ser Ser His Gly
465 470 475 480 Pro Ser Asp Pro Thr Asp Arg Ala Glu Val Glu Lys Asp
Ser Gly His 485 490 495 Gly Ser Thr Ser Val Asp Ser Glu Gly Phe Ser
Ile Pro Asp Thr Gly 500 505 510 Ser His Cys Ser Ser Glu Tyr Ala Ala
Ser Ser Pro Gly Asp Arg Gly 515 520 525 Ser Gln Glu His Val Asp Ser
Gln Glu Lys Ala Pro Glu Thr Asp Asp 530 535 540 Ser Phe Ser Asp Val
Asp Cys His Ser Asn Gln Glu Asp Thr Gly Cys 545 550 555 560 Lys Phe
Arg Val Leu Pro Gln Pro Thr Asn Leu Ala Thr Pro Asn Thr 565 570 575
Lys Arg Phe Lys Lys Glu Glu Ile Leu Ser Ser Ser Asp Ile Cys Gln 580
585 590 Lys Leu Val Asn Thr Gln Asp Met Ser Ala Ser Gln Val Asp Val
Ala 595 600 605 Val Lys Ile Asn Lys Lys Val Val Pro Leu Asp Phe Ser
Met Ser Ser 610 615 620 Leu Ala Lys Arg Ile Lys Gln Leu His His Glu
Ala Gln Gln Ser Glu 625 630 635 640 Gly Glu Gln Asn Tyr Arg Lys Phe
Arg Ala Lys Ile Cys Pro Gly Glu 645 650 655 Asn Gln Ala Ala Glu Asp
Glu Leu Arg Lys Glu Ile Ser Lys Thr Met 660 665 670 Phe Ala Glu Met
Glu Ile Ile Gly Gln Phe Asn Leu Gly Phe Ile Ile 675 680 685 Thr Lys
Leu Asn Glu Asp Ile Phe Ile Val Asp Gln His Ala Thr Asp 690 695 700
Glu Lys Tyr Asn Phe Glu Met Leu Gln Gln His Thr Val Leu Gln Gly 705
710 715 720 Gln Arg Leu Ile Ala Pro Gln Thr Leu Asn Leu Thr Ala Val
Asn Glu 725 730 735 Ala Val Leu Ile Glu Asn Leu Glu Ile Phe Arg Lys
Asn Gly Phe Asp 740 745 750 Phe Val Ile Asp Glu Asn Ala Pro Val Thr
Glu Arg Ala Lys Leu Ile 755 760 765 Ser Leu Pro Thr Ser Lys Asn Trp
Thr Phe Gly Pro Gln Asp Val Asp 770 775 780 Glu Leu Ile Phe Met Leu
Ser Asp Ser Pro Gly Val Met Cys Arg Pro 785 790 795 800 Ser Arg Val
Lys Gln Met Phe Ala Ser Arg Ala Cys Arg Lys Ser Val 805 810 815 Met
Ile Gly Thr Ala Leu Asn Thr Ser Glu Met Lys Lys Leu Ile Thr 820 825
830 His Met Gly Glu Met Asp His Pro Trp Asn Cys Pro His Gly Arg Pro
835 840 845 Thr Met Arg His Ile Ala Asn Leu Gly Val Ile Ser Gln Asn
850 855 860 10 2771 DNA Homo sapiens 10 cgaggcggat cgggtgttgc
atccatggag cgagctgaga gctcgagtac agaacctgct 60 aaggccatca
aacctattga tcggaagtca gtccatcaga tttgctctgg gcaggtggta 120
ctgagtctaa gcactgcggt aaaggagtta gtagaaaaca gtctggatgc tggtgccact
180 aatattgatc taaagcttaa ggactatgga gtggatctta ttgaagtttc
agacaatgga 240 tgtggggtag aagaagaaaa cttcgaaggc ttaactctga
aacatcacac atctaagatt 300 caagagtttg ccgacctaac tcaggttgaa
acttttggct ttcgggggga agctctgagc 360 tcactttgtg cactgagcga
tgtcaccatt tctacctgcc acgcatcggc gaaggttgga 420 actcgactga
tgtttgatca caatgggaaa attatccaga aaacccccta cccccgcccc 480
agagggacca cagtcagcgt
gcagcagtta ttttccacac tacctgtgcg ccataaggaa 540 tttcaaagga
atattaagaa ggagtatgcc aaaatggtcc aggtcttaca tgcatactgt 600
atcatttcag caggcatccg tgtaagttgc accaatcagc ttggacaagg aaaacgacag
660 cctgtggtat gcacaggtgg aagccccagc ataaaggaaa atatcggctc
tgtgtttggg 720 cagaagcagt tgcaaagcct cattcctttt gttcagctgc
cccctagtga ctccgtgtgt 780 gaagagtacg gtttgagctg ttcggatgct
ctgcataatc ttttttacat ctcaggtttc 840 atttcacaat gcacgcatgg
agttggaagg agttcaacag acagacagtt tttctttatc 900 aaccggcggc
cttgtgaccc agcaaaggtc tgcagactcg tgaatgaggt ctaccacatg 960
tataatcgac accagtatcc atttgttgtt cttaacattt ctgttgattc agaatgcgtt
1020 gatatcaatg ttactccaga taaaaggcaa attttgctac aagaggaaaa
gcttttgttg 1080 gcagttttaa agacctcttt gataggaatg tttgatagtg
atgtcaacaa gctaaatgtc 1140 agtcagcagc cactgctgga tgttgaaggt
aacttaataa aaatgcatgc agcggatttg 1200 gaaaagccca tggtagaaaa
gcaggatcaa tccccttcat taaggactgg agaagaaaaa 1260 aaagacgtgt
ccatttccag actgcgagag gccttttctc ttcgtcacac aacagagaac 1320
aagcctcaca gcccaaagac tccagaacca agaaggagcc ctctaggaca gaaaaggggt
1380 atgctgtctt ctagcacttc aggtgccatc tctgacaaag gcgtcctgag
acctcagaaa 1440 gaggcagtga gttccagtca cggacccagt gaccctacgg
acagagcgga ggtggagaag 1500 gactcggggc acggcagcac ttccgtggat
tctgaggggt tcagcatccc agacacgggc 1560 agtcactgca gcagcgagta
tgcggccagc tccccagggg acaggggctc gcaggaacat 1620 gtggactctc
aggagaaagc gcctgaaact gacgactctt tttcagatgt ggactgccat 1680
tcaaaccagg aagataccgg atgtaaattt cgagttttgc ctcagccaac taatctcgca
1740 accccaaaca caaagcgttt taaaaaagaa gaaattcttt ccagttctga
catttgtcaa 1800 aagttagtaa atactcagga catgtcagcc tctcaggttg
atgtagctgt gaaaattaat 1860 aagaaagttg tgcccctgga cttttctatg
agttctttag ctaaacgaat aaagcagtta 1920 catcatgaag cacagcaaag
tgaaggggaa cagaattaca ggaagtttag ggcaaagatt 1980 tgtcctggag
aaaatcaagc agccgaagat gaactaagaa aagagataag taaaacgatg 2040
tttgcagaaa tggaaatcat tggtcagttt aacctgggat ttataataac caaactgaat
2100 gaggatatct tcatagtgga ccagcatgcc acggacgaga agtataactt
cgagatgctg 2160 cagcagcaca ccgtgctcca ggggcagagg ctcatagcac
ctcagactct caacttaact 2220 gctgttaatg aagctgttct gatagaaaat
ctggaaatat ttagaaagaa tggctttgat 2280 tttgttatcg atgaaaatgc
tccagtcact gaaagggcta aactgatttc cttgccaact 2340 agtaaaaact
ggaccttcgg accccaggac gtcgatgaac tgatcttcat gctgagcgac 2400
agccctgggg tcatgtgccg gccttcccga gtcaagcaga tgtttgcctc cagagcctgc
2460 cggaagtcgg tgatgattgg gactgctctt aacacaagcg agatgaagaa
actgatcacc 2520 cacatggggg agatggacca cccctggaac tgtccccatg
gaaggccaac catgagacac 2580 atcgccaacc tgggtgtcat ttctcagaac
tgaccgtagt cactgtatgg aataattggt 2640 tttatcgcag atttttatgt
tttgaaagac agagtcttca ctaacctttt ttgttttaaa 2700 atgaaacctg
ctacttaaaa aaaatacaca tcacacccat ttaaaagtga tcttgagaac 2760
cttttcaaac c 2771 11 932 PRT Homo sapiens 11 Met Lys Gln Leu Pro
Ala Ala Thr Val Arg Leu Leu Ser Ser Ser Gln 1 5 10 15 Ile Ile Thr
Ser Val Val Ser Val Val Lys Glu Leu Ile Glu Asn Ser 20 25 30 Leu
Asp Ala Gly Ala Thr Ser Val Asp Val Lys Leu Glu Asn Tyr Gly 35 40
45 Phe Asp Lys Ile Glu Val Arg Asp Asn Gly Glu Gly Ile Lys Ala Val
50 55 60 Asp Ala Pro Val Met Ala Met Lys Tyr Tyr Thr Ser Lys Ile
Asn Ser 65 70 75 80 His Glu Asp Leu Glu Asn Leu Thr Thr Tyr Gly Phe
Arg Gly Glu Ala 85 90 95 Leu Gly Ser Ile Cys Cys Ile Ala Glu Val
Leu Ile Thr Thr Arg Thr 100 105 110 Ala Ala Asp Asn Phe Ser Thr Gln
Tyr Val Leu Asp Gly Ser Gly His 115 120 125 Ile Leu Ser Gln Lys Pro
Ser His Leu Gly Gln Gly Thr Thr Val Thr 130 135 140 Ala Leu Arg Leu
Phe Lys Asn Leu Pro Val Arg Lys Gln Phe Tyr Ser 145 150 155 160 Thr
Ala Lys Lys Cys Lys Asp Glu Ile Lys Lys Ile Gln Asp Leu Leu 165 170
175 Met Ser Phe Gly Ile Leu Lys Pro Asp Leu Arg Ile Val Phe Val His
180 185 190 Asn Lys Ala Val Ile Trp Gln Lys Ser Arg Val Ser Asp His
Lys Met 195 200 205 Ala Leu Met Ser Val Leu Gly Thr Ala Val Met Asn
Asn Met Glu Ser 210 215 220 Phe Gln Tyr His Ser Glu Glu Ser Gln Ile
Tyr Leu Ser Gly Phe Leu 225 230 235 240 Pro Lys Cys Asp Ala Asp His
Ser Phe Thr Ser Leu Ser Thr Pro Glu 245 250 255 Arg Ser Phe Ile Phe
Ile Asn Ser Arg Pro Val His Gln Lys Asp Ile 260 265 270 Leu Lys Leu
Ile Arg His His Tyr Asn Leu Lys Cys Leu Lys Glu Ser 275 280 285 Thr
Arg Leu Tyr Pro Val Phe Phe Leu Lys Ile Asp Val Pro Thr Ala 290 295
300 Asp Val Asp Val Asn Leu Thr Pro Asp Lys Ser Gln Val Leu Leu Gln
305 310 315 320 Asn Lys Glu Ser Val Leu Ile Ala Leu Glu Asn Leu Met
Thr Thr Cys 325 330 335 Tyr Gly Pro Leu Pro Ser Thr Asn Ser Tyr Glu
Asn Asn Lys Thr Asp 340 345 350 Val Ser Ala Ala Asp Ile Val Leu Ser
Lys Thr Ala Glu Thr Asp Val 355 360 365 Leu Phe Asn Lys Val Glu Ser
Ser Gly Lys Asn Tyr Ser Asn Val Asp 370 375 380 Thr Ser Val Ile Pro
Phe Gln Asn Asp Met His Asn Asp Glu Ser Gly 385 390 395 400 Lys Asn
Thr Asp Asp Cys Leu Asn His Gln Ile Ser Ile Gly Asp Phe 405 410 415
Gly Tyr Gly His Cys Ser Ser Glu Ile Ser Asn Ile Asp Lys Asn Thr 420
425 430 Lys Asn Ala Phe Gln Asp Ile Ser Met Ser Asn Val Ser Trp Glu
Asn 435 440 445 Ser Gln Thr Glu Tyr Ser Lys Thr Cys Phe Ile Ser Ser
Val Lys His 450 455 460 Thr Gln Ser Glu Asn Gly Asn Lys Asp His Ile
Asp Glu Ser Gly Glu 465 470 475 480 Asn Glu Glu Glu Ala Gly Leu Glu
Asn Ser Ser Glu Ile Ser Ala Asp 485 490 495 Glu Trp Ser Arg Gly Asn
Ile Leu Lys Asn Ser Val Gly Glu Asn Ile 500 505 510 Glu Pro Val Lys
Ile Leu Val Pro Glu Lys Ser Leu Pro Cys Lys Val 515 520 525 Ser Asn
Asn Asn Tyr Pro Ile Pro Glu Gln Met Asn Leu Asn Glu Asp 530 535 540
Ser Cys Asn Lys Lys Ser Asn Val Ile Asp Asn Lys Ser Gly Lys Val 545
550 555 560 Thr Ala Tyr Asp Leu Leu Ser Asn Arg Val Ile Lys Lys Pro
Met Ser 565 570 575 Ala Ser Ala Leu Phe Val Gln Asp His Arg Pro Gln
Phe Leu Ile Glu 580 585 590 Asn Pro Lys Thr Ser Leu Glu Asp Ala Thr
Leu Gln Ile Glu Glu Leu 595 600 605 Trp Lys Thr Leu Ser Glu Glu Glu
Lys Leu Lys Tyr Glu Glu Lys Ala 610 615 620 Thr Lys Asp Leu Glu Arg
Tyr Asn Ser Gln Met Lys Arg Ala Ile Glu 625 630 635 640 Gln Glu Ser
Gln Met Ser Leu Lys Asp Gly Arg Lys Lys Ile Lys Pro 645 650 655 Thr
Ser Ala Trp Asn Leu Ala Gln Lys His Lys Leu Lys Thr Ser Leu 660 665
670 Ser Asn Gln Pro Lys Leu Asp Glu Leu Leu Gln Ser Gln Ile Glu Lys
675 680 685 Arg Arg Ser Gln Asn Ile Lys Met Val Gln Ile Pro Phe Ser
Met Lys 690 695 700 Asn Leu Lys Ile Asn Phe Lys Lys Gln Asn Lys Val
Asp Leu Glu Glu 705 710 715 720 Lys Asp Glu Pro Cys Leu Ile His Asn
Leu Arg Phe Pro Asp Ala Trp 725 730 735 Leu Met Thr Ser Lys Thr Glu
Val Met Leu Leu Asn Pro Tyr Arg Val 740 745 750 Glu Glu Ala Leu Leu
Phe Lys Arg Leu Leu Glu Asn His Lys Leu Pro 755 760 765 Ala Glu Pro
Leu Glu Lys Pro Ile Met Leu Thr Glu Ser Leu Phe Asn 770 775 780 Gly
Ser His Tyr Leu Asp Val Leu Tyr Lys Met Thr Ala Asp Asp Gln 785 790
795 800 Arg Tyr Ser Gly Ser Thr Tyr Leu Ser Asp Pro Arg Leu Thr Ala
Asn 805 810 815 Gly Phe Lys Ile Lys Leu Ile Pro Gly Val Ser Ile Thr
Glu Asn Tyr 820 825 830 Leu Glu Ile Glu Gly Met Ala Asn Cys Leu Pro
Phe Tyr Gly Val Ala 835 840 845 Asp Leu Lys Glu Ile Leu Asn Ala Ile
Leu Asn Arg Asn Ala Lys Glu 850 855 860 Val Tyr Glu Cys Arg Pro Arg
Lys Val Ile Ser Tyr Leu Glu Gly Glu 865 870 875 880 Ala Val Arg Leu
Ser Arg Gln Leu Pro Met Tyr Leu Ser Lys Glu Asp 885 890 895 Ile Gln
Asp Ile Ile Tyr Arg Met Lys His Gln Phe Gly Asn Glu Ile 900 905 910
Lys Glu Cys Val His Gly Arg Pro Phe Phe His His Leu Thr Tyr Leu 915
920 925 Pro Glu Thr Thr 930 12 3063 DNA Homo sapiens 12 ggcacgagtg
gctgcttgcg gctagtggat ggtaattgcc tgcctcgcgc tagcagcaag 60
ctgctctgtt aaaagcgaaa atgaaacaat tgcctgcggc aacagttcga ctcctttcaa
120 gttctcagat catcacttcg gtggtcagtg ttgtaaaaga gcttattgaa
aactccttgg 180 atgctggtgc cacaagcgta gatgttaaac tggagaacta
tggatttgat aaaattgagg 240 tgcgagataa cggggagggt atcaaggctg
ttgatgcacc tgtaatggca atgaagtact 300 acacctcaaa aataaatagt
catgaagatc ttgaaaattt gacaacttac ggttttcgtg 360 gagaagcctt
ggggtcaatt tgttgtatag ctgaggtttt aattacaaca agaacggctg 420
ctgataattt tagcacccag tatgttttag atggcagtgg ccacatactt tctcagaaac
480 cttcacatct tggtcaaggt acaactgtaa ctgctttaag attatttaag
aatctacctg 540 taagaaagca gttttactca actgcaaaaa aatgtaaaga
tgaaataaaa aagatccaag 600 atctcctcat gagctttggt atccttaaac
ctgacttaag gattgtcttt gtacataaca 660 aggcagttat ttggcagaaa
agcagagtat cagatcacaa gatggctctc atgtcagttc 720 tggggactgc
tgttatgaac aatatggaat cctttcagta ccactctgaa gaatctcaga 780
tttatctcag tggatttctt ccaaagtgtg atgcagacca ctctttcact agtctttcaa
840 caccagaaag aagtttcatc ttcataaaca gtcgaccagt acatcaaaaa
gatatcttaa 900 agttaatccg acatcattac aatctgaaat gcctaaagga
atctactcgt ttgtatcctg 960 ttttctttct gaaaatcgat gttcctacag
ctgatgttga tgtaaattta acaccagata 1020 aaagccaagt attattacaa
aataaggaat ctgttttaat tgctcttgaa aatctgatga 1080 cgacttgtta
tggaccatta cctagtacaa attcttatga aaataataaa acagatgttt 1140
ccgcagctga catcgttctt agtaaaacag cagaaacaga tgtgcttttt aataaagtgg
1200 aatcatctgg aaagaattat tcaaatgttg atacttcagt cattccattc
caaaatgata 1260 tgcataatga tgaatctgga aaaaacactg atgattgttt
aaatcaccag ataagtattg 1320 gtgactttgg ttatggtcat tgtagtagtg
aaatttctaa cattgataaa aacactaaga 1380 atgcatttca ggacatttca
atgagtaatg tatcatggga gaactctcag acggaatata 1440 gtaaaacttg
ttttataagt tccgttaagc acacccagtc agaaaatggc aataaagacc 1500
atatagatga gagtggggaa aatgaggaag aagcaggtct tgaaaactct tcggaaattt
1560 ctgcagatga gtggagcagg ggaaatatac ttaaaaattc agtgggagag
aatattgaac 1620 ctgtgaaaat tttagtgcct gaaaaaagtt taccatgtaa
agtaagtaat aataattatc 1680 caatccctga acaaatgaat cttaatgaag
attcatgtaa caaaaaatca aatgtaatag 1740 ataataaatc tggaaaagtt
acagcttatg atttacttag caatcgagta atcaagaaac 1800 ccatgtcagc
aagtgctctt tttgttcaag atcatcgtcc tcagtttctc atagaaaatc 1860
ctaagactag tttagaggat gcaacactac aaattgaaga actgtggaag acattgagtg
1920 aagaggaaaa actgaaatat gaagagaagg ctactaaaga cttggaacga
tacaatagtc 1980 aaatgaagag agccattgaa caggagtcac aaatgtcact
aaaagatggc agaaaaaaga 2040 taaaacccac cagcgcatgg aatttggccc
agaagcacaa gttaaaaacc tcattatcta 2100 atcaaccaaa acttgatgaa
ctccttcagt cccaaattga aaaaagaagg agtcaaaata 2160 ttaaaatggt
acagatcccc ttttctatga aaaacttaaa aataaatttt aagaaacaaa 2220
acaaagttga cttagaagag aaggatgaac cttgcttgat ccacaatctc aggtttcctg
2280 atgcatggct aatgacatcc aaaacagagg taatgttatt aaatccatat
agagtagaag 2340 aagccctgct atttaaaaga cttcttgaga atcataaact
tcctgcagag ccactggaaa 2400 agccaattat gttaacagag agtcttttta
atggatctca ttatttagac gttttatata 2460 aaatgacagc agatgaccaa
agatacagtg gatcaactta cctgtctgat cctcgtctta 2520 cagcgaatgg
tttcaagata aaattgatac caggagtttc aattactgaa aattacttgg 2580
aaatagaagg aatggctaat tgtctcccat tctatggagt agcagattta aaagaaattc
2640 ttaatgctat attaaacaga aatgcaaagg aagtttatga atgtagacct
cgcaaagtga 2700 taagttattt agagggagaa gcagtgcgtc tatccagaca
attacccatg tacttatcaa 2760 aagaggacat ccaagacatt atctacagaa
tgaagcacca gtttggaaat gaaattaaag 2820 agtgtgttca tggtcgccca
ttttttcatc atttaaccta tcttccagaa actacatgat 2880 taaatatgtt
taagaagatt agttaccatt gaaattggtt ctgtcataaa acagcatgag 2940
tctggtttta aattatcttt gtattatgtg tcacatggtt attttttaaa tgaggattca
3000 ctgacttgtt tttatattga aaaaagttcc acgtattgta gaaaacgtaa
ataaactaat 3060 aac 3063 13 934 PRT Homo sapiens 13 Met Ala Val Gln
Pro Lys Glu Thr Leu Gln Leu Glu Ser Ala Ala Glu 1 5 10 15 Val Gly
Phe Val Arg Phe Phe Gln Gly Met Pro Glu Lys Pro Thr Thr 20 25 30
Thr Val Arg Leu Phe Asp Arg Gly Asp Phe Tyr Thr Ala His Gly Glu 35
40 45 Asp Ala Leu Leu Ala Ala Arg Glu Val Phe Lys Thr Gln Gly Val
Ile 50 55 60 Lys Tyr Met Gly Pro Ala Gly Ala Lys Asn Leu Gln Ser
Val Val Leu 65 70 75 80 Ser Lys Met Asn Phe Glu Ser Phe Val Lys Asp
Leu Leu Leu Val Arg 85 90 95 Gln Tyr Arg Val Glu Val Tyr Lys Asn
Arg Ala Gly Asn Lys Ala Ser 100 105 110 Lys Glu Asn Asp Trp Tyr Leu
Ala Tyr Lys Ala Ser Pro Gly Asn Leu 115 120 125 Ser Gln Phe Glu Asp
Ile Leu Phe Gly Asn Asn Asp Met Ser Ala Ser 130 135 140 Ile Gly Val
Val Gly Val Lys Met Ser Ala Val Asp Gly Gln Arg Gln 145 150 155 160
Val Gly Val Gly Tyr Val Asp Ser Ile Gln Arg Lys Leu Gly Leu Cys 165
170 175 Glu Phe Pro Asp Asn Asp Gln Phe Ser Asn Leu Glu Ala Leu Leu
Ile 180 185 190 Gln Ile Gly Pro Lys Glu Cys Val Leu Pro Gly Gly Glu
Thr Ala Gly 195 200 205 Asp Met Gly Lys Leu Arg Gln Ile Ile Gln Arg
Gly Gly Ile Leu Ile 210 215 220 Thr Glu Arg Lys Lys Ala Asp Phe Ser
Thr Lys Asp Ile Tyr Gln Asp 225 230 235 240 Leu Asn Arg Leu Leu Lys
Gly Lys Lys Gly Glu Gln Met Asn Ser Ala 245 250 255 Val Leu Pro Glu
Met Glu Asn Gln Val Ala Val Ser Ser Leu Ser Ala 260 265 270 Val Ile
Lys Phe Leu Glu Leu Leu Ser Asp Asp Ser Asn Phe Gly Gln 275 280 285
Phe Glu Leu Thr Thr Phe Asp Phe Ser Gln Tyr Met Lys Leu Asp Ile 290
295 300 Ala Ala Val Arg Ala Leu Asn Leu Phe Gln Gly Ser Val Glu Asp
Thr 305 310 315 320 Thr Gly Ser Gln Ser Leu Ala Ala Leu Leu Asn Lys
Cys Lys Thr Pro 325 330 335 Gln Gly Gln Arg Leu Val Asn Gln Trp Ile
Lys Gln Pro Leu Met Asp 340 345 350 Lys Asn Arg Ile Glu Glu Arg Leu
Asn Leu Val Glu Ala Phe Val Glu 355 360 365 Asp Ala Glu Leu Arg Gln
Thr Leu Gln Glu Asp Leu Leu Arg Arg Phe 370 375 380 Pro Asp Leu Asn
Arg Leu Ala Lys Lys Phe Gln Arg Gln Ala Ala Asn 385 390 395 400 Leu
Gln Asp Cys Tyr Arg Leu Tyr Gln Gly Ile Asn Gln Leu Pro Asn 405 410
415 Val Ile Gln Ala Leu Glu Lys His Glu Gly Lys His Gln Lys Leu Leu
420 425 430 Leu Ala Val Phe Val Thr Pro Leu Thr Asp Leu Arg Ser Asp
Phe Ser 435 440 445 Lys Phe Gln Glu Met Ile Glu Thr Thr Leu Asp Met
Asp Gln Val Glu 450 455 460 Asn His Glu Phe Leu Val Lys Pro Ser Phe
Asp Pro Asn Leu Ser Glu 465 470 475 480 Leu Arg Glu Ile Met Asn Asp
Leu Glu Lys Lys Met Gln Ser Thr Leu 485 490 495 Ile Ser Ala Ala Arg
Asp Leu Gly Leu Asp Pro Gly Lys Gln Ile Lys 500 505 510 Leu Asp Ser
Ser Ala Gln Phe Gly Tyr Tyr Phe Arg Val Thr Cys Lys 515 520 525 Glu
Glu Lys Val Leu Arg Asn Asn Lys Asn Phe Ser Thr Val Asp Ile 530 535
540 Gln Lys Asn Gly Val Lys Phe Thr Asn Ser Lys Leu Thr Ser Leu Asn
545 550 555 560 Glu Glu Tyr Thr Lys Asn Lys Thr Glu Tyr Glu Glu Ala
Gln Asp Ala 565 570 575 Ile Val Lys Glu Ile Val Asn Ile Ser Ser Gly
Tyr Val Glu Pro Met 580 585 590 Gln Thr Leu Asn Asp Val Leu Ala Gln
Leu Asp Ala Val Val Ser Phe 595 600 605 Ala His Val Ser Asn Gly Ala
Pro Val Pro Tyr Val Arg Pro Ala Ile 610 615
620 Leu Glu Lys Gly Gln Gly Arg Ile Ile Leu Lys Ala Ser Arg His Ala
625 630 635 640 Cys Val Glu Val Gln Asp Glu Ile Ala Phe Ile Pro Asn
Asp Val Tyr 645 650 655 Phe Glu Lys Asp Lys Gln Met Phe His Ile Ile
Thr Gly Pro Asn Met 660 665 670 Gly Gly Lys Ser Thr Tyr Ile Arg Gln
Thr Gly Val Ile Val Leu Met 675 680 685 Ala Gln Ile Gly Cys Phe Val
Pro Cys Glu Ser Ala Glu Val Ser Ile 690 695 700 Val Asp Cys Ile Leu
Ala Arg Val Gly Ala Gly Asp Ser Gln Leu Lys 705 710 715 720 Gly Val
Ser Thr Phe Met Ala Glu Met Leu Glu Thr Ala Ser Ile Leu 725 730 735
Arg Ser Ala Thr Lys Asp Ser Leu Ile Ile Ile Asp Glu Leu Gly Arg 740
745 750 Gly Thr Ser Thr Tyr Asp Gly Phe Gly Leu Ala Trp Ala Ile Ser
Glu 755 760 765 Tyr Ile Ala Thr Lys Ile Gly Ala Phe Cys Met Phe Ala
Thr His Phe 770 775 780 His Glu Leu Thr Ala Leu Ala Asn Gln Ile Pro
Thr Val Asn Asn Leu 785 790 795 800 His Val Thr Ala Leu Thr Thr Glu
Glu Thr Leu Thr Met Leu Tyr Gln 805 810 815 Val Lys Lys Gly Val Cys
Asp Gln Ser Phe Gly Ile His Val Ala Glu 820 825 830 Leu Ala Asn Phe
Pro Lys His Val Ile Glu Cys Ala Lys Gln Lys Ala 835 840 845 Leu Glu
Leu Glu Glu Phe Gln Tyr Ile Gly Glu Ser Gln Gly Tyr Asp 850 855 860
Ile Met Glu Pro Ala Ala Lys Lys Cys Tyr Leu Glu Arg Glu Gln Gly 865
870 875 880 Glu Lys Ile Ile Gln Glu Phe Leu Ser Lys Val Lys Gln Met
Pro Phe 885 890 895 Thr Glu Met Ser Glu Glu Asn Ile Thr Ile Lys Leu
Lys Gln Leu Lys 900 905 910 Ala Glu Val Ile Ala Lys Asn Asn Ser Phe
Val Asn Glu Ile Ile Ser 915 920 925 Arg Ile Lys Val Thr Thr 930 14
3145 DNA Homo sapiens 14 ggcgggaaac agcttagtgg gtgtggggtc
gcgcattttc ttcaaccagg aggtgaggag 60 gtttcgacat ggcggtgcag
ccgaaggaga cgctgcagtt ggagagcgcg gccgaggtcg 120 gcttcgtgcg
cttctttcag ggcatgccgg agaagccgac caccacagtg cgccttttcg 180
accggggcga cttctatacg gcgcacggcg aggacgcgct gctggccgcc cgggaggtgt
240 tcaagaccca gggggtgatc aagtacatgg ggccggcagg agcaaagaat
ctgcagagtg 300 ttgtgcttag taaaatgaat tttgaatctt ttgtaaaaga
tcttcttctg gttcgtcagt 360 atagagttga agtttataag aatagagctg
gaaataaggc atccaaggag aatgattggt 420 atttggcata taaggcttct
cctggcaatc tctctcagtt tgaagacatt ctctttggta 480 acaatgatat
gtcagcttcc attggtgttg tgggtgttaa aatgtccgca gttgatggcc 540
agagacaggt tggagttggg tatgtggatt ccatacagag gaaactagga ctgtgtgaat
600 tccctgataa tgatcagttc tccaatcttg aggctctcct catccagatt
ggaccaaagg 660 aatgtgtttt acccggagga gagactgctg gagacatggg
gaaactgaga cagataattc 720 aaagaggagg aattctgatc acagaaagaa
aaaaagctga cttttccaca aaagacattt 780 atcaggacct caaccggttg
ttgaaaggca aaaagggaga gcagatgaat agtgctgtat 840 tgccagaaat
ggagaatcag gttgcagttt catcactgtc tgcggtaatc aagtttttag 900
aactcttatc agatgattcc aactttggac agtttgaact gactactttt gacttcagcc
960 agtatatgaa attggatatt gcagcagtca gagcccttaa cctttttcag
ggttctgttg 1020 aagataccac tggctctcag tctctggctg ccttgctgaa
taagtgtaaa acccctcaag 1080 gacaaagact tgttaaccag tggattaagc
agcctctcat ggataagaac agaatagagg 1140 agagattgaa tttagtggaa
gcttttgtag aagatgcaga attgaggcag actttacaag 1200 aagatttact
tcgtcgattc ccagatctta accgacttgc caagaagttt caaagacaag 1260
cagcaaactt acaagattgt taccgactct atcagggtat aaatcaacta cctaatgtta
1320 tacaggctct ggaaaaacat gaaggaaaac accagaaatt attgttggca
gtttttgtga 1380 ctcctcttac tgatcttcgt tctgacttct ccaagtttca
ggaaatgata gaaacaactt 1440 tagatatgga tcaggtggaa aaccatgaat
tccttgtaaa accttcattt gatcctaatc 1500 tcagtgaatt aagagaaata
atgaatgact tggaaaagaa gatgcagtca acattaataa 1560 gtgcagccag
agatcttggc ttggaccctg gcaaacagat taaactggat tccagtgcac 1620
agtttggata ttactttcgt gtaacctgta aggaagaaaa agtccttcgt aacaataaaa
1680 actttagtac tgtagatatc cagaagaatg gtgttaaatt taccaacagc
aaattgactt 1740 ctttaaatga agagtatacc aaaaataaaa cagaatatga
agaagcccag gatgccattg 1800 ttaaagaaat tgtcaatatt tcttcaggct
atgtagaacc aatgcagaca ctcaatgatg 1860 tgttagctca gctagatgct
gttgtcagct ttgctcacgt gtcaaatgga gcacctgttc 1920 catatgtacg
accagccatt ttggagaaag gacaaggaag aattatatta aaagcatcca 1980
ggcatgcttg tgttgaagtt caagatgaaa ttgcatttat tcctaatgac gtatactttg
2040 aaaaagataa acagatgttc cacatcatta ctggccccaa tatgggaggt
aaatcaacat 2100 atattcgaca aactggggtg atagtactca tggcccaaat
tgggtgtttt gtgccatgtg 2160 agtcagcaga agtgtccatt gtggactgca
tcttagcccg agtaggggct ggtgacagtc 2220 aattgaaagg agtctccacg
ttcatggctg aaatgttgga aactgcttct atcctcaggt 2280 ctgcaaccaa
agattcatta ataatcatag atgaattggg aagaggaact tctacctacg 2340
atggatttgg gttagcatgg gctatatcag aatacattgc aacaaagatt ggtgcttttt
2400 gcatgtttgc aacccatttt catgaactta ctgccttggc caatcagata
ccaactgtta 2460 ataatctaca tgtcacagca ctcaccactg aagagacctt
aactatgctt tatcaggtga 2520 agaaaggtgt ctgtgatcaa agttttggga
ttcatgttgc agagcttgct aatttcccta 2580 agcatgtaat agagtgtgct
aaacagaaag ccctggaact tgaggagttt cagtatattg 2640 gagaatcgca
aggatatgat atcatggaac cagcagcaaa gaagtgctat ctggaaagag 2700
agcaaggtga aaaaattatt caggagttcc tgtccaaggt gaaacaaatg ccctttactg
2760 aaatgtcaga agaaaacatc acaataaagt taaaacagct aaaagctgaa
gtaatagcaa 2820 agaataatag ctttgtaaat gaaatcattt cacgaataaa
agttactacg tgaaaaatcc 2880 cagtaatgga atgaaggtaa tattgataag
ctattgtctg taatagtttt atattgtttt 2940 atattaaccc tttttccata
gtgttaactg tcagtgccca tgggctatca acttaataag 3000 atatttagta
atattttact ttgaggacat tttcaaagat ttttattttg aaaaatgaga 3060
gctgtaactg aggactgttt gcaattgaca taggcaataa taagtgatgt gctgaatttt
3120 ataaataaaa tcatgtagtt tgtgg 3145 15 756 PRT Homo sapiens 15
Met Ser Phe Val Ala Gly Val Ile Arg Arg Leu Asp Glu Thr Val Val 1 5
10 15 Asn Arg Ile Ala Ala Gly Glu Val Ile Gln Arg Pro Ala Asn Ala
Ile 20 25 30 Lys Glu Met Ile Glu Asn Cys Leu Asp Ala Lys Ser Thr
Ser Ile Gln 35 40 45 Val Ile Val Lys Glu Gly Gly Leu Lys Leu Ile
Gln Ile Gln Asp Asn 50 55 60 Gly Thr Gly Ile Arg Lys Glu Asp Leu
Asp Ile Val Cys Glu Arg Phe 65 70 75 80 Thr Thr Ser Lys Leu Gln Ser
Phe Glu Asp Leu Ala Ser Ile Ser Thr 85 90 95 Tyr Gly Phe Arg Gly
Glu Ala Leu Ala Ser Ile Ser His Val Ala His 100 105 110 Val Thr Ile
Thr Thr Lys Thr Ala Asp Gly Lys Cys Ala Tyr Arg Ala 115 120 125 Ser
Tyr Ser Asp Gly Lys Leu Lys Ala Pro Pro Lys Pro Cys Ala Gly 130 135
140 Asn Gln Gly Thr Gln Ile Thr Val Glu Asp Leu Phe Tyr Asn Ile Ala
145 150 155 160 Thr Arg Arg Lys Ala Leu Lys Asn Pro Ser Glu Glu Tyr
Gly Lys Ile 165 170 175 Leu Glu Val Val Gly Arg Tyr Ser Val His Asn
Ala Gly Ile Ser Phe 180 185 190 Ser Val Lys Lys Gln Gly Glu Thr Val
Ala Asp Val Arg Thr Leu Pro 195 200 205 Asn Ala Ser Thr Val Asp Asn
Ile Arg Ser Ile Phe Gly Asn Ala Val 210 215 220 Ser Arg Glu Leu Ile
Glu Ile Gly Cys Glu Asp Lys Thr Leu Ala Phe 225 230 235 240 Lys Met
Asn Gly Tyr Ile Ser Asn Ala Asn Tyr Ser Val Lys Lys Cys 245 250 255
Ile Phe Leu Leu Phe Ile Asn His Arg Leu Val Glu Ser Thr Ser Leu 260
265 270 Arg Lys Ala Ile Glu Thr Val Tyr Ala Ala Tyr Leu Pro Lys Asn
Thr 275 280 285 His Pro Phe Leu Tyr Leu Ser Leu Glu Ile Ser Pro Gln
Asn Val Asp 290 295 300 Val Asn Val His Pro Thr Lys His Glu Val His
Phe Leu His Glu Glu 305 310 315 320 Ser Ile Leu Glu Arg Val Gln Gln
His Ile Glu Ser Lys Leu Leu Gly 325 330 335 Ser Asn Ser Ser Arg Met
Tyr Phe Thr Gln Thr Leu Leu Pro Gly Leu 340 345 350 Ala Gly Pro Ser
Gly Glu Met Val Lys Ser Thr Thr Ser Leu Thr Ser 355 360 365 Ser Ser
Thr Ser Gly Ser Ser Asp Lys Val Tyr Ala His Gln Met Val 370 375 380
Arg Thr Asp Ser Arg Glu Gln Lys Leu Asp Ala Phe Leu Gln Pro Leu 385
390 395 400 Ser Lys Pro Leu Ser Ser Gln Pro Gln Ala Ile Val Thr Glu
Asp Lys 405 410 415 Thr Asp Ile Ser Ser Gly Arg Ala Arg Gln Gln Asp
Glu Glu Met Leu 420 425 430 Glu Leu Pro Ala Pro Ala Glu Val Ala Ala
Lys Asn Gln Ser Leu Glu 435 440 445 Gly Asp Thr Thr Lys Gly Thr Ser
Glu Met Ser Glu Lys Arg Gly Pro 450 455 460 Thr Ser Ser Asn Pro Arg
Lys Arg His Arg Glu Asp Ser Asp Val Glu 465 470 475 480 Met Val Glu
Asp Asp Ser Arg Lys Glu Met Thr Ala Ala Cys Thr Pro 485 490 495 Arg
Arg Arg Ile Ile Asn Leu Thr Ser Val Leu Ser Leu Gln Glu Glu 500 505
510 Ile Asn Glu Gln Gly His Glu Val Leu Arg Glu Met Leu His Asn His
515 520 525 Ser Phe Val Gly Cys Val Asn Pro Gln Trp Ala Leu Ala Gln
His Gln 530 535 540 Thr Lys Leu Tyr Leu Leu Asn Thr Thr Lys Leu Ser
Glu Glu Leu Phe 545 550 555 560 Tyr Gln Ile Leu Ile Tyr Asp Phe Ala
Asn Phe Gly Val Leu Arg Leu 565 570 575 Ser Glu Pro Ala Pro Leu Phe
Asp Leu Ala Met Leu Ala Leu Asp Ser 580 585 590 Pro Glu Ser Gly Trp
Thr Glu Glu Asp Gly Pro Lys Glu Gly Leu Ala 595 600 605 Glu Tyr Ile
Val Glu Phe Leu Lys Lys Lys Ala Glu Met Leu Ala Asp 610 615 620 Tyr
Phe Ser Leu Glu Ile Asp Glu Glu Gly Asn Leu Ile Gly Leu Pro 625 630
635 640 Leu Leu Ile Asp Asn Tyr Val Pro Pro Leu Glu Gly Leu Pro Ile
Phe 645 650 655 Ile Leu Arg Leu Ala Thr Glu Val Asn Trp Asp Glu Glu
Lys Glu Cys 660 665 670 Phe Glu Ser Leu Ser Lys Glu Cys Ala Met Phe
Tyr Ser Ile Arg Lys 675 680 685 Gln Tyr Ile Ser Glu Glu Ser Thr Leu
Ser Gly Gln Gln Ser Glu Val 690 695 700 Pro Gly Ser Ile Pro Asn Ser
Trp Lys Trp Thr Val Glu His Ile Val 705 710 715 720 Tyr Lys Ala Leu
Arg Ser His Ile Leu Pro Pro Lys His Phe Thr Glu 725 730 735 Asp Gly
Asn Ile Leu Gln Leu Ala Asn Leu Pro Asp Leu Tyr Lys Val 740 745 750
Phe Glu Arg Cys 755 16 2484 DNA Homo sapiens 16 cttggctctt
ctggcgccaa aatgtcgttc gtggcagggg ttattcggcg gctggacgag 60
acagtggtga accgcatcgc ggcgggggaa gttatccagc ggccagctaa tgctatcaaa
120 gagatgattg agaactgttt agatgcaaaa tccacaagta ttcaagtgat
tgttaaagag 180 ggaggcctga agttgattca gatccaagac aatggcaccg
ggatcaggaa agaagatctg 240 gatattgtat gtgaaaggtt cactactagt
aaactgcagt cctttgagga tttagccagt 300 atttctacct atggctttcg
aggtgaggct ttggccagca taagccatgt ggctcatgtt 360 actattacaa
cgaaaacagc tgatggaaag tgtgcataca gagcaagtta ctcagatgga 420
aaactgaaag cccctcctaa accatgtgct ggcaatcaag ggacccagat cacggtggag
480 gacctttttt acaacatagc cacgaggaga aaagctttaa aaaatccaag
tgaagaatat 540 gggaaaattt tggaagttgt tggcaggtat tcagtacaca
atgcaggcat tagtttctca 600 gttaaaaaac aaggagagac agtagctgat
gttaggacac tacccaatgc ctcaaccgtg 660 gacaatattc gctccatctt
tggaaatgct gttagtcgag aactgataga aattggatgt 720 gaggataaaa
ccctagcctt caaaatgaat ggttacatat ccaatgcaaa ctactcagtg 780
aagaagtgca tcttcttact cttcatcaac catcgtctgg tagaatcaac ttccttgaga
840 aaagccatag aaacagtgta tgcagcctat ttgcccaaaa acacacaccc
attcctgtac 900 ctcagtttag aaatcagtcc ccagaatgtg gatgttaatg
tgcaccccac aaagcatgaa 960 gttcacttcc tgcacgagga gagcatcctg
gagcgggtgc agcagcacat cgagagcaag 1020 ctcctgggct ccaattcctc
caggatgtac ttcacccaga ctttgctacc aggacttgct 1080 ggcccctctg
gggagatggt taaatccaca acaagtctga cctcgtcttc tacttctgga 1140
agtagtgata aggtctatgc ccaccagatg gttcgtacag attcccggga acagaagctt
1200 gatgcatttc tgcagcctct gagcaaaccc ctgtccagtc agccccaggc
cattgtcaca 1260 gaggataaga cagatatttc tagtggcagg gctaggcagc
aagatgagga gatgcttgaa 1320 ctcccagccc ctgctgaagt ggctgccaaa
aatcagagct tggaggggga tacaacaaag 1380 gggacttcag aaatgtcaga
gaagagagga cctacttcca gcaaccccag aaagagacat 1440 cgggaagatt
ctgatgtgga aatggtggaa gatgattccc gaaaggaaat gactgcagct 1500
tgtacccccc ggagaaggat cattaacctc actagtgttt tgagtctcca ggaagaaatt
1560 aatgagcagg gacatgaggt tctccgggag atgttgcata accactcctt
cgtgggctgt 1620 gtgaatcctc agtgggcctt ggcacagcat caaaccaagt
tataccttct caacaccacc 1680 aagcttagtg aagaactgtt ctaccagata
ctcatttatg attttgccaa ttttggtgtt 1740 ctcaggttat cggagccagc
accgctcttt gaccttgcca tgcttgcctt agatagtcca 1800 gagagtggct
ggacagagga agatggtccc aaagaaggac ttgctgaata cattgttgag 1860
tttctgaaga agaaggctga gatgcttgca gactatttct ctttggaaat tgatgaggaa
1920 gggaacctga ttggattacc ccttctgatt gacaactatg tgcccccttt
ggagggactg 1980 cctatcttca ttcttcgact agccactgag gtgaattggg
acgaagaaaa ggaatgtttt 2040 gaaagcctca gtaaagaatg cgctatgttc
tattccatcc ggaagcagta catatctgag 2100 gagtcgaccc tctcaggcca
gcagagtgaa gtgcctggct ccattccaaa ctcctggaag 2160 tggactgtgg
aacacattgt ctataaagcc ttgcgctcac acattctgcc tcctaaacat 2220
ttcacagaag atggaaatat cctgcagctt gctaacctgc ctgatctata caaagtcttt
2280 gagaggtgtt aaatatggtt atttatgcac tgtgggatgt gttcttcttt
ctctgtattc 2340 cgatacaaag tgttgtatca aagtgtgata tacaaagtgt
accaacataa gtgttggtag 2400 cacttaagac ttatacttgc cttctgatag
tattccttta tacacagtgg attgattata 2460 aataaataga tgtgtcttaa cata
2484 17 133 PRT Homo sapiens 17 Met Glu Arg Ala Glu Ser Ser Ser Thr
Glu Pro Ala Lys Ala Ile Lys 1 5 10 15 Pro Ile Asp Arg Lys Ser Val
His Gln Ile Cys Ser Gly Gln Val Val 20 25 30 Leu Ser Leu Ser Thr
Ala Val Lys Glu Leu Val Glu Asn Ser Leu Asp 35 40 45 Ala Gly Ala
Thr Asn Ile Asp Leu Lys Leu Lys Asp Tyr Gly Val Asp 50 55 60 Leu
Ile Glu Val Ser Asp Asn Gly Cys Gly Val Glu Glu Glu Asn Phe 65 70
75 80 Glu Gly Leu Thr Leu Lys His His Thr Ser Lys Ile Gln Glu Phe
Ala 85 90 95 Asp Leu Thr Gln Val Glu Thr Phe Gly Phe Arg Gly Glu
Ala Leu Ser 100 105 110 Ser Leu Cys Ala Leu Ser Asp Val Thr Ile Ser
Thr Cys His Ala Ser 115 120 125 Ala Lys Val Gly Thr 130 18 426 DNA
Homo sapiens 18 cgaggcggat cgggtgttgc atccatggag cgagctgaga
gctcgagtac agaacctgct 60 aaggccatca aacctattga tcggaagtca
gtccatcaga tttgctctgg gcaggtggta 120 ctgagtctaa gcactgcggt
aaaggagtta gtagaaaaca gtctggatgc tggtgccact 180 aatattgatc
taaagcttaa ggactatgga gtggatctta ttgaagtttc agacaatgga 240
tgtggggtag aagaagaaaa cttcgaaggc ttaactctga aacatcacac atctaagatt
300 caagagtttg ccgacctaac tcaggttgaa acttttggct ttcgggggga
agctctgagc 360 tcactttgtg cactgagcga tgtcaccatt tctacctgcc
acgcatcggc gaaggttgga 420 acttga 426
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