U.S. patent application number 10/646950 was filed with the patent office on 2004-04-15 for afc1 and rce1: isoprenylated caax processing enzymes.
Invention is credited to Ashby, Matthew N., Boyartchuk, Victor L., Rine, Jasper D..
Application Number | 20040072296 10/646950 |
Document ID | / |
Family ID | 32074190 |
Filed Date | 2004-04-15 |
United States Patent
Application |
20040072296 |
Kind Code |
A1 |
Rine, Jasper D. ; et
al. |
April 15, 2004 |
AFC1 and RCE1: isoprenylated CAAX processing enzymes
Abstract
Two genes which encode polypeptides that mediate
post-prenylation processing steps in CAAX polypeptides such as Ras
are provided. The two genes (AFC1 and RCE1) encode polypeptides
that mediate the removal of the AAX tripeptide from the CAAX
polypeptide following prenylation. The genes and encoded
polypeptides provide assays for testing compounds for an effect on
post-prenylation processing steps. A heat shock assay for assessing
Ras activity is also provided.
Inventors: |
Rine, Jasper D.; (Moraga,
CA) ; Boyartchuk, Victor L.; (Berkeley, CA) ;
Ashby, Matthew N.; (Mill Valley, CA) |
Correspondence
Address: |
RICHARD ARON OSMAN
SCIENCE AND TECHNOLOGY LAW GROUP
242 AVE VISTA DEL OCEANO
SAN CLEMEMTE
CA
92672
US
|
Family ID: |
32074190 |
Appl. No.: |
10/646950 |
Filed: |
August 21, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10646950 |
Aug 21, 2003 |
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09165460 |
Oct 2, 1998 |
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09165460 |
Oct 2, 1998 |
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08902774 |
Jul 30, 1997 |
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60023491 |
Aug 7, 1996 |
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Current U.S.
Class: |
435/69.1 ;
435/226; 435/320.1; 435/325; 536/23.2 |
Current CPC
Class: |
A61K 48/00 20130101;
C12N 9/60 20130101 |
Class at
Publication: |
435/069.1 ;
435/226; 435/320.1; 435/325; 536/023.2 |
International
Class: |
C12N 009/64; C07H
021/04; C12P 021/02; C12N 005/06 |
Goverment Interests
[0002] This invention was made with support pursuant to Grant
(Contract) No. GM 35827 awarded by the National Institutes of
Health and with support pursuant to Grant (Contract) Nos. 4FT-0083
and 1RT-0026 awarded by the Tobacco-Related Disease Research
Program.
Claims
What is claimed is:
1. A recombinant expression vector comprising a heterologous
promoter operably linked to an expressed polynucleotide which
naturally encodes an Afc1 polypeptide, wherein said polypeptide
mediates the proteolytic removal of an AAX tripeptide from a
prenylated CAAX protein.
2. A vector according to claim 1, wherein the polynucleotide
comprises SEQ ID NO:1.
3. A vector according to claim 1, wherein the polypeptide comprises
SEQ ID NO:2.
4. An isolated polynucleotide comprising SEQ ID NO:6 hybridized to
an Afc1 transcript.
5. A recombinant expression vector comprising a promoter operably
linked to an expressed polynucleotide which naturally encodes an
Rce1 polypeptide, wherein said polypeptide mediates the proteolytic
removal of an AAX tripeptide from a prenylated CAAX protein.
6. A vector according to claim 5, wherein the polynucleotide
comprises SEQ ID NO:3.
7. A vector according to claim 5, wherein the polypeptide comprises
SEQ ID NO:4.
8. An isolated polynucleotide comprising SEQ ID NO:5 hybridized to
an Rce1 transcript.
9. A recombinant cell transduced with the vector of claim 1.
10. A recombinant cell transduced with the polynucleotide of claim
4.
11. A recombinant cell transduced with the vector of claim 5.
12. A recombinant cell transduced with the polynucleotide of claim
8.
13. A method for making a polynucleotide according to claim 4, the
method comprising the step of hybridizing a polynucleotide
comprising SEQ ID NO:6 with an Afc1 transcript to form a
polynucleotide according to claim 4.
14. A method for making a polynucleotide according to claim 8, the
method comprising the step of hybridizing a polynucleotide
comprising SEQ ID NO:5 with an RCE1 transcript to form a
polynucleotide according to claim 8.
15. A method of identifying a compound which inhibits the
proteolytic removal of an AAX tripeptide of a CAAX protein in a
cell, the method comprising steps: contacting a sample comprising a
recombinant cell according to claim 9, or lysate thereof with a
test compound; and measuring activity or expression of the Afc1p or
Rce1p expressed by the cell, wherein compound-dependent inhibition
of the activity or expression indicates that the compound inhibits
the proteolytic removal of the AAX tripeptide.
16. A method of identifying a compound which inhibits the
proteolytic removal of an AAX tripeptide of a CAAX protein in a
cell, the method comprising steps: contacting a sample comprising a
recombinant cell according to claim 10, or lysate thereof with a
test compound; and measuring activity or expression of the Afc1p or
Rce1p expressed by the cell, wherein compound-dependent inhibition
of the activity or expression indicates that the compound inhibits
the proteolytic removal of the AAX tripeptide.
17. A method of identifying a compound which inhibits the
proteolytic removal of an AAX tripeptide of a CAAX protein in a
cell, the method comprising steps: contacting a sample comprising a
recombinant cell according to claim 11, or lysate thereof with a
test compound; and measuring activity or expression of the Afc1p or
Rce1p expressed by the cell, wherein compound-dependent inhibition
of the activity or expression indicates that the compound inhibits
the proteolytic removal of the AAX tripeptide.
18. A method of identifying a compound which inhibits the
proteolytic removal of an AAX tripeptide of a CAAX protein in a
cell, the method comprising steps: contacting a sample comprising a
recombinant cell according to claim 12, or lysate thereof with a
test compound; and measuring activity or expression of the Afc1p or
Rce1p expressed by the cell, wherein compound-dependent inhibition
of the activity or expression indicates that the compound inhibits
the proteolytic removal of the AAX tripeptide.
19. A method of identifying a compound which inhibits Rce1p
activity or Afc1p activity, the method comprising steps: expressing
from a polynucleotide according to claim 4 an Afc1 or Rce1
polypeptide; isolating the polypeptide; contacting a test compound
to a sample comprising the isolated polypeptide; and measuring an
activity selected from the group consisting of Afc1p activity,
Rce1p activity, Afc1p expression, and Rce1p expression, wherein
compound-dependent inhibition of the activity indicates that the
compound inhibits Rce1p activity or Afc1p activity.
20. A method of identifying a compound which inhibits Rce1p
activity or Afc1p activity, the method comprising steps: expressing
from a polynucleotide according to claim 8 an Afc1 or Rce1
polypeptide; isolating the polypeptide; contacting a test compound
to a sample comprising the isolated polypeptide; and measuring an
activity selected from the group consisting of Afc1p activity,
Rce1p activity, Afc1p expression, and Rce1p expression, wherein
compound-dependent inhibition of the activity indicates that the
compound inhibits Rce1p activity or Afc1p activity.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of and claims priority
under 35 U.S.C. .sctn. 120 to 09/165,460, filed Oct. 2, 1998, which
is a divisional of 08/902,774, filed Jul. 30, 1997, abandoned,
which claims the benefit of 60/023,491, filed Aug. 7, 1996, all of
which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] A cell possesses thousands of unique proteins that serve
structural, enzymatic or signaling functions. The intracellular
environment is composed of a myriad of structures and
membrane-enclosed compartments. The correct subcellular
localization is critical for the proper functioning of many
proteins. Proteins situated at the lipid bilayer membrane are
classified as peripheral proteins, whereas proteins situated within
the lipid bilayer membrane are classified as integral membrane
proteins. Integral membrane proteins possess intrinsic hydrophobic
regions which are inserted into the lipid bilayer as they are
synthesized. Typically, peripheral membrane proteins are less
tightly associated with membranes and are localized to the lipid
bilayer by protein-protein interactions, by intrinsic hydrophobic
properties or by the post-translational addition of a lipid
group.
[0004] A major class of peripheral membrane proteins, known as
prenylated proteins, are modified by isoprenoids on a so-called
CAaa.sub.1Aaa.sub.2Xaa (CAAX) motif, wherein C is cysteine,
Aaa.sub.1 and Aaa.sub.2 are aliphatic amino acids and Xaa is any
amino acid. This tetra-peptide sequence is located at the proteins'
carboxyl termini and triggers a series of modification reactions.
Of the approximately 30 known CAAX-containing proteins, the Ras
family of small GTP-binding proteins are major constituents. Ras
proteins localize at the inner surface of the plasma membrane where
they function as key components of various signal transduction
pathways or participate in cytoskeletal organization and
establishment of cell polarity. The critical role of the Ras
proto-oncogene in controlling cell division is exemplified by the
participation of mutated forms of the Ras protein in a variety of
human tumors, including colorectal carcinoma, exocrine pancreatic
carcinoma and myeloid leukemias. Forms of Ras in cancer cells have
mutations that distinguish the protein from Ras in normal
cells.
[0005] The presence of the CAaa.sub.1Aaa.sub.2Xaa motif sequence
targets the protein for at least 3 post-translational
modifications. Generally, such modifications include prenylation of
the cysteine amino acid, proteolytic removal of the terminal three
amino acids (i.e., the Aaa.sub.1Aaa.sub.2Xaa tripeptide) and
methylesterification of the prenylated cysteine, i.e., the
C-terminus. More particularly, in the first step, a 15 carbon
farnesyl or a 20 carbon geranylgeranyl isoprenyl lipid is added to
the cysteine residue. The lipid which is added depends upon the
amino acid at the "X" position. Following prenylation, the terminal
tripeptide, i.e., the Aaa.sub.1Aaa.sub.2Xaa tripeptide, is removed
by a membrane-bound endoprotease. Thereafter, the resulting
C-terminal isoprenylated cysteine is methylesterified.
[0006] It has been determined that prenylation of the CAAX motif is
essential for the proper functioning of every prenylated protein
that has been tested to date. However, the functional requirement
of CAAX proteolysis has not been rigorously evaluated because the
gene encoding the protease has been elusive. This is true despite
the fact that the entire yeast genome has been sequenced and the
sequences deposited in GenBank. Unfortunately, elucidation of the
complete yeast genome in the absence of functional information for
each yeast gene is insufficient for identification of any
particular gene. Although many open reading frames (ORFs) have been
identified, it is not known whether these ORFs encode functional
mRNAs.
[0007] Kato, et al. (Proc. Natl. Acad. Sci. USA, 89:9554-9558
(1992)) monitored foci formation of NIH3T3 cells transformed with
activated forms of Ras with altered CAAX sequences. They found that
one sequence, CVYS, when substituted for the normal Ras CAAX
sequence appeared not to have undergone proteolysis and resulted in
approximately 50% reduction in foci formation. Unfortunately, the
design of this experiment was not ideal because it relied on the
heterologous expression of Ras from an SV40 promoter, which
resulted in a considerably higher expression level than the
physiological Ras promoter. Moreover, the CAAX sequence CVYS
displayed a prenylation defect.
[0008] In view of the foregoing, there remains a need in the art
for the identification of the genes encoding the polypeptides that
participate in the post-prenylation modification reactions so that
the functional importance of such enzymes can be elucidated.
SUMMARY OF THE INVENTION
[0009] The present invention includes the discovery of two families
of genes which encode polypeptides that mediate the proteolytic
removal of an AAX tripeptide from a prenylated CAAX protein in a
cell. In yeast, the families of genes are represented by the genes
AFC1 and RCE1 which encode the polypeptides Afc1p and Rce1p,
respectively.
[0010] Accordingly, the invention provides vectors that includes a
nucleic acid sequence which encodes an Afc1p or Rce1p polypeptide
(or both polypeptides), or conservatively modified variations of
Afc1p or Rce1p. Exemplar nucleic acids which encode Afc1p, or Rce1p
include those set forth in SEQ ID NO:1 and SEQ ID NO:2. Recombinant
cells, including recombinant yeast cells, which comprise a vector
nucleic acid of the invention are also provided.
[0011] In one class of embodiments, the vector of the invention
provides a nucleic acid sequence which hybridizes under stringent
conditions to a nucleic acid selected from the group consisting of
the AFC1, and RCE1 genes. Exemplar nucleic acids with the desired
hybridization properties include those represented by the sequences
of SEQ ID NO:1 and SEQ ID NO:3.
[0012] The invention provides isolated polypeptides, such as Afc1p
and Rce1p, encoded by the vectors of the invention. Exemplar
polypeptides include those represented by SEQ ID NO:2 and SEQ ID
NO:4. Antibodies which specifically bind to the polypeptides of the
invention are also provided.
[0013] In addition to nucleic acids, cells, polypeptides and
antibodies, a variety of useful methods and assays are provided by
the present invention. In one embodiment, the invention provides
methods for inhibiting the proteolytic removal of an AAX tripeptide
from a prenylated CAAX protein in a cell. Exemplar prenylated CAAX
proteins include the Ras protein, a-factor, and the .gamma.-subunit
of the heterotrimeric G-protein. In these methods, a mutation is
introduced into an AFC1 and/or RCE1 gene.
[0014] In one class of embodiments, the invention provides methods
for inhibiting the proteolytic removal of an AAX tripeptide from a
prenylated CAAX protein in a cell. In this class of embodiments,
the activity of the Afc1p or Rce1p protein is blocked using an
inhibitor. Exemplar inhibitors include 1,10-phenanthroline and NME
181.
[0015] The invention provides assays for testing the inhibitory
activity of a potential inhibitor of the Afc1p or Rce1p proteases,
which are responsible for the proteolytic removal of an AAX
tripeptide of a CAAX protein in a cell. In the assay method, a test
compound to be tested for inhibitory activity is provided. The test
compound is contacted to a cell expressing either the AFC1 or RCE1
genes, or both. The transcriptional or translational activity of
the genes or, alternatively, the activity of the encoded proteins,
is measured, and typically compared to a reference, such as a
control assay which establishes the activity of the measured
activity in the absence of the test compound. One convenient
activity which is mediated by the AFC1 and RCE1 genes is heat shock
sensitivity of cells. Accordingly, in one embodiment, the measured
activity is heat shock sensitivity. In a second convenient assay,
the level of Afc1p or Rce1p protein in a population of cells is
measured in a standard immunological assay, such as an ELISA.
[0016] In addition, the present invention provides an improved
method for monitoring heat shock sensitivity, particularly in
yeast, is provided. In this method, a plurality of aliquoted yeast
strains in liquid are provided. Each strain is separated into a
test population of cells and a control population of cells. The
test population of cells is heated to a heat shock temperature of
between about 40.degree. C. and about 60.degree. C. for a time
period of between about 30 seconds and about 10 minutes, followed
by cooling to a temperature of between about 0.degree. C. and about
35.degree. C. In a preferred embodiment, the test population of
cells is heated and cooled in a PCR thermocycler to allow for
better temperature control. The test population of cells and the
control population of cells are grown on growth media and
quantitated. The number of test and control cells are compared. The
comparison of the number of cells in the test population and the
control population provides a measure of heat shock sensitivity.
Exemplar yeast strains include .DELTA.afc1, .DELTA.rce1, and the
double deletion strain Aafcl-Arcel.
Definitions
[0017] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Singleton et al. (1994) Dictionary of Microbiology and Molecular
Biology, second edition, John Wiley and Sons (New York); Walker
(ed) (1988) The Cambridge Dictionary of Science and Technology, The
press syndicate of the University of Cambridge, NY; and Hale and
Marham (1991) The Harper Collins Dictionary of Biology Harper
Perennial, N.Y. provide one of skill with a general dictionary of
many of the terms used in reference to this invention. Although any
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, certain preferred methods and materials are described in
detail. For purposes of the present invention, the following terms
are defined below.
[0018] "CAaa.sub.1Aaa.sub.2Xaa" or, interchangeably, "CAAX," as
used herein, refers to a carboxy-terminal motif sequence, wherein C
is cysteine, Aaa.sub.1 and Aaa.sub.2 are aliphatic amino acids and
Xaa is any one of a number of different amino acids. The presence
of the CAaa.sub.1Aaa.sub.2Xaa motif sequence targets the protein
for at least 3 post-translational modifications. Such modifications
include prenylation of the cysteine amino acid, proteolytic removal
of the terminal three amino acids (i.e., the Aaa.sub.1Aaa.sub.2Xaa
tripeptide) and methylesterification of the prenylated cysteine,
i.e., the C-terminus. Examples of CAAX-containing proteins include,
but are not limited to, fungal mating pheromones, RAS proteins,
nuclear lamins and the .gamma.-subunit of trimeric G-proteins (see,
e.g., Hrycyna, et al., EMBO Journal, 10(7): 1699-1709 (1991)).
[0019] An "Aaa.sub.1Aaa.sub.2Xaa tripeptide," as used herein refers
to the terminal three amino acids of the CAaa.sub.1Aaa.sub.2Xaa
motif sequence.
[0020] A "prenylated CAaa.sub.1Aaa.sub.2Xaa protein" refers to a
protein containing a CAaa.sub.1Aaa.sub.2Xaa motif sequence, wherein
the cysteine amino acid has been prenylated by the addition of a
geranyl, farnesyl or geranylgeranyl lipid.
[0021] "Proteolytic cleavage," as used herein, refers to the
removal of the terminal three amino acids of the
CAaa.sub.1Aaa.sub.2Xaa motif sequence through the cleavage of a
peptide bond by a protease.
[0022] "Inhibit" or, interchangeably, "antagonize," or "blocking
the activity" as used herein, refers to the reduction or prevention
of a reaction or process.
[0023] A "yeast strain" is a population of yeast cells, each of
which share a particular phenotype or a particular genotype.
[0024] A "test population of cells" is a population of cells to be
characterized, e.g., in a method or an assay. A "control"
population of cells is a population of cells which are used to
determine that an observed effect in a method or assay is the
result of experimental manipulation, and not the result of an
unknown or unintended environmental parameter.
[0025] A "vector" is a composition which can transduce, transfect,
transform or infect a cell, thereby causing the cell to replicate
or express nucleic acids and/or proteins other than those native to
the cell, or in a manner not native to the cell. A cell is
"transduced" by a nucleic acid when the nucleic acid is
translocated into the cell from the extracellular environment. Any
method of transferring a nucleic acid into the cell may be used;
the term, unless otherwise indicated, does not imply any particular
method of delivering a nucleic acid into a cell, nor that any
particular cell type is the subject of transduction. A cell is
"transformed" by a nucleic acid when the nucleic acid is transduced
into the cell and stably replicated. A vector includes a nucleic
acid (ordinarily RNA or DNA) to be expressed by the cell. This
nucleic acid is optionally referred to as a "vector nucleic acid."
A vector optionally includes materials to aid in achieving entry of
the nucleic acid into the cell, such as a viral particle, liposome,
protein coating or the like. A "cell transduction vector" is a
vector which encodes a nucleic acid which is expressed in a cell
once the nucleic acid is transduced into the cell.
[0026] A "promoter" is an array of nucleic acid control sequences
which direct transcription of a nucleic acid. As used herein, a
promoter includes necessary nucleic acid sequences near the start
site of transcription, such as, in the case of a polymerase II type
promoter, a TATA element. A promoter also optionally includes
distal enhancer or repressor elements which can be located as much
as several thousand base pairs from the start site of
transcription. A "constitutive" promoter is a promoter which is
active under most environmental and developmental conditions. An
"inducible" promoter is a promoter which is under environmental or
developmental regulation. A "tissue specific" promoter is active in
certain tissue types of an organism, but not in other tissue types
from the same organism.
[0027] The term "operably linked" refers to functional linkage
between a nucleic acid expression control sequence (such as a
promoter, or array of transcription factor binding sites) and a
second nucleic acid sequence, wherein the expression control
sequence directs transcription of the nucleic acid corresponding to
the second sequence.
[0028] The term "nucleic acid" refers to a deoxyribonucleotide or
ribonucleotide polymer in either single- or double-stranded form,
and unless otherwise limited, encompasses known analogues of
natural nucleotides that hybridize to nucleic acids in manner
similar to naturally occurring nucleotides. Unless otherwise
indicated, a particular nucleic acid sequence optionally includes
the complementary sequence thereof.
[0029] The term "subsequence" in the context of a particular
nucleic acid sequence refers to a region of the nucleic acid equal
to or smaller than the specified nucleic acid. A "recombinant
nucleic acid" comprises or is encoded by one or more nucleic acids
that are derived from a nucleic acid which was artificially
constructed. For example, the nucleic acid can comprise or be
encoded by a cloned nucleic acid formed by joining heterologous
nucleic acids as taught, e.g., in Berger and Kimmel, Guide to
Molecular Cloning Techniques, Methods in Enzymology volume 152
Academic Press, Inc., San Diego, Calif. (Berger) and in Sambrook et
al. (1989) Molecular Cloning--A Laboratory Manual (2nd ed.) Vol.
1-3 (Sambrook). Alternatively, the nucleic acid can be synthesized
chemically. The term "recombinant" when used with reference to a
cell indicates that the cell replicates or expresses a nucleic
acid, or expresses a peptide or protein encoded by a nucleic acid
whose origin is exogenous to the cell. Recombinant cells can
express genes that are not found within the native
(non-recombinant) form of the cell. Recombinant cells can also
express genes found in the native form of the cell wherein the
genes are re-introduced into the cell or a progenitor of the cell
by artificial means.
[0030] The terms "isolated" or "biologically pure" refer to
material which is substantially or essentially free from components
which normally accompany it as found in its native state.
[0031] "Stringent hybridization" and "Stringent hybridization wash
conditions" in the context of nucleic acid hybridization
experiments such as Southern and northern hybridizations are
sequence dependent, and are different under different environmental
parameters. An extensive guide to the hybridization of nucleic
acids is found in Tijssen (1993) Laboratory Techniques in
Biochemistry and Molecular Biology--Hybridization with Nucleic Acid
Probes part I chapter 2 "overview of principles of hybridization
and the strategy of nucleic acid probe assays", Elsevier, N.Y.
Generally, highly stringent hybridization and wash conditions are
selected to be about 5.degree. C. lower than the thermal melting
point (T.sub.m) for the specific sequence at a defined ionic
strength and pH. The T.sub.m is the temperature (under defined
ionic strength and pH) at which 50% of the target sequence
hybridizes to a perfectly matched probe. Very stringent conditions
are selected to be equal to the T.sub.m for a particular probe.
[0032] An example of stringent hybridization conditions for
hybridization of complementary nucleic acids which have more than
100 complementary residues on a filter in a Southern or northern
blot is 50% formamide with 1 mg of heparin per 50 mL at 42.degree.
C., with the hybridization being carried out overnight. An example
of stringent wash conditions is a 0.2.times.SSC wash at 65.degree.
C. for 15 minutes (see, Sambrook, supra for a description of SSC
buffer). Often the high stringency wash is preceded by a low
stringency wash to remove background probe signal. An example
medium stringency wash for a duplex of, e.g., more than 100
nucleotides, is 1.times.SSC at 45.degree. C. for 15 minutes. An
example low stringency wash for a duplex of, e.g., more than 100
nucleotides, is 4.times.SSC at 40.degree. C. for 15 minutes. In
general, a signal to noise ratio of 2.times.(or higher) than that
observed for an unrelated probe in the particular hybridization
assay indicates detection of a specific hybridization.
[0033] Nucleic acids which do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides which they encode are substantially identical. This
occurs, e.g., when a copy of a nucleic acid is created using the
maximum codon degeneracy permitted by the genetic code.
[0034] The term "identical" in the context of two nucleic acid or
polypeptide sequences refers to the residues in the two sequences
which are the same when aligned for maximum correspondence. When
percentage of sequence identity is used in reference to proteins or
peptides it is recognized that residue positions which are not
identical often differ by conservative amino acid substitutions,
where amino acid residues are substituted for other amino acid
residues with similar chemical properties (e.g. charge or
hydrophobicity) and therefore do not change the functional
properties of the molecule. Where sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Means for making this adjustment are well known to those of skill
in the art. Typically this involves scoring a conservative
substitution as a partial rather than a full mismatch, thereby
increasing the percentage sequence identity. Thus, for example,
where an identical amino acid is given a score of 1 and a
non-conservative substitution is given a score of zero, a
conservative substitution is given a score between zero and 1. The
scoring of conservative substitutions is calculated, e.g.,
according to known algorithm. See, e.g., Myers and Miller, Comput
Appl Biosci (now Bioinformatics), 4: 11-17 (1988); Smith and
Waterman (1981) Adv. Appl. Math. 2: 482; Needleman and Wunsch
(1970) J. Mol. Biol. 48: 443; Pearson and Lipman (1988) Proc. Natl.
Acad. Sci. USA 85: 2444; Higgins and Sharp (1988) Gene, 73: 237-244
and Higgins and Sharp (1989) CABIOS 5: 151-153; Corpet, et al.
(1988) Nucleic Acids Research 16, 10881-90; Huang, et al. (1992)
Computer Applications in the Biosciences 8, 155-65, and Pearson, et
al. (1994) Methods in Molecular Biology 24, 307-31. Alignment is
also often performed by inspection and manual alignment.
[0035] "Conservatively modified variations" of a particular nucleic
acid sequence refers to those nucleic acids which encode identical
or essentially identical amino acid sequences, or where the nucleic
acid does not encode an amino acid sequence, to essentially
identical sequences. Because of the degeneracy of the genetic code,
a large number of functionally identical nucleic acids encode any
given polypeptide. For instance, the codons CGU, CGC, CGA, CGG,
AGA, and AGG all encode the amino acid arginine. Thus, at every
position where an arginine is specified by a codon, the codon can
be altered to any of the corresponding codons described without
altering the encoded polypeptide. Such nucleic acid variations are
"silent variations," which are one species of "conservatively
modified variations." Every nucleic acid sequence herein which
encodes a polypeptide also describes every possible silent
variation. One of skill will recognize that each codon in a nucleic
acid (except AUG, which is ordinarily the only codon for
methionine) can be modified to yield a functionally identical
molecule by standard techniques. Accordingly, each "silent
variation" of a nucleic acid which encodes a polypeptide is
implicit in each described sequence. Furthermore, one of skill will
recognize that individual substitutions, deletions or additions
which alter, add or delete a single amino acid or a small
percentage of amino acids (typically less than 5%, more typically
less than 1%) in an encoded sequence are "conservatively modified
variations" where the alterations result in the substitution of an
amino acid with a chemically similar amino acid. Conservative
substitution tables providing functionally similar amino acids are
well known in the art. The following six groups each contain amino
acids that are conservative substitutions for one another:
[0036] 1) Alanine (A), Serine (S), Threonine (T);
[0037] 2) Aspartic acid (D), Glutamic acid (E);
[0038] 3) Asparagine (N), Glutamine (Q);
[0039] 4) Arginine (R), Lysine (K);
[0040] 5) Isoleucine (1), Leucine (L), Methionine (M), Valine (V);
and
[0041] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0042] The term "antibody" refers to a polypeptide substantially
encoded by an immunoglobulin gene or immunoglobulin genes, or
fragments thereof. The recognized immunoglobulin genes include the
kappa, lambda, alpha, gamma, delta, epsilon and mu constant region
genes, as well as myriad immunoglobulin variable region genes.
Light chains are classified as either kappa or lambda. Heavy chains
are classified as gamma, mu, alpha, delta, or epsilon, which in
turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively.
[0043] An exemplar immunoglobulin (antibody) structural unit
comprises a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each
chain defines a variable region of about 100 to 110 or more amino
acids primarily responsible for antigen recognition. The terms
variable light chain (V.sub.L) and variable heavy chain (V.sub.H)
refer to these light and heavy chains respectively.
[0044] Antibodies exist, for example, as intact immunoglobulins or
as a number of well characterized fragments produced by digestion
with various peptidases. Thus, for example, pepsin digests an
antibody below the disulfide linkages in the hinge region to
produce F(ab)'.sub.2, a dimer of Fab which itself is a light chain
joined to V.sub.H-C.sub.H1 by a disulfide bond. The F(ab)'.sub.2
may be reduced under mild conditions to break the disulfide linkage
in the hinge region thereby converting the F(ab)'.sub.2 dimer into
an Fab' monomer. The Fab' monomer is essentially an Fab with part
of the hinge region (see, Fundamental Immunology, Third Edition, W.
E. Paul, ed., Raven Press, N.Y. (1993), which is incorporated
herein by reference, for a more detailed description of other
antibody fragments). While various antibody fragments are defined
in terms of the digestion of an intact antibody, one of skill will
appreciate that such Fab' fragments may be synthesized de novo
either chemically or by utilizing recombinant DNA methodology.
Thus, the term antibody, as used herein, also includes antibody
fragments either produced by the modification of whole antibodies
or those synthesized de novo using recombinant DNA methodologies.
Antibodies in the context of the present invention are optionally
derived from libraries of recombinant antibodies in phage or
similar vectors (see, e.g., Huse et al. (1989) Science 246:
1275-1281; and Ward, et al. (1989) Nature 341: 544-546; and Vaughan
et al. (1996) Nature Biotechnology, 14: 309-314).
DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS
[0045] Proteins with a CAAX motif, such as Ras, a-factor and the
.gamma.-subunit of heterotrimeric G-protein, are
post-translationally modified by prenylation of the cysteine,
proteolytic removal of the terminal three residues and
methylation-esterification of the newly-formed carboxyl group. A
novel, farnesylation-dependent endoproteolytic activity named RACE
(Ras and a-factor Converting Enzyme) was previously discovered.
Numerous efforts to identify a CAAX protease gene responsible for
the activity were unsuccessful. An autocrine arrest, sensitized
selection for such mutants was developed. It involved ectopic
expression of the a-factor receptor in a cells and utilized a CAAX
sequence permutation defective for proteolysis. 127 mutants were
isolated and characterized. 24 of these mutants had altered
substrate specificity, of which 2 had novel alleles of RAM1. The
remaining 22 had mutations in a single new gene, AFC1 (a-Factor
Convertase).
[0046] Afc1p is the first identified farnesylation-dependent zinc
metalloprotease. It is an integral membrane protein localized to
internal membranes. Mutations in the HEXXH motif found in the
protein, and characteristic of zinc metalloproteases, destroy Afc1p
function. Null alleles of AFC1 are viable and produce lowered
levels of mature a-factor. The residual pheromone produced by these
cells implies the existence of multiple prenyl-dependent proteases.
Ras2p with a proteolysis defective C-terminus has altered
biological properties, suggesting that CAAX proteolysis is
important for Ras function. Accordingly, AFC1 and the proteins
encoded by the protein are attractive targets for therapeutics
against Ras dependent cancers. Because of this, assays which can be
used to detect the inhibition of AFC1 activity are of immediate
commercial value to pharmaceutical companies.
[0047] RCE1 encodes a 315 amino acids long protein. Extensive
searches against public molecular biology databases revealed no
significant homologies to any known gene. Conceptual translation of
the sequence obtained by mouse cDNA sequencing project (XREF Clone
ID 331228, GenBank accession W14344, NCBI ID 521315) shows 46%
identity on 49 aa stretch (63% positives) to Rce1p. The Rce1
protein sequence has limited similarity to sequence blocks
characteristic of signal peptidases type II (SPase II), class A8
(lipoprotein signal peptidase, which recognize a conserved sequence
and cuts in front of a cysteine to which a glyceride-fatty acid
lipid is attached). Even though there is limited similarity, a
consensus sequence for A8 SPase II is not present. Analysis of the
Rce1 protein sequence predicts the presence of a number of
transmembrane domains, suggesting that Rce1p is an integral
membrane protein.
[0048] A deletion of RCE1 in haploid yeast cells of a-mating type
has no effect on viability; however, it results in reduced a-factor
halo size. Protease assays (see, Ashby and Rine "Ras and a-Factor
Converting Enzyme" (1995) Methods in Enzymology 230:235) detect
reduced proteolytic activity in the membrane preparations from the
RCE1 null strain.
[0049] RCE1 deletion, combined with a deletion of AFC1, causes
complete sterility of a cells due to a complete bloc in proteolytic
processing of a-factor.
[0050] RCE1 seems to affect activity of yeast homologues of Ras
oncoproteins. When deletion of RCE1 is combined with
temperature-sensitive alleles of RAS2, it further decreases the
viability of the yeast cells at elevated temperatures. The same
deletion decreases the heat shock sensitivity of the yeast strains
carrying an activated form of Ras protein RAS.sup.val19. This
allele is analogous to the mutation in the mammalian
Ras-Ras.sup.val12 found in a number of cancers. Yeast strains
carrying the deletion also show increased intracellular
localization of Ras2p (presumably to internal membranes) as
measured by completely functional GFP-Ras2 fusions. All of this
shows that RCE1 modulates activity of the yeast homologue of Ras
oncoproteins, without completely inhibiting its activity. RCE1,
therefore, is an attractive target for pharmaceutical treatments
directed on reduction of elevated Ras activity, found in a many
malignancies. Because of this, assays which can be used to detect
the inhibition of RCE1 activity are of immediate commercial value
to pharmaceutical companies for the identification of therapeutic
compounds against Ras-mediated cancers.
[0051] Vectors, Cloning, Nucleic Acids and Proteins
[0052] The vectors of the invention include a vector nucleic acid,
and optionally include components for packaging the vector nucleic
acid to facilitate entry of the nucleic acid into a cell. The
vector nucleic acid includes a nucleic acid subsequence which
encodes a nucleic acid or protein of the invention. The subsequence
is typically cloned into a cloning site in the vector nucleic acid
which is designed to facilitate recombinant manipulation. A variety
of commercially or commonly available vectors and vector nucleic
acids can be converted into a vector of the invention by cloning a
nucleic acid encoding a protein of the invention into the
commercially or commonly available vector. A variety of common
vectors suitable for this purpose are well known in the art. For
cloning in bacteria, common vectors include pBR322 derived vectors
such as pBLUESCRIPT.TM., and .lambda.-phage derived vectors. In
yeast, vectors include Yeast Integrating plasmids (e.g., YIp5) and
Yeast Replicating plasmids (the YRp series plasmids), Yeast
Centromeric plasmids (the YCp series of plasmids) and pGPD-2.
Expression in mammalian cells can be achieved using a variety of
plasmids, including pSV2, pBC12BI, and p91023, as well as lytic
virus vectors (e.g., vaccinia virus, adeno virus, and
bacculovirus), episomal virus vectors (e.g., bovine
papillomavirus), and retroviral vectors (e.g., murine
retroviruses).
[0053] The nucleic acid sequence encoding a selected polypeptide is
placed under the control of a promoter. A extremely wide variety of
promoters are well known, and can be used in the vectors of the
invention, depending on the particular application. Ordinarily, the
promoter selected depends upon the cell in which the promoter is to
be active. Other expression control sequences such as ribosome
binding sites, transcription termination sites and the like are
optionally included. For E. coli, example control sequences include
the T7, trp, or lambda promoters, a ribosome binding site and
preferably a transcription termination signal. For eukaryotic
cells, the control sequences typically include a promoter which
optionally includes an enhancer derived from immunoglobulin genes,
SV40, cytomegalovirus, etc., and a polyadenylation sequence, and
may include splice donor and acceptor sequences. In yeast,
convenient promoters include GAL1,10 (Johnson and Davies (1984)
Mol. Cell. Biol. 4:1440-1448) ADH2 (Russell et al. (1983) J. Biol.
Chem. 258:2674-2682), PH05 (EMBO J. (1982) 6:675-680), and
MF.alpha.1 (Herskowitz and Oshima (1982) in The Molecular Biology
of the Yeast Saccharomyces (eds. Strathern, Jones, and Broach) Cold
Spring Harbor Lab., Cold Spring Harbor, N.Y., pp. 181-209).
Multicopy plasmids with selective markers, such as LEU2, URA3,
TRP1, and HIS3 is also commonly used. A number of yeast expression
plasmids such as YEp6, YEp13, YEp4 can be used as expression
vectors. A gene of interest can be fused, e.g., to any of the
promoters in known yeast vectors. The above-mentioned plasmids have
been fully described in the literature (Botstein et al. (1979) Gene
8:17-24; Broach, et al. (1979) Gene, 8:121-133). For a discussion
of yeast expression plasmids, see, e.g., Parents, B., YEAST (1985),
and Ausbel, Sambrook and Berger, all supra).
[0054] Given the strategy for making the vectors and nucleic acids
of the present invention, one of skill can construct a variety of
vectors and nucleic acid clones containing functionally equivalent
nucleic acids. Cloning methodologies to accomplish these ends, and
sequencing methods to verify the sequence of nucleic acids are well
known in the art. Examples of appropriate cloning and sequencing
techniques, and instructions sufficient to direct persons of skill
through many cloning exercises are found in Berger and Kimmel,
Guide to Molecular Cloning Techniques, Methods in Enzymology volume
152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et
al. (1989) Molecular Cloning--A Laboratory Manual (2nd ed.) Vol.
1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY,
(Sambrook); and Current Protocols in Molecular Biology, F. M.
Ausubel et al., eds., Current Protocols, a joint venture between
Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,
(1994 Supplement) (Ausubel). Product information from manufacturers
of biological reagents and experimental equipment also provide
information useful in known biological methods. Such manufacturers
include the SIGMA chemical company (Saint Louis, Mo.), R&D
systems (Minneapolis, Minn.), Pharmacia LKB Biotechnology
(Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto,
Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee,
Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc.
(Gaithersberg, Md.), Fluka Chemica-Biochemika Analytika (Fluka
Chemie AG, Buchs, Switzerland), Invitrogen, San Diego, Calif., and
Applied Biosystems (Foster City, Calif.), as well as many other
commercial sources known to one of skill.
[0055] The nucleic acids provided by this invention, whether RNA,
cDNA, genomic DNA, or a hybrid of the various combinations, are
isolated from biological sources or synthesized in vitro. The
nucleic acids and vectors of the invention are present in
transformed or transfected whole cells, in transformed or
transfected cell lysates, or in a partially purified or
substantially pure form.
[0056] In vitro amplification techniques suitable for amplifying
sequences to provide a nucleic acid, or for subsequent analysis,
sequencing or subcloning are known. Examples of techniques
sufficient to direct persons of skill through such in vitro
amplification methods, including the polymerase chain reaction
(PCR) the ligase chain reaction (LCR), Q.beta.-replicase
amplification and other RNA polymerase mediated techniques (e.g.,
NASBA) are found in Berger, Sambrook, and Ausubel, as well as
Mullis et al., (1987) U.S. Pat. No. 4,683,202; PCR Protocols A
Guide to Methods and Applications (Innis et al. eds) Academic Press
Inc. San Diego, Calif. (1990) (Innis); Arnheim & Levinson (Oct.
1, 1990) C&EN 36-47; The Journal Of NIH Research (1991) 3,
81-94; (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86, 1173;
Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lomell
et al. (1989) J. Clin. Chem 35, 1826; Landegren et al., (1988)
Science 241, 1077-1080; Van Brunt (1990) Biotechnology 8, 291-294
Wu and Wallace, (1989) Gene 4, 560; Barringer et al. (1990) Gene
89, 117, and Sooknanan and Malek (1995) Biotechnology 13: 563-564.
Improved methods of cloning in vitro amplified nucleic acids are
described in Wallace et al., U.S. Pat. No. 5,426,039. Improved
methods of amplifying large nucleic acids are summarized in Cheng
et al. (1994) Nature 369: 684-685 and the references therein. One
of skill will appreciate that essentially any RNA can be converted
into a double stranded DNA suitable for restriction digestion, PCR
expansion and sequencing using reverse transcriptase and a
polymerase. See, Ausbel, Sambrook and Berger, all supra.
[0057] Oligonucleotides for in vitro amplification methods or for
use as gene probes, for example, are typically chemically
synthesized according to the solid phase phosphoramidite triester
method described by Beaucage and Caruthers (1981), Tetrahedron
Letts., 22(20): 1859-1862, e.g., using an automated synthesizer, as
described in Needham-VanDevanter et al. (1984) Nucleic Acids Res.,
12:6159-6168. Purification of oligonucleotides, where necessary, is
typically performed by either native acrylamide gel electrophoresis
or by anion-exchange HPLC as described in Pearson and Regnier
(1983) J. Chrom. 255:137-149. The sequence of the synthetic
oligonucleotides can be verified using the chemical degradation
method of Maxam and Gilbert (1980) in Grossman and Moldave (eds.)
Academic Press, New York, Methods in Enzymology 65:499-560.
[0058] The polypeptides of the invention can be synthetically
prepared in a wide variety of well-know ways. For instance,
polypeptides of relatively short length can be synthesized in
solution or on a solid support in accordance with conventional
techniques. See, e.g., Merrifield (1963) J. Am. Chem. Soc.
85:2149-2154. Various automatic synthesizers are commercially
available and can be used in accordance with known protocols. See,
e.g., Stewart and Young (1984) Solid Phase Peptide Synthesis, 2d.
ed., Pierce Chemical Co. As described in more detail herein, the
polypeptide of the invention are most preferably made using
recombinant techniques, by expressing the polypeptides in host
cells and purifying the expressed proteins.
[0059] In a preferred embodiment, the polypeptides, or subsequences
thereof, are synthesized using recombinant DNA methodology.
Generally this involves creating a DNA sequence that encodes the
protein, through recombinant, synthetic, or in vitro amplification
techniques, placing the DNA in an expression cassette under the
control of a particular promoter, expressing the protein in a host
cell, isolating the expressed protein and, if required, renaturing
the protein.
[0060] Once a nucleic acid encoding a polypeptide of the invention
is isolated and cloned, the nucleic acid is optionally expressed in
recombinantly engineered cells known to those of skill in the art.
Examples of such cells include bacteria, yeast, plant, filamentous
fungi, insect (especially employing baculoviral vectors), and
mammalian cells. The recombinant nucleic acids are operably linked
to appropriate control sequences for expression in the selected
host. For E. coli, example control sequences include the T7, trp,
or lambda promoters, a ribosome binding site and preferably a
transcription termination signal. For eukaryotic cells, the control
sequences typically include a promoter and preferably an enhancer
derived from immunoglobulin genes, SV40, cytomegalovirus, etc., and
a polyadenylation sequence, and optionally include splice donor and
acceptor sequences.
[0061] Plasmids of the invention can be transferred into the chosen
host cell by well-known methods such as calcium chloride
transformation for E. coli and calcium phosphate treatment or
electroporation for mammalian cells. Yeast transformation is
conveniently performed by one of two common procedures. In one
procedure, yeast cells are first converted into protoplasts using
zymolyase, lyticase or glusulase, followed by addition of DNA and
polyethylene glycol (PEG). The PEG-treated protoplasts are then
regenerated in a 3% agar medium under selective conditions. Details
of this procedure are given in Beggs (1978) Nature (London)
275:104-109, and Hinnen, et al. (1978) Proc. Natl. Acad. Sci. USA
75:1929-1933. The second procedure does not involve removal of the
cell wall. Instead, the cells are treated, e.g., with lithium
chloride or acetate and PEG and put on selective plates (Ito, et
al. (1983) J. Bact. 153:163-168).
[0062] Cells transformed by plasmids can be selected by resistance
to antibiotics conferred by genes contained on the plasmids, such
as the amp, gpt, neo and hyg genes. Viral vectors of the invention
transduce nucleic acids into cells within the host range of the
viral vector.
[0063] Once expressed, the recombinant polypeptides can be purified
according to standard procedures of the art, including ammonium
sulfate precipitation, affinity columns, column chromatography, gel
electrophoresis and the like (see, generally, R. Scopes,
Polypeptide Purification, Springer-Verlag, N.Y. (1982), Deutscher,
Methods in Enzymology Vol. 182: Guide to Polypeptide Purification.,
Academic Press, Inc. N.Y. (1990)). Once purified, partially or to
homogeneity as desired, the polypeptides may then be used (e.g., as
immunogens for antibody production).
[0064] After chemical synthesis, biological expression or
purification, the polypeptide(s) may possess a conformation
substantially different than the native conformations of the
constituent polypeptides. In this case, it is helpful to denature
and reduce the polypeptide and then to cause the polypeptide to
re-fold into the preferred conformation. Methods of reducing and
denaturing polypeptides and inducing re-folding are well known to
those of skill in the art (See, Debinski et al. (1993) J. Biol.
Chem., 268: 14065-14070; Kreitman and Pastan (1993) Bioconjug.
Chem., 4: 581-585; and Buchner, et al., (1992) Anal. Biochem., 205:
263-270). Debinski et al., for example, describe the denaturation
and reduction of inclusion body polypeptides in guanidine-DTE. The
polypeptide is then refolded in a redox buffer containing oxidized
glutathione and L-arginine.
[0065] One of skill will recognize that modifications can be made
to the polypeptides without diminishing their biological activity.
Some modifications may be made to facilitate the cloning,
expression, or incorporation of the targeting molecule into a
fusion polypeptide. Such modifications are well known to those of
skill in the art and include, for example, a methionine added at
the amino terminus to provide an initiation site, or additional
amino acids (e.g., poly His) placed on either terminus to create
conveniently located restriction sites or termination codons or
purification sequences.
[0066] Making Conservative Modifications of the Nucleic Acids and
Polypeptides of the Invention.
[0067] One of skill will appreciate that many conservative
variations of the polypeptides and vectors disclosed yield
essentially identical polypeptides and vectors. For example, due to
the degeneracy of the genetic code, "silent substitutions" (i.e.,
substitutions of a nucleic acid sequence which do not result in an
alteration in an encoded polypeptide) are an implied feature of
every nucleic acid sequence which encodes an amino acid. Similarly,
"conservative amino acid substitutions," in one or a few amino
acids in an amino acid sequence are substituted with different
amino acids with highly similar properties (see, the definitions
section, supra), are also readily identified as being highly
similar to a disclosed amino acid sequence, or to a disclosed
nucleic acid sequence which encodes an amino acid. Such
conservatively substituted variations of each disclosed sequence
are a feature of the present invention.
[0068] One of skill will recognize many ways of generating
alterations (introducing mutations) in a given nucleic acid
sequence. Such well-known methods include site-directed
mutagenesis, PCR amplification using degenerate oligonucleotides,
exposure of cells containing the nucleic acid to mutagenic agents
or radiation, chemical synthesis of a desired oligonucleotide
(e.g., in conjunction with ligation and/or cloning to generate
large nucleic acids) and other well-known techniques. See, Giliman
and Smith (1979) Gene 8:81-97; Roberts et al. (1987) Nature
328:731-734 and Sambrook, Innis, Ausbel, Berger, Needham
VanDevanter and Mullis (all supra).
[0069] Most commonly, amino acid sequences are altered by altering
the corresponding nucleic acid sequence and expressing the
polypeptide. However, polypeptide sequences are also optionally
generated synthetically on commercially available peptide
synthesizers to produce any desired polypeptide (see, Merrifield,
and Stewart and Young, supra). General knowledge regarding the
nature of proteins and nucleic acids allows one of skill to select
appropriate sequences with activity similar or equivalent to the
nucleic acids, vectors and polypeptides disclosed herein. The
definitions section herein describes exemplar conservative amino
acid substitutions.
[0070] Most modifications to nucleic acids and polypeptides are
evaluated by routine screening techniques in suitable assays for
the desired characteristic. For instance, changes in the
immunological character of a polypeptide can be detected by an
appropriate immunological assay. Modifications of other properties
such as nucleic acid hybridization to a target nucleic acid, redox
or thermal stability of a protein, hydrophobicity, susceptibility
to proteolysis, or the tendency to aggregate are all assayed
according to standard techniques.
[0071] Human Homologues of AFC1 and RCE1
[0072] The present invention provides for mammalian homologues to
the yeast AFC1 and RCE1 genes. A Genbank search for nucleic acids
encoding sequences similar to the proteins encoded by the
respective genes revealed expression sequence tags (ESTs) with
homology to the yeast genes. The entries are found at accession
number z43273 (SEQ ID NO:5; a partial cDNA encoding a human protein
with similarity to AFC1) and w14344 (SEQ ID NO:6; a partial cDNA
encoding a mouse protein with similarity to RCE1).
[0073] Complete mammalian homologues to AFC1 and RCE1 are isolated
in a variety of ways. In one embodiment, the Afc1p or Rce1p
polypeptides, or polypeptides encoded by the identified mammalian
sequences, are used to raise antibodies as described herein. The
antibodies are used to screen expression libraries for polypeptides
with homology to the immunogen used to raise the antibody. Thus,
the invention provides an isolated clone encoding a polypeptide
which binds to an antibody encoded by a mammalian AFC1 or RCE1
homologue.
[0074] In another embodiment, the nucleic acids encoded by the
yeast AFC1 or RCE1 genes, or mammalian nucleic acids encoded by the
GenBank nos z43273 or w14344 are labeled and hybridized to a
mammalian cDNA or genomic DNA library under increasingly stringent
conditions. Clones which hybridize under moderate to stringent
conditions are homologous to the probe sequences. Preferred clones
hybridize to the selected labeled nucleic acid under stringent
conditions. Mammalian cDNA and genomic libraries are widely
available, and methods of hybridizing nucleic acids to the
libraries are well known.
[0075] In yet another embodiment, the invention provides PCR probes
which are used to amplify a mammalian AFC1 or RCE1 homologue from a
library or tissue sample. Most typically, amplification primers are
between 8 and 100 nucleotides in length, and preferably between
about 10 and 30 nucleotides in length. More typically, the primers
are between about 15 and 25 nucleic acids in length. One of skill
will recognize that the 3' end of an amplification primer is more
important for PCR than the 5' end. Investigators have reported PCR
products where only a few nucleotides at the 3' end of an
amplification primer were complementary to a DNA to be amplified.
In this regard, nucleotides at the 5' end of a primer can
incorporate structural features unrelated to the target nucleic
acid; for instance, in one embodiment, a sequencing primer
hybridization site (or a complement to such as primer, depending on
the application) is incorporated into the amplification primer,
where the sequencing primer is derived from a primer used in a
standard sequencing kit, such as one using a biotinylated or
dye-labeled universal M13 or SP6 primer. Alternatively, the primers
optionally incorporate restriction endonuclease sites. The primers
are selected so that there is no complementarity between any known
sequence which is likely to occur in the sample to be amplified and
any constant primer region. One of skill will appreciate that
constant regions in primer sequences are optional.
[0076] Typically, all primer sequences are selected to hybridize
only to a perfectly complementary DNA, with the nearest mismatch
hybridization possibility from known DNA sequences which are likely
to occur in the sample to be amplified having at least about 50 to
70% hybridization mismatches to sequences which are known to be in
the sample and which do not encode a nucleic acid of the invention,
and preferably 100% mismatches for the terminal 5 nucleotides at
the 3' end of the primer. Alternatively, a series of degenerate
primers with universal base acceptors at ambiguous codon positions
are used in parallel reactions to amplify a nucleic acid.
[0077] The primers are selected so that no secondary structure
forms within the primer. Self-complementary primers have poor
hybridization properties, because the complementary portions of the
primers self hybridize (i.e., form hairpin structures). The primers
are also selected so that the primers do not hybridize to each
other, thereby preventing duplex formation of the primers in
solution, and possible concatenation of the primers during PCR. If
there is more than one constant region in the primer, the constant
regions of the primer are selected so that they do not
self-hybridize or form hairpin structures.
[0078] Where sets of amplification primers (i.e., the 5' and 3'
primers used for exponential amplification) are of a single length,
the primers are selected so that they have roughly the same, and
preferably exactly the same overall base composition (i.e., the
same A+T to G+C ratio of nucleic acids). Where the primers are of
differing lengths, the A+T to G+C ratio is determined by selecting
a thermal melting temperature for the primer-DNA hybridization, and
selecting an A+T to G+C ratio and probe length for each primer
which has approximately the selected thermal melting
temperature.
[0079] One of skill will recognize that there are a variety of
possible ways of performing the above selection steps, and that
variations on the steps are appropriate.
[0080] Most typically, selection steps are performed using simple
computer programs to perform the selection as outlined above;
however, all of the steps are optionally performed manually. One
available computer program for primer selection is the MacVector
program from Kodak. In addition to commercially available programs
for primer selection, one of skill can easily design simple
programs for any of the preferred selection steps. Amplification
primers can be selected to provide amplification products that span
specific deletions, truncations, and insertions in an amplification
target, thereby facilitating the detection of specific
abnormalities such as a transposon insertion as described
herein.
[0081] Antibodies to Afc1p and to Rce1p
[0082] Antibodies are raised to Afc1p and Rce1p polypeptides of the
present invention, including individual, allelic, strain, or
species variants, and fragments thereof, both in their naturally
occurring (full-length) forms and in recombinant forms.
Additionally, antibodies are raised to these polypeptides in either
their native configurations or in non-native configurations.
Anti-idiotypic antibodies can also be generated. Many methods of
making antibodies are known to persons of skill. The following
discussion is presented as a general overview of the techniques
available; however, one of skill will recognize that many
variations upon the following methods are known.
[0083] A number of immunogens are optionally used to produce
antibodies specifically reactive with Afc1p and Rce1p polypeptides.
Recombinant or synthetic polypeptides of 10 amino acids in length,
or greater, typically 20 amino acids in length, or greater, more
typically 30 amino acids in length, or greater, selected from amino
acid subsequences of Afc1p and Rce1p are the preferred polypeptide
immunogen for the production of monoclonal or polyclonal
antibodies. In one class of preferred embodiments, an immunogenic
peptide conjugate is also included as an immunogen. Naturally
occurring polypeptides are also used either in pure or partially
pure form.
[0084] Recombinant polypeptides are expressed in eukaryotic or
prokaryotic cells and purified using standard techniques. The
polypeptide, or a synthetic version thereof, is then injected into
an animal capable of producing antibodies. Either monoclonal or
polyclonal antibodies can be generated for subsequent use in
immunoassays to measure the presence and quantity of the
polypeptide.
[0085] Methods of producing polyclonal antibodies are known to
those of skill in the art. In brief, an immunogen (antigen),
preferably a purified polypeptide, a polypeptide coupled to an
appropriate carrier (e.g., GST, keyhole limpet hemanocyanin, etc.),
or a polypeptide incorporated into an immunization vector, such as
a recombinant vaccinia virus (see, U.S. Pat. No. 4,722,848) is
mixed with an adjuvant and animals are immunized with the mixture.
The animal's immune response to the immunogen preparation is
monitored by taking test bleeds and determining the titer of
reactivity to the polypeptide of interest. When appropriately high
titers of antibody to the immunogen are obtained, blood is
collected from the animal and antisera are prepared. Further
fractionation of the antisera to enrich for antibodies reactive to
the polypeptide is performed where desired (see, e.g., Coligan
(1991) Current Protocols in Immunology Wiley/Greene, NY; and Harlow
and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor
Press, NY).
[0086] Antibodies, including binding fragments and single chain
recombinant versions thereof, against whole or predetermined
fragments of Rce1p or Rce1p are raised by immunizing animals, e.g.,
with conjugates of the fragments with carrier proteins as described
above. Typically, the immunogen of interest is a peptide of at
least about 10 amino acids, more typically the peptide is 20 amino
acids in length, generally the fragment is 25 amino acids in length
and often the fragment is 30 amino acids in length or greater. The
peptides are optionally coupled to a carrier protein (e.g., as a
fusion protein), or are recombinantly expressed in an immunization
vector. Antigenic determinants on selected peptides to which
antibodies bind are typically 3 to 10 amino acids in length.
[0087] Monoclonal antibodies are prepared from cells secreting the
desired antibody. These antibodies are screened for binding to
normal or modified polypeptides, or screened for agonistic or
antagonistic activity, e.g., activity mediated through a selected
polypeptide. Specific monoclonal and polyclonal antibodies will
usually bind with a K.sub.D of at least about 0.1 mM, more usually
at least about 50 .mu.M, and preferably at least about 1 .mu.M or
better.
[0088] In some instances, it is desirable to prepare monoclonal
antibodies from various mammalian hosts, such as mice, rodents,
primates, humans, etc. Description of techniques for preparing such
monoclonal antibodies are found in, e.g., Stites et al. (eds.)
Basic and Clinical Immunology (4th ed.) Lange Medical Publications,
Los Altos, Calif., and references cited therein; Harlow and Lane,
Supra; Goding (1986) Monoclonal Antibodies: Principles and Practice
(2d ed.) Academic Press, New York, N.Y.; and Kohler and Milstein
(1975) Nature 256: 495-497. Summarized briefly, this method
proceeds by injecting an animal with an immunogen. The animal is
then sacrificed and cells taken from its spleen, which are fused
with myeloma cells. The result is a hybrid cell or "hybridoma" that
is capable of reproducing in vitro. The population of hybridomas is
then screened to isolate individual clones, each of which secretes
a single antibody species to the immunogen. In this manner, the
individual antibody species obtained are the products of
immortalized and cloned single B cells from the immune animal
generated in response to a specific site recognized on the
immunogenic substance.
[0089] Alternative methods of immortalization include
transformation with Epstein Barr Virus, oncogenes, retroviruses, or
other methods known in the art. Colonies arising from single
immortalized cells are screened for production of antibodies of the
desired specificity and affinity for the antigen, and yield of the
monoclonal antibodies produced by such cells is enhanced by various
techniques, including injection into the peritoneal cavity of a
vertebrate (preferably mammalian) host. The polypeptides and
antibodies of the present invention are used with or without
modification, and include chimeric antibodies such as humanized
murine antibodies.
[0090] Other suitable techniques involve selection of libraries of
recombinant antibodies in phage or similar vectors (see, e.g., Huse
et al. (1989) Science 246: 1275-1281; and Ward, et al. (1989)
Nature 341: 544-546; and Vaughan et al. (1996) Nature
Biotechnology, 14: 309-314).
[0091] Frequently, the polypeptides and antibodies will be labeled
by joining, either covalently or non-covalently, a substance which
provides for a detectable signal. A wide variety of labels and
conjugation techniques are known and are reported extensively in
both the scientific and patent literature. Suitable labels include
radionucleotides, enzymes, substrates, cofactors, inhibitors,
fluorescent moieties, chemiluminescent moieties, magnetic
particles, and the like. Patents teaching the use of such labels
include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;
4,277,437; 4,275,149; and 4,366,241. Also, recombinant
immunoglobulins may be produced. See, Cabilly, U.S. Pat. No.
4,816,567; and Queen et al. (1989) Proc. Nat'l Acad. Sci. USA 86:
10029-10033.
[0092] The antibodies of this invention are also used for affinity
chromatography in isolating natural or recombinant Afc1p or Rce1p
polypeptides. Columns are prepared, e.g., with the antibodies
linked to a solid support, e.g., particles, such as agarose,
Sephadex, or the like, where a cell lysate is passed through the
column, washed, and treated with increasing concentrations of a
mild denaturant, whereby purified polypeptides are released.
[0093] In one highly preferred embodiment, the antibodies are used
to screen expression libraries for particular expression products
such as homologous proteins to the yeast Afc1p or Rce1p proteins,
e.g., in an expression library from human or other mammalian
tissue. Optionally, the antibodies in such a procedure are labeled
with a moiety allowing easy detection of presence of antigen by
antibody binding.
[0094] Antibodies raised against polypeptides can also be used to
raise anti-idiotypic antibodies. These are useful for detecting
abnormal growth related to the presence of the respective
polypeptides.
[0095] Antibodies are optionally humanized. Humanized (chimeric)
antibodies are immunoglobulin molecules comprising a human and
non-human portion. The antigen combining region (or variable
region) of a humanized chimeric antibody is derived from a
non-human source (e.g., murine) and the constant region of the
chimeric antibody (which confers biological effector function, such
as cytotoxicity, to the immunoglobulin) is derived from a human
source. The humanized chimeric antibody has the antigen binding
specificity of the non-human antibody molecule and the effector
function conferred by the human antibody molecule. A large number
of methods of generating chimeric antibodies are well known to
those of skill in the art (see, e.g., U.S. Pat. Nos. 5,502,167,
5,500,362, 5,491,088, 5,482,856, 5,472,693, 5,354,847, 5,292,867,
5,231,026, 5,204,244, 5,202,238, 5,169,939, 5,081,235, 5,075,431,
and 4,975,369).
[0096] In another embodiment, this invention provides for fully
human antibodies against Afc1p or Rce1p polypeptides. Human
antibodies consist entirely of characteristically human
immunoglobulin sequences. The human antibodies of this invention
can be produced in using a wide variety of methods (see, e.g.,
Larrick et al., U.S. Pat. No. 5,001,065, for a review). A general
approach for producing human antibodies by trioma technology is
described by Ostberg et al. (1983), Hybridoma 2: 361-367, Ostberg,
U.S. Pat. No. 4,634,664, and Engelman et al., U.S. Pat. No.
4,634,666. Other approaches include immunization of mice
transformed to express human immunoglobulin genes, and phage
display screening (Vaughan et al. supra.).
[0097] Afc1p and Rce1p Polypeptide Assays.
[0098] The expression of selected polypeptides (e.g., Afc1p, Rce1p
and conservative modifications thereof) can also be detected and/or
quantified by detecting or quantifying the expressed polypeptide.
As described herein, detection of the various polypeptides of the
invention is a feature of certain assays of the invention.
[0099] The polypeptides can be detected and quantified by any of a
number of means well known to those of skill in the art. These
include analytic biochemical methods, such as electrophoresis,
capillary electrophoresis, high performance liquid chromatography
(HPLC), thin layer chromatography (TLC), hyperdiffusion
chromatography, and the like, or various immunological methods such
as fluid or gel precipitin reactions, immunodiffusion (single or
double), immunoelectrophoresis, radioimmunoassay (RIA),
enzyme-linked immunosorbent assays (ELISAs), immunofluorescent
assays, western blotting, and the like.
[0100] In a particularly preferred embodiment, the polypeptides are
detected in an electrophoretic protein separation, more preferably
in a two-dimensional electrophoresis, while in a most preferred
embodiment, the polypeptides are detected using an immunoassay.
[0101] As used herein, an immunoassay is an assay that utilizes an
antibody to specifically bind to the analyte (e.g., selected
polypeptide, such as Afc1p or Rce1p). The immunoassay is thus
characterized by detection of specific binding of a polypeptide to
an anti-polypeptide antibody, as opposed to the use of other
physical or chemical properties to isolate, target, and quantify
the analyte.
[0102] As indicated above, the presence or absence of polypeptides
in a biological sample can be determined using electrophoretic
methods. Means of detecting proteins using electrophoretic
techniques are well known to those of skill in the art (see
generally, R. Scopes (1982) Protein Purification, Springer-Verlag,
N.Y.; Deutscher, (1990) Methods in Enzymology Vol. 182: Guide to
Protein Purification., Academic Press, Inc., N.Y.).
[0103] In a preferred embodiment, the polypeptides are detected
and/or quantified using any of a number of well recognized
immunological binding assays (see, e.g., U.S. Pat. Nos. 4,366,241;
4,376,110; 4,517,288; and 4,837,168). For a review of the general
immunoassays, see also Methods in Cell Biology Volume 37:
Antibodies in Cell Biology, Asai, ed. Academic Press, Inc. New York
(1993); Basic and Clinical Immunology 7th Edition, Stites &
Terr, eds. (1991). Immunological binding assays (or immunoassays)
typically utilize a "capture agent" to specifically bind to and
often immobilize the analyte. The capture agent is a moiety that
specifically binds to the analyte. In a preferred embodiment, the
capture agent is an antibody that specifically binds polypeptide(s)
or polypeptide subsequences (e.g., antigenic domains which
specifically bind to the antibody). In a second preferred
embodiment, the capture agent is the polypeptide and the analyte is
antisera comprising an antibody which specifically binds to the
polypeptide.
[0104] Immunoassays often utilize a labeling agent to specifically
bind to and label the binding complex formed by the capture agent
and the analyte. The labeling agent may itself be one of the
moieties comprising the antibody/analyte complex. Thus, the
labeling agent may be a labeled polypeptide or a labeled
anti-polypeptide antibody. Alternatively, the labeling agent may be
a third moiety, such as another antibody, that specifically binds
to the antibody/polypeptide complex.
[0105] In a preferred embodiment, the labeling agent is a second
antibody bearing a label. Alternatively, the second antibody may
lack a label, but it may, in turn, be bound by a labeled third
antibody specific to antibodies of the species from which the
second antibody is derived. The second antibody can be modified
with a detectable moiety, such as biotin, to which a third labeled
molecule can specifically bind, such as enzyme-labeled
streptavidin.
[0106] Other proteins capable of specifically binding
immunoglobulin constant regions, such as streptococcal protein A or
protein G may also be used as the label agent. These proteins are
normal constituents of the cell walls of streptococcal bacteria.
They exhibit a strong non-immunogenic reactivity with
immunoglobulin constant regions from a variety of species (see,
generally Kronval, et al. (1973) J. Immunol., 111: 1401-1406, and
Akerstrom, et al. (1985) J. Immunol., 135: 2589-2542).
[0107] Throughout the assays, incubation and/or washing steps are
optionally performed after each combination of reagents. Incubation
steps can vary from about 5 seconds to several hours, preferably
from about 5 minutes to about 24 hours. However, the incubation
time will depend upon the assay format, analyte, volume of
solution, concentrations, and the like. Usually, the assays will be
carried out at ambient temperature, although they can be conducted
over a range of temperatures, such as 10.degree. C. to 40.degree.
C.
[0108] Immunoassays for detecting polypeptides may be either
competitive or noncompetitive. Noncompetitive immunoassays are
assays in which the amount of captured analyte is directly
measured. In one preferred "sandwich" assay, for example, the
capture agent can be bound directly to a solid substrate where they
are immobilized. The immobilized capture agent then captures
analyte present in the test sample. The analyte thus immobilized is
then bound by a labeling agent, such as a second antibody bearing a
label. Alternatively, the second antibody may lack a label, but it
may, in turn, be bound by a labeled third antibody specific to
antibodies of the species from which the second antibody is
derived. The second antibody can be modified with a detectable
moiety, such as biotin, to which a third labeled molecule can
specifically bind, such as enzyme-labeled streptavidin.
[0109] In competitive assays, the initial amount of analyte present
in the sample is measured indirectly by measuring the amount of an
added (exogenous) analyte displaced (or competed away) from a
capture agent by the analyte present in the sample. In one
competitive assay, a known amount of, in this case, analyte is
added to the sample and the sample is then contacted with a capture
agent. The amount of exogenous analyte bound to the capture agent
is inversely proportional to the initial analyte present in the
sample.
[0110] In a preferred embodiment, western blot (immunoblot)
analysis is used to detect and quantify the presence of selected
polypeptides in the sample. The technique generally comprises
separating sample proteins by gel electrophoresis on the basis of
molecular weight, transferring the separated proteins to a suitable
solid support (such as a nitrocellulose filter, a nylon filter, or
derivatized nylon filter) and incubating the sample with the
antibodies that specifically bind the selected polypeptide. The
antibodies specifically bind to polypeptide on the solid support.
These antibodies are optionally directly labeled or alternatively
are optionally subsequently detected using labeled antibodies
(e.g., labeled sheep anti-mouse antibodies) that specifically bind
to the selected polypeptide.
[0111] Other assay formats include liposome immunoassays (LIA),
which use liposomes designed to bind specific molecules (e.g.,
antibodies) and release encapsulated reagents or markers. The
released chemicals are then detected according to standard
techniques (see, Monroe et al. (1986) Amer. Clin. Prod. Rev.
5:34-41). Enzyme linked assays (e.g., ELISA assays) are also
preferred.
[0112] The assays of this invention as scored (as positive or
negative for a selected polypeptide) according to standard methods
well known to those of skill in the art. The particular method of
scoring will depend on the assay format and choice of label. For
example, a western blot assay can be scored by visualizing the
colored product produced by the enzymatic label. A clearly visible
colored band or spot at the correct molecular weight is scored as a
positive result, while the absence of a clearly visible spot or
band is scored as a negative. In a preferred embodiment, a positive
test will show a signal intensity (e.g., polypeptide quantity) at
least twice that of the background and/or control and more
preferably at least 3 times or even at least 5 times greater than
the background and/or negative control.
[0113] One of skill in the art will appreciate that it is often
desirable to reduce nonspecific binding in immunoassays.
Particularly, where the assay involves an antigen or antibody
immobilized on a solid substrate it is desirable to minimize the
amount of nonspecific binding to the substrate. Means of reducing
such non-specific binding are well known to those of skill in the
art. Typically, this involves coating the substrate with a
proteinaceous composition. In particular, protein compositions such
as bovine serum albumin (BSA), nonfat powdered milk, and
gelatin.
[0114] The particular label or detectable group used in the assay
is not a critical aspect of the invention, so long as it does not
significantly interfere with the specific binding of the antibody
used in the assay. The detectable group can be any material having
a detectable physical or chemical property. Such detectable labels
have been well-developed in the field of immunoassays and, in
general, most any label useful in such methods can be applied to
the present invention. Thus, a label is any composition detectable
by spectroscopic, photochemical, biochemical, immunochemical,
electrical, optical or chemical means. Useful labels in the present
invention include magnetic beads (e.g. Dynabeads.TM.), fluorescent
dyes (e.g., fluorescein isothiocyanate, texas red, rhodamine, and
the like), radiolabels (e.g., .sup.3H, .sup.125I, .sup.35S,
.sup.14C, or .sup.32P), enzymes (e.g., horse radish peroxidase,
alkaline phosphatase and others commonly used in an ELISA), and
colorimetric labels such as colloidal gold or colored glass or
plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.
[0115] The label may be coupled directly or indirectly to the
desired component of the assay according to methods well known in
the art. As indicated above, a wide variety of labels may be used,
with the choice of label depending on sensitivity required, ease of
conjugation with the compound, stability requirements, available
instrumentation, and disposal provisions.
[0116] Non-radioactive labels are often attached by indirect means.
Generally, a ligand molecule (e.g., biotin) is covalently bound to
the molecule. The ligand then binds to an anti-ligand (e.g.,
streptavidin) molecule which is either inherently detectable or
covalently bound to a signal system, such as a detectable enzyme, a
fluorescent compound, or a chemiluminescent compound. A number of
ligands and anti-ligands can be used. Where a ligand has a natural
anti-ligand, for example, biotin, thyroxine, and cortisol, it can
be used in conjunction with the labeled, naturally occurring
anti-ligands. Alternatively, any haptenic or antigenic compound can
be used in combination with an antibody.
[0117] The molecules can also be conjugated directly to signal
generating compounds, e.g., by conjugation with an enzyme or
fluorophore. Enzymes of interest as labels will primarily be
hydrolases, particularly phosphatases, esterases and glycosidases,
or oxidoreductases, particularly peroxidases. Fluorescent compounds
include fluorescein and its derivatives, rhodamine and its
derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds
include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol.
For a review of various labeling or signal producing systems which
may be used, see, U.S. Pat. No. 4,391,904).
[0118] Means of detecting labels are well known to those of skill
in the art. Thus, for example, where the label is a radioactive
label, means for detection include a scintillation counter or
photographic film as in autoradiography. Where the label is a
fluorescent label, it may be detected by exciting the fluorochrome
with the appropriate wavelength of light and detecting the
resulting fluorescence. The fluorescence may be detected visually,
by means of photographic film, by the use of electronic detectors
such as charge coupled devices (CCDs) or photomultipliers and the
like. Similarly, enzymatic labels may be detected by providing the
appropriate substrates for the enzyme and detecting the resulting
reaction product. Finally simple calorimetric labels may be
detected simply by observing the color associated with the label.
Thus, in various dipstick assays, conjugated gold often appears
pink, while various conjugated beads appear the color of the
bead.
[0119] Some assay formats do not require the use of labeled
components. For instance, agglutination assays can be used to
detect the presence of the target antibodies. In this case,
antigen-coated particles are agglutinated by samples comprising the
target antibodies. In this format, none of the components need be
labeled and the presence of the target antibody is detected by
simple visual inspection.
[0120] As mentioned above, depending upon the assay, various
components, including the antigen, target antibody, or
anti-antibody, may be bound to a solid surface. Many methods for
immobilizing biomolecules to a variety of solid surfaces are known
in the art. For instance, the solid surface may be a membrane
(e.g., nitrocellulose), a microtiter dish (e.g., PVC,
polypropylene, or polystyrene), a test tube (glass or plastic), a
dipstick (e.g. glass, PVC, polypropylene, polystyrene, latex, and
the like), a microcentrifuge tube, or a glass or plastic bead. The
desired component may be covalently bound or noncovalently attached
through nonspecific bonding.
[0121] A wide variety of organic and inorganic polymers, both
natural and synthetic may be employed as the material for the solid
surface. Illustrative polymers include polyethylene, polypropylene,
poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene
terephthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidene
difluoride (PVDF), silicones, polyformaldehyde, cellulose,
cellulose acetate, nitrocellulose, and the like. Other materials
which may be employed, include paper, glasses, ceramics, metals,
metalloids, semiconductive materials, cements or the like. In
addition, are included substances that form gels, such as proteins
(e.g., gelatins), lipopolysaccharides, silicates, agarose and
polyacrylamides can be used. Polymers which form several aqueous
phases, such as dextrans, polyalkylene glycols or surfactants, such
as phospholipids, long chain (12-24 carbon atoms) alkyl ammonium
salts and the like are also suitable. Where the solid surface is
porous, various pore sizes may be employed depending upon the
nature of the system.
[0122] In preparing the surface, a plurality of different materials
may be employed, particularly as laminates, to obtain various
properties. For example, protein coatings, such as gelatin can be
used to avoid non-specific binding, simplify covalent conjugation,
enhance signal detection or the like.
[0123] If covalent bonding between a compound and the surface is
desired, the surface will usually be polyfunctional or be capable
of being polyfunctionalized. Functional groups which may be present
on the surface and used for linking can include carboxylic acids,
aldehydes, amino groups, cyano groups, ethylenic groups, hydroxyl
groups, mercapto groups and the like. The manner of linking a wide
variety of compounds to various surfaces is well known and is amply
illustrated in the literature. See, for example, Immobilized
Enzymes, Ichiro Chibata, Halsted Press, New York, 1978, and
Cuatrecasas (1970) J. Biol. Chem. 245 3059).
[0124] In addition to covalent bonding, various methods for
noncovalently binding an assay component can be used. Noncovalent
binding is typically nonspecific absorption of a compound to the
surface. Typically, the surface is blocked with a second compound
to prevent nonspecific binding of labeled assay components.
Alternatively, the surface is designed such that it nonspecifically
binds one component but does not significantly bind another. For
example, a surface bearing a lectin such as Concanavalin A will
bind a carbohydrate containing compound but not a labeled protein
that lacks glycosylation. Various solid surfaces for use in
noncovalent attachment of assay components are reviewed in U.S.
Pat. Nos. 4,447,576 and 4,254,082.
[0125] Screening for Nucleic Acids and the Use of Nucleic Acids as
Molecular Probes
[0126] The nucleic acids of the invention (e.g., AFC1, RCE1,
homologoues thereof, and conservative modifications thereof) are
useful as molecular probes, in addition to their utility in
encoding the polypeptides described herein. As set forth supra,
certain assays of the invention include the detection of AFC1 or
RCE1 expression.
[0127] Typically, probes derived from the exemplar nucleic acids
are used to detect and/or quantitate the presence of complementary
nucleic acid in a selected biological sample. A wide variety of
formats and labels are available and appropriate for nucleic acid
hybridization, including those reviewed in Tijssen (1993)
Laboratory Techniques in biochemistry and molecular
biology--hybridization with nucleic acid probes parts I and II,
Elsevier, N.Y. and Choo (ed) (1994) Methods In Molecular Biology
Volume 33-In Situ Hybridization Protocols Humana Press Inc., New
Jersey (see also, other books in the Methods in Molecular Biology
series); see especially, Chapter 21 of Choo (id) "Detection of
Virus Nucleic Acids by Radioactive and Nonisotopic in Situ
Hybridization".
[0128] For instance, PCR is routinely used to detect nucleic acids
in biological samples (see, Innis, supra, for a general description
of PCR techniques). Accordingly, in one class of embodiments, the
nucleic acids of the invention are used as PCR primers or
templates, or as positive controls in PCR reactions for the
detection of in a biological sample. Briefly, nucleic acids with
sequence identity or complementarity to an exemplar sequence is
used as templates to synthetically produce oligonucleotides of
about 15-25 nucleotides with sequences similar or identical to the
complement of a the selected nucleic acid subsequence. The
oligonucleotides are then used as primers in PCR reactions to
detect selected nucleic acids in biological samples. A nucleic acid
of the invention (i.e., a cloned nucleic acid corresponding to the
region to be amplified) is also optionally used as an amplification
template in a separate reactions as a positive control to determine
that the PCR reagents and hybridization conditions are
appropriate.
[0129] Other methods for the detection of nucleic acids in
biological samples using nucleic acids of the invention include
Southern blots, northern blots, in situ hybridization (including
Fluorescent in situ hybridization (FISH), and a variety of other
techniques overviewed in Choo (supra)). A variety of automated
solid-phase detection techniques are also appropriate. For
instance, very large scale immobilized polymer arrays (VLSIPS.TM.)
are used for the detection of nucleic acids. See, Tijssen (supra),
Fodor et al. (1991) Science, 251: 767-777; Sheldon et al. (1993)
Clinical Chemistry 39(4): 718-719 and Kozal et al. (1996) Nature
Medicine 2(7): 753-759.
[0130] Therapeutic Uses for Afc1p and Rce1p Inhibitors
[0131] In one aspect, the present invention provides therapeutics
for treating cancers such as tumors which are correlated to Ras
activation. In the therapeutic methods of the invention, a small
molecule inhibitor of a mammalian Rce1p or RCE1p protein, such as
NMe 181, is administered to a patient suffering from cancer and, in
particular, cancer associated with an activated Ras oncogene.
[0132] Administration is by any of the routes normally used for
introducing a molecule into ultimate contact with blood or tissue
cells. Administration is made in any suitable manner, preferably
with pharmaceutically acceptable excipients. Suitable methods of
administering inhibitors in the context of the present invention to
a patient are available. Intra-muscular, subcutaneous and
parenteral administration such as intravenous administration are
suitable methods of administration. Where the inhibitor is
administered to inhibit growth of a tumor, the inhibitor is often
administered to the site of the tumor, rather than by systemic
introduction. However, systemic introduction is optionally used.
Formulations of compositions to be administered can be presented in
unit-dose or multi-dose sealed containers, such as ampules and
vials.
[0133] Pharmaceutically acceptable excipients are determined in
part by the particular composition being administered, as well as
by the particular method used to administer the composition.
Accordingly, there is a wide variety of suitable formulations of
pharmaceutical compositions of the present invention. Formulations
suitable for parenteral administration, such as, for example, by
intraarticular (in the joints), intravenous, intramuscular,
intradermal, intraperitoneal, and subcutaneous routes, include
aqueous and non-aqueous, isotonic sterile injection solutions,
which can contain antioxidants, buffers, bacteriostats, and solutes
that render the formulation isotonic with the blood of the intended
recipient, and aqueous and non-aqueous sterile suspensions that can
include suspending agents, solubilizers, thickening agents,
stabilizers, and preservatives.
[0134] The dose administered to a patient, in the context of the
present invention should be sufficient to effect a beneficial
therapeutic response in the patient over time, such as a reduction
in the rate of tumor growth, or more preferably, a reduction in
tumor size. The dose will be determined by the efficacy of the
particular inhibitor employed and the condition of the patient, as
well as the body weight or surface area of the patient to be
treated. The size of the dose also will be determined by the
existence, nature, and extent of any adverse side-effects that
accompany the administration of a particular inhibitor.
[0135] In determining the effective amount of the inhibitor, the
physician evaluates circulating plasma levels of the inhibitor,
toxicities, progression of the disease, and the production of
antibodies to the particular inhibitor.
[0136] For administration, inhibitors can be administered at a rate
determined by the LD-50 of the inhibitor and the side-effects of
the inhibitor at various concentrations, as applied to the mass and
overall health of the patient. Administration can be accomplished
via single or divided doses.
[0137] If a patient undergoing infusion of an inhibitor develops
fevers, chills, or muscle aches, he/she typically receives the
appropriate dose of aspirin, ibuprofen or acetaminophen. Patients
who experience reactions to the infusion such as fever, muscle
aches, and chills are premedicated 30 minutes prior to the future
infusions with either aspirin, acetaminophen, or diphenhydramine.
Meperidine is used for more severe chills and muscle aches that do
not quickly respond to antipyretics and antihistamines. Infusion is
slowed or discontinued depending upon the severity of the
reaction.
[0138] The effect of the therapeutic inhibitors are measured by
monitoring the size of a tumor or the extent of metastasis before
treatment, and comparing the size of the tumor or extent of
metastasis in a patient over time. Typically, measurements are
taken before, during and after the therapeutic regimen.
[0139] Kits
[0140] The present invention provides a variety of kits for the
detection of AFC1 or RCE1 nucleic acids or proteins, and for the
testing compounds for their ability to inhibit AFC1 or RCE1
expression, or Afc1p or Rce1p protein activity.
[0141] Detection kits preferably include one or more reagents for
determining the presence or absence of a selected nucleic acid or
protein, i.e., any of the nucleic acids or proteins described
herein. Preferred reagents include nucleic acid probes that
specifically hybridize to the exemplar sequences, or subsequence
thereof; probes that specifically bind to an abnormal genes (e.g.,
one containing premature truncations, insertions, or deletions),
and antibodies that specifically bind to polypeptides or
subsequences thereof. The antibody or hybridization probe may be
free or immobilized on a solid support such as a test tube, a
microtiter plate, a dipstick and the like. The kit optionally
include instructional materials teaching the use of the antibody or
hybridization probe in an assay for the detection of the relevant
nucleic acid or protein, a container or other packaging material or
the like.
[0142] The kits optionally include alternatively, or in combination
with any of the other components described herein, an antibody
which specifically binds a polypeptide of the invention. The
antibody can be monoclonal or polyclonal. The antibody can be
conjugated to another moiety such as a label and/or it can be
immobilized on a solid support (substrate).
[0143] The kits can also optionally include a second antibody for
detection of polypeptide/antibody complexes or for detection of
hybridized nucleic acid probes. The kits optionally include
appropriate reagents for detection of labels, positive and negative
controls, washing solutions, dilution buffers and the like.
[0144] Kits testing for inhibition of RCE1 or AFC1 expression
optionally include any of the components described above for
detecting nucleic acids or proteins. Kits testing for Afc1p or
Rce1p activity can monitor the proteolytic cleavage of the terminal
AAX peptide from a relevant CAAX protein such as Ras. This can be
accomplished by monitoring the change in electrophoretic mobility,
e.g., in a western blot or ELISA assay. These kits optionally
include any of the following: reagents for detecting a CAAX protein
(e.g., anti-Ras antibodies), electrophoretic equipment,
instructions in the detection of AAX cleavage or the like. Other
assays for monitoring protease activity are described in Ashby and
Rine "Ras and a-Factor Converting Enzyme" (1995) Methods in
Enzymology 230:235.
[0145] In addition to monitoring AAX cleavage directly, the present
invention provides for the detection of AAX cleavage using a
functional assay. For example, as described herein, a heat-shock
assay can be used to monitor prenylation-dependent Ras activity.
Kits optionally include any of the components used in such an
assay, including yeast strains such as .DELTA.AFC1 or .DELTA.RCE1,
instructions, containers, growth media, control yeast strains,
thermocycling equipment, water baths for administering the heat
shock, and the like.
[0146] Discussion of the Accompanying Sequence Listing
[0147] SEQ ID NO: 1 provides the sequence of the AFC1 gene from
yeast. SEQ ID NO:3 provides the sequence of the RCE1 gene from
yeast. In each case, the information is presented as a DNA
sequence. One of skill will readily understand that the sequence
also describes the corresponding RNA (i.e., by substitution of the
T residues with U residues) and a variety of conservatively
modified variations thereof. In addition, the nucleic acid sequence
provides the corresponding amino acid sequence by translating the
given DNA sequence using the genetic code.
[0148] SEQ ID NO:2 provides the protein sequence of the Afc1p
protein from yeast. SEQ ID NO:4 provides the sequence of the Rce1p
protein from yeast. In each case, the information is presented as a
polypeptide sequence. One of skill will readily understand that the
sequences also describe all of the corresponding RNA and DNA
sequences which encode the protein, by conversion of the amino acid
sequence into the corresponding nucleotide sequence using the
genetic code, by alternately assigning each possible codon in each
possible codon position. The sequences also provides a variety of
conservatively modified variations by substituting appropriate
residues with the exemplar conservative amino acid substitutions
provided, e.g., in the Definitions section above.
EXAMPLES
[0149] The following examples are provided by way of illustration
only and not by way of limitation. Those of skill will readily
recognize a variety of noncritical parameters which can be changed
or modified to yield essentially similar results.
[0150] To evaluate the functional role of Ras CAAX proteolysis,
naturally occurring CAAX sequences from rabbit skeletal muscle
phosphorylase kinase were utilized. The .alpha. and .beta. subunits
of this holoenzyme contain CAAX sequences that in vivo are
prenylated, yet do not undergo further processing (Heilmeyer, et
al., Proc. Natl. Acad. Sci. USA, 89(20):9554-9558 (1992)). It has
now been demonstrated that peptides containing these sequences are
not proteolytically cleaved in vitro by the membrane-bound CAAX
protease. These same peptides do, however, bind to the protease as
defined by their ability to compete for proteolysis of peptides
with normally processed CAAX sequences.
[0151] The issue regarding whether CAAX proteolysis and methylation
are required for yeast Ras2p function has been addressed. To do so,
a highly sensitive and reproducible assay of Ras2p function has
been developed. One of the hallmark phenotypes of activating
mutations in yeast Ras2p is the inability of the cells to properly
enter stationary phase resulting in poor viability and heat shock
sensitivity. Current heat shock assays to assess Ras activation
utilize plate assays where a large number of cells being tested are
transferred to plates that have been prewarmed to 55.degree. C. The
plates are then incubated at 55.degree. C. for various times up to
one hour. After a subsequent incubation at 30.degree. C. for about
18 hours, the strains are scored for yeast death or growth and,
thus, heat shock sensitivity or resistance, respectively. This
assay is adequate for crude measurements of heat shock sensitivity,
but, unfortunately, it is clearly inadequate for quantitating
intermediate degrees of heat shock sensitivity.
[0152] As such, an improved assay of Ras2p function has been
developed. This assay utilizes liquid-based heat shock of the yeast
strains in, for example, a programmable thermocycle (PCR) machine
or other water-bath. Generally, cells are grown in a liquid medium
into a stationary phase. The liquid medium can be water or some
other growth medium, such as the commercially available Yeast
Minimal Medium (See, e.g., Atlas, et al., HANDBOOK OF
MICROBIOLOGICAL MEDIA (CRC Press, Ann Arbor, Mich. (1993); see
also, Sambrook, Ausbel and Berger, all supra). Equal amounts of
cells are added to tubes containing the liquid medium, preferably
water. At the same time, portions of these suspensions are plated
on the growth media to score the viability before the heat shock,
i.e., to serve as controls. The tubes are then placed in a PCR
machine or a water-bath at a temperature ranging between about
40.degree. C. and about 60.degree. C., more preferably at a
temperature between 50.degree. C. and 55.degree. C., and heat
shocked for a time period ranging from about 0.5 minute to about 20
minutes, more preferably for a time period from about 3 minutes to
about 12 minutes. It will be readily apparent to those of skill in
the art that the specific reaction conditions employed will depend
upon the amount of cells in the suspension and upon how deep into
the stationary phase the tested cells are.
[0153] The heat-treated cells are then cooled down to a temperature
ranging from 0.degree. C. to about 30.degree. C., more preferably
to about room temperature (20.degree. C.), and plated on the growth
media to score viability after the heat shock. The ratio of the
number of survivors in the tested strain (e.g., a strain carrying
deletions in AFC1 or RCE1 or alterations in the RAS2 C-terminus
CAMQ sequence) to the number of survivors in the control strain
(i.e., the strain which has no deletions or alterations) defines
the level of suppression of heat shock sensitivity. At the same
time, the strains with plasmids carrying the wild type RAS2 gene
are also tested for heat shock sensitivity to determine if the
introduced changes by themselves have any effect on the rate of
survival.
[0154] To determine the functional impact of blocking Ras2p CAAX
proteolysis, the normal Ras2p CAAX sequence CIIS with replaced with
CAMQ or CLVS using site-directed mutagenesis. These
non-proteolyzable sequences reduced the activated phenotype of
cells approximately 100-fold. In contrast, the presence of these
non-proteolyzable CAAX sequences did not have any detectable
phenotype in non-activated forms of Ras2p. These results are the
first demonstration of a functional requirement of Ras-CAAX
proteolysis and indicate that this aspect of Ras processing
represents a pharmacological target in tumors containing activating
mutations in Ras.
[0155] In addition to testing the effects of the non-proteolyzable
rabbit phosphorylase kinase CAAX sequences CAMQ and CLVS on Ras2p
function, the importance of Ras2p C-terminal methylation was also
tested. The STE14 gene product has been shown to encode the
prenylated protein C-terminal methyltransferase that processes both
the Ras2p protein and the pheromone a-factor (Hrycyna, et al., EMBO
J., 10:1699-1709 (1991)). Deletion of the STE14 structural gene
results in an absence of detectable Ras2p methylation, but does not
effect activated Ras2p heat shock sensitivity when evaluated with
the standard plate heat shock assay (Id.).
[0156] The importance of C-terminal methylation of activated
Ras2.sup.val19 was assessed in a strain in which the STE14 gene was
replaced with the LEU2 gene (ste14.DELTA.::LEU2) in the
liquid-based heat shock assay described above. As was the case with
blocking CAAX proteolysis with the CAMQ or CLVS substitution,
preventing C-terminal methylation reduced heat shock sensitivity
greater than 50-fold. This result indicated that blocking activated
Ras2p methylation significantly reduced Ras2p function and, thus,
attenuated the activated phenotype. This result is the first
demonstration of the functional relevance of Ras2p farnesylated
cysteine methylation.
[0157] In addition to the foregoing, the issue of whether the
effects of blocking Ras2p proteolysis and blocking Ras2p
methylation would be additive when combined was tested. For this
experiment, heat shock sensitivities in strains with wild-type or
activated forms of Ras2p with various CAAX sequences in the
presence or absence of the STE14 gene were compared. The results
indicate that the heat shock resistance conferred by the
non-proteolyzable CAAX sequence together with the absence of the
STE14 gene (ste14.DELTA.::LEU2) does not result in an increased
heat shock resistance beyond that of each component alone. These
data therefore support the notion that CAMQ and CLVS represent
non-proteolyzable sequences and that the two phenotypes, namely
heat shock resistance in Ras2.sup.val19 CAMQ or CLVS and heat shock
resistance in the Ras2.sup.val19 (ste14.DELTA.::LEU2) strains, are
epistatic.
[0158] As a means to isolate mutants in CAAX processing, a powerful
genetic selection for mutants defective in a-factor processing was
performed. The a-factor pheromone is another CAAX containing
peptide that is processed by the same protein:farnesyltransferase
and methyltransferase enzymes as Ras2p. The genetic selection
produced a novel mutant that displayed a differential phenotype
depending upon the particular AAX extension of the CAAX sequence.
This mutant possessed a single mutation that was responsible for
the phenotype and the corresponding gene was cloned by
complementation. The gene, named AFC1 for a-Factor Convertase,
contains an amino acid sequence motif identified as the active site
in several characterized zinc metalloproteases. This property is
consistent with both the in vitro proteolysis defect of the
corresponding mutant and the o-phenanthroline sensitivity of this
activity in wild-type extracts. Furthermore, this a-factor CAAX
protease was dependent upon a farnesylated substrate as expected of
a protease that processes a-factor in vivo. The isolation of AFC1
represents the first CAAX protease to be identified. A genetic
knockout of AFC1 resulted in a significant, but not complete,
reduction of a-factor production, thereby exposing the presence of
a second CAAX protease. Using a similar approach, a second CAAX
protease was identified. This gene for the second protease has been
named RCE1 (Ras and a-Factor Converting Enzyme).
Example 1
Autocrine Arrest Selection
[0159] Autocrine arrest selection was designed to simplify the task
of identifying mutants that are defective in the production of
yeast mating pheromone- a-factor. This assay relies on the fact
that haploid yeast cells of both a and a mating types share the
same mating signal transduction pathway. The mating specificity is
defined only by the type of mating pheromone receptor that is
expressed on the surface of the cell. Expression of the a-factor
receptor in a cell producing biologically active a-factor will lead
to autocrine arrest. Mutants having mutations in the genes
responsible for either a-factor production or for transduction of
the mating signal will be able to escape the arrest, grow and form
colonies.
[0160] A. Strain Design
[0161] The SST2 gene (mutations in this gene confer
supersensitivity to the mating pheromone, thereby allowing tighter
arrest) was deleted using a two-step gene replacement in the
JRY3658 strain (W303 HML.DELTA.p, HMR.DELTA.p, MAT.DELTA.p). A
plasmid carrying the MAT.alpha. locus was introduced in the JRY3658
.DELTA.sst2 strain allowing it to mate as an .alpha. strain.
JRY3658 .DELTA.sst2 pMAT.alpha. was crossed to JRY527
.DELTA.mfa1::hisG, mfa2.DELTA.::hisG. The diploid was sporulated
and the segregant of the following genotype was identified:
HMLa.DELTA.p, HMRa.DELTA.p, MATa.DELTA.p, .DELTA.mfa1::hisG,
mfa2.DELTA.::hisG, .DELTA.sst2, his3, lys2, leu2-3, 112, ura3.
[0162] The gene encoding the a-factor receptor (i.e., STE3) was
placed under the control of the inducible GAL promoter, thereby
allowing the expression of the gene to be controlled by the type of
carbon source in the growth medium. This construct was integrated
in the STE3 locus of the strain described above. A plasmid carrying
MFA1 gene with the altered C-terminal CAAX sequence, i.e., CAMQ,
was used as a source of the mating pheromone. This sequence was
introduced by site directed mutagenesis.
[0163] B. Selection In each round of selection, 20 independent
colonies were picked and grown at 30.degree. C. in the supplemented
minimal media. Equal numbers of cells from each culture were
mutagenized by UV light in liquid. The mutagenized cells were
transferred back into the growth media and grown overnight at room
temperature to overcome the phenotypic lag. Cells were plated on
plates containing galactose to induce expression of the a-factor
receptor construct, and incubated at room temperature.
[0164] C. Identification of the Mutants
[0165] All colonies formed on galactose-containing media were
scored for a-factor production in halo assays. Mutant strains which
produced no or very little of the a-factor were used in a secondary
screen. In the secondary screen, patches of cells were treated with
synthetic alpha factor to eliminate all the mutants in the mating
signal transduction pathway. Strains which were able to arrest
(indicating that they were able to grow on galactose not because
they couldn't respond to the pheromone) were used in
complementation tests. The mutants were crossed to the strains with
defects in the genes known to be involved in production of a-factor
(i.e., ram1--a subunit of the farnesyltransferase,
ste14--methyltransferase and ste6--a-factor transporter). The
strains which were complemented by all the mutant strains were used
in another complementation test, using plasmids carrying the
a-factor processing genes (i.e., RAM1, RAM2, STE14 and STE6).
[0166] The mutant strains which passed both complementation tests
were analyzed for substrate specific processing defects. The
plasmid with the MFA1 (CAMQ) variant, used in the selection, was
cured from the strains and a wild type MFA1 (CVIA) gene carrying
plasmid was introduced. The mutant strains which produced no
a-factor with MFA1 (CAMQ) plasmid and which were able to produce
detectable amounts of biologically active a-factor with MFA1 (CVIA)
formed a large complementation group termed AFC1 (a-Factor
Convertase). The gene defined by this complementation group was
cloned using a multicopy genomic library by complementation of the
mutant phenotype (a-factor production). The RCE1 (Ras and a-Factor
Converting Enzyme) was identified as a partial multicopy suppressor
of the afc1 MFA1 (CAMQ) dependent mutant phenotype.
Example 2
Heat Shock Sensitivity Assay
[0167] The heat shock sensitivity assay is used to determine if
changes introduced in the yeast strain have any effects on Ras
signaling. This assay utilizes a dominant, hyperactivated allele of
RAS2, i.e., RAS2.sup.val19. Cells carrying the RAS2.sup.Val19
allele on a CEN plasmid are significantly more sensitive to the
short term heat treatment then wild type strains. The assay can be
performed in many different ways. The goal of the assay is to
evaluate the number of cells able to survive the heat shock.
[0168] The original assay involves streaking similar numbers of
cells on a plate which is then placed in a water-bath at a
temperature of about 50.degree. to 55.degree. C. for a time period
of about 10 to 30 min. Unfortunately, this assay can reveal only
major differences in heat shock sensitivity. It has, however, been
discovered that modifications introduced to this assay allow more
precise quantitative evaluation of the differences in the survival
rate. In the modified assay, cells are grown in a liquid medium
into a stationary phase. The liquid medium can be water or some
other growth medium, such as the commercially available Yeast
Minimal Medium (See, e.g., Atlas, et al., HANDBOOK OF
MICROBIOLOGICAL MEDIA (CRC Press, Ann Arbor, Mich. (1993)). Equal
amounts of cells are added to tubes containing the liquid medium,
preferably water. At the same time, portions of these suspensions
are plated on the growth media to score the viability before the
heat shock, i.e., to serve as controls. The tubes are then placed
in a PCR machine or a water-bath at a temperature ranging between
about 40.degree. C. and about 60.degree. C., more preferably at a
temperature between 50.degree. C. and 55.degree. C., and heat
shocked for a time period ranging from about 0.5 minute to about 20
minutes, more preferably for a time period from about 3 minutes to
about 12 minutes. It will be readily apparent to those of skill in
the art that the specific reaction conditions employed will depend
upon the amount of cells in the suspension and upon how deep into
the stationary phase the tested cells are.
[0169] The heat-treated cells are then cooled down to a temperature
ranging from 0.degree. C. to about 30.degree. C., more preferably
to about room temperature (20.degree. C.), and plated on the growth
media to score viability after the heat shock. The ratio of the
number of survivors in the tested strain (e.g., a strain carrying
deletions in AFC1 or RCE1 or alterations in the RAS2 C-terminus
CAMQ sequence) to the number of survivors in the control strain
(i.e., the strain which has no deletions or alterations) defines
the level of suppression of heat shock sensitivity. At the same
time, the strains with plasmids carrying the wild type RAS2 gene
are also tested for heat shock sensitivity to determine if the
introduced changes by themselves have any effect on the rate of
survival.
[0170] In a variation of the above assay, all tested strains are
transferred to a 96-well microtiter plate and serially diluted
5-fold 5 times. An aliquot (about 6 .mu.l) from each well is placed
on the growth media to determine the viability of the strain. The
plate is placed in a water-bath at a temperature of about
50.degree. C. to about 55.degree. C. Aliquots are removed after 7
and 12 minutes of heat treatment. The heat shock sensitivity is
scored by determining which of the lowest dilutions still has
survivors.
[0171] An additional assay to measure RCE1 function in Ras
processing was to test growth at semi-permissive conditions of
cells contains the Ras temperature sensitive mutant. RCE1 mutants
decreased the maximum permissive temperature for growth. In one of
the experiments carried out, 2 ODs (.sup.-2.times.10.sup.7 cells)
of cells without the RAS2.sup.val19 plasmid and 3.5 ODs of cells
with the RAS2.sup.val19 plasmid were placed in the first row of the
plate. Each row thereafter represents a 5-fold dilution. Cells were
incubated at a temperature of about 50.degree. C. for about 7
minutes. Longer incubation times showed even more dramatic effects.
Heat shock sensitivity was reduced 5-fold. The following assay was
used to measure suppression. Strains were grown in YM+ supplements
for about 2.5 days to an OD of about 2.6.times.10.sup.6. Cells from
each strain (in duplicate) carrying the RAS2.sup.Val19 plasmid were
resuspended in 1 ml of water. 50 .mu.l of each of the suspension
was plated for viability. The tubes were then incubated for about
10 min at a temperature of about 50.degree. C. After being cooled
down to about room temperature, 650 .mu.l and 150 .mu.l aliquots
were plated on the growth media to score the rate of survival.
Example 3
Yeasts Strains Used In The Various Assays
[0172] A. Strains Used In Temperature Sensitivity Experiments
[0173] The strains used in the temperature sensitivity experiments
were obtained by crossing the W303 MAT.alpha. .DELTA.afc1,
.DELTA.rce1 strain to W303 MAT.alpha. ras1::HIS3, ras2-23.sup.ts
(PHY 1150).
[0174] Analysis of the segregants from the cross yielded the
following strains:
1 Wild type: W303, MATa, ras1::HIS3, ras2-23.sup.ts, ade2, leu2,
his3, trp1, ura3 .DELTA.afc1: W303, MATa, ras1::HIS3,
ras2-23.sup.ts, ade2, .DELTA.afc1::HIS3, leu2, his3, trp1, ura3
.DELTA.rce1: W303, MATa, ras1::HIS3, ras2-23.sup.ts, ade2,
.DELTA.rce1::TRP1, leu2, his3, trp1, ura3 .DELTA.afc1, .DELTA.rce1:
W303, MATa, ras1::HIS3, ras2-23.sup.ts, ade2, .DELTA.afc1::HIS3,
.DELTA.rce1::TRP1, leu2, his3, trp1, ura3
[0175] B. Strains Used In Heat Shock Assays:
[0176] All strains were derived from the common laboratory strain
W303 (JRY2334). AFC1 was deleted by homologous recombination using
the construct in which a part of the open reading frame (ORF) is
replaces with the HIS3 gene. For a discussion regarding the use of
homologous recombination, see, e.g., Scherer, S., et al.,
"Replacement of chromosome segments with altered DNA sequences
constructed in vitro," Proc. Natl. Acad. Sci. USA, 76(10): 4951-5
(1979). RCE1 was deleted using homologous recombination using a PCR
product in which the coding region of TRP1 gene is flanked by
.sup.-40 bp sequences homologous to the sequences just upstream and
downstream of the ORF encoding the RCE1 gene.
[0177] A double deletion strain was obtained by crossing
.DELTA.afc1 MAT.alpha. strain to .DELTA.rce1 MAT.alpha. strain,
followed by dissection of the sporulated diploid and analysis of
the segregants.
2 Genotypes: Wild type: W303, MATa, leu2, his3, trp1, ura3
.DELTA.afc1: W303, MATa, .DELTA.afc1::HIS3, leu2, his3, trp1, ura3
.DELTA.rce1: W303, MATa, .DELTA.rcel::TRP1, leu2, his3, trp1, ura3
.DELTA.afc1, .DELTA.rce1: W303, MATa, .DELTA.afc1::HIS3,
.DELTA.rce1::TRP1, leu2, his3, trp1, ura3
[0178] The RAS2.sup.val19 allele was introduced in these strains on
URA3 CEN plasmid. Control strains carried URA3 CEN plasmid with
wild type RAS2 gene.
[0179] The above examples are provided to illustrate the invention
but not to limit its scope. Other variants of the invention will be
readily apparent to one of ordinary skill in the art and are
encompassed by the appended claims. All publications, patents, and
patent applications cited herein are hereby incorporated by
reference for all purposes.
Sequence CWU 1
1
6 1 1825 DNA Saccharomyces cerevisiae CDS (343)..(1701) 1
acctaccttt ttttctatct tcaacaacga aacgccttac acacacacac acatacatct
60 acatacatac atacaaatat acatatatgt aaacttgtat attcattcct
attaaccaaa 120 aagaggcaat taaacttttc cctctttttc tacgtcattt
actcaaaaac tctaattcct 180 tcgtctctgt tctgccattt tctccagaaa
aaaatcgacg ggaaataaaa aaaaaaagac 240 aacgaacaag agaaaaagtt
cgcgaattat aaaccacttc tataattaac aggaaaagga 300 aggaaaaaaa
aggaggaaat agaaaactgc aggcctttat tc atg ttt gat ctt 354 Met Phe Asp
Leu 1 aag acg att ctc gac cat cct aat atc ccg tgg aaa tta atc att
tct 402 Lys Thr Ile Leu Asp His Pro Asn Ile Pro Trp Lys Leu Ile Ile
Ser 5 10 15 20 ggg ttc tcg att gcc caa ttt tct ttc gaa tct tac ttg
acg tac aga 450 Gly Phe Ser Ile Ala Gln Phe Ser Phe Glu Ser Tyr Leu
Thr Tyr Arg 25 30 35 cag tac cag aag cta tct gaa aca aag ttg cca
cct gtg ctg gaa gac 498 Gln Tyr Gln Lys Leu Ser Glu Thr Lys Leu Pro
Pro Val Leu Glu Asp 40 45 50 gaa att gat gat gaa act ttt cat aaa
tca agg aac tac tcc cgg gcc 546 Glu Ile Asp Asp Glu Thr Phe His Lys
Ser Arg Asn Tyr Ser Arg Ala 55 60 65 aag gcc aag ttc tcc att ttc
ggt gac gtc tat aac cta gcc caa aag 594 Lys Ala Lys Phe Ser Ile Phe
Gly Asp Val Tyr Asn Leu Ala Gln Lys 70 75 80 cta gtt ttc atc aaa
tac gac ctc ttc cct aaa atc tgg cac atg gcc 642 Leu Val Phe Ile Lys
Tyr Asp Leu Phe Pro Lys Ile Trp His Met Ala 85 90 95 100 gtt tct
tta ttg aat gca gtc ctg cca gtc aga ttt cat atg gtc tcc 690 Val Ser
Leu Leu Asn Ala Val Leu Pro Val Arg Phe His Met Val Ser 105 110 115
act gtc gca cag agt tta tgc ttc ttg ggt ctc tta tcc agt ttg tct 738
Thr Val Ala Gln Ser Leu Cys Phe Leu Gly Leu Leu Ser Ser Leu Ser 120
125 130 acc ttg gtt gat ttg cca ctc tct tac tat agc cat ttt gtc ctg
gaa 786 Thr Leu Val Asp Leu Pro Leu Ser Tyr Tyr Ser His Phe Val Leu
Glu 135 140 145 gaa aaa ttt ggt ttc aat aaa ttg acc gtc caa cta tgg
atc acc gat 834 Glu Lys Phe Gly Phe Asn Lys Leu Thr Val Gln Leu Trp
Ile Thr Asp 150 155 160 atg atc aag agt ctg act ttg gcg tat gct att
ggt ggc cca atc ctt 882 Met Ile Lys Ser Leu Thr Leu Ala Tyr Ala Ile
Gly Gly Pro Ile Leu 165 170 175 180 tac ctg ttc ctt aag atc ttt gat
aaa ttc cct act gat ttc ctt tgg 930 Tyr Leu Phe Leu Lys Ile Phe Asp
Lys Phe Pro Thr Asp Phe Leu Trp 185 190 195 tac att atg gtc ttc ttg
ttc gtt gtc caa atc tta gcc atg aca atc 978 Tyr Ile Met Val Phe Leu
Phe Val Val Gln Ile Leu Ala Met Thr Ile 200 205 210 att cca gtc ttc
atc atg ccc atg ttt aat aag ttc act cca ttg gag 1026 Ile Pro Val
Phe Ile Met Pro Met Phe Asn Lys Phe Thr Pro Leu Glu 215 220 225 gac
ggt gaa ctg aaa aaa tct att gaa agt ttg gcc gat aga gtt ggg 1074
Asp Gly Glu Leu Lys Lys Ser Ile Glu Ser Leu Ala Asp Arg Val Gly 230
235 240 ttc cct cta gat aag att ttt gtc att gac ggc tca aaa aga tct
tct 1122 Phe Pro Leu Asp Lys Ile Phe Val Ile Asp Gly Ser Lys Arg
Ser Ser 245 250 255 260 cat tca aac gca tat ttc aca ggt ttg cca ttc
acc tcc aag aga att 1170 His Ser Asn Ala Tyr Phe Thr Gly Leu Pro
Phe Thr Ser Lys Arg Ile 265 270 275 gtt ttg ttc gac act tta gtg aac
agt aat tct act gat gaa att acg 1218 Val Leu Phe Asp Thr Leu Val
Asn Ser Asn Ser Thr Asp Glu Ile Thr 280 285 290 gct gtt ttg gcc cat
gaa atc ggt cac tgg caa aaa aac cac atc gtt 1266 Ala Val Leu Ala
His Glu Ile Gly His Trp Gln Lys Asn His Ile Val 295 300 305 aat atg
gtc atc ttt agt caa ttg cac acc ttc ctc att ttc tcc ctt 1314 Asn
Met Val Ile Phe Ser Gln Leu His Thr Phe Leu Ile Phe Ser Leu 310 315
320 ttc acc agc atc tac aga aat aca tca ttt tac aac acc ttc ggc ttt
1362 Phe Thr Ser Ile Tyr Arg Asn Thr Ser Phe Tyr Asn Thr Phe Gly
Phe 325 330 335 340 ttc tta gag aag tcc act ggc agt ttt gtt gat ccc
gtt atc act aag 1410 Phe Leu Glu Lys Ser Thr Gly Ser Phe Val Asp
Pro Val Ile Thr Lys 345 350 355 gaa ttc ccc att atc att gga ttt atg
tta ttt aac gac tta tta act 1458 Glu Phe Pro Ile Ile Ile Gly Phe
Met Leu Phe Asn Asp Leu Leu Thr 360 365 370 cca ctc gaa tgt gcc atg
caa ttc gtg atg agt tta att tcc aga act 1506 Pro Leu Glu Cys Ala
Met Gln Phe Val Met Ser Leu Ile Ser Arg Thr 375 380 385 cat gaa tat
caa gct gat gct tat gct aaa aaa ttg ggc tac aag caa 1554 His Glu
Tyr Gln Ala Asp Ala Tyr Ala Lys Lys Leu Gly Tyr Lys Gln 390 395 400
aat cta tgt agg gct cta att gat cta caa atc aaa aac ctt tcc acc
1602 Asn Leu Cys Arg Ala Leu Ile Asp Leu Gln Ile Lys Asn Leu Ser
Thr 405 410 415 420 atg aat gta gat cct ctg tat tct agc tat cat tat
tcc cat cca act 1650 Met Asn Val Asp Pro Leu Tyr Ser Ser Tyr His
Tyr Ser His Pro Thr 425 430 435 cta gct gaa aga tcg acc gct cta gac
tat gtt agt gaa aag aag aaa 1698 Leu Ala Glu Arg Ser Thr Ala Leu
Asp Tyr Val Ser Glu Lys Lys Lys 440 445 450 aac taatctatag
agtacacata ttagcatgta ccgttaaatt cagcttcgtt 1751 Asn atgtctatat
ctacatacat acacaggtat ctactataag aataaaggaa agaaaaaata 1811
aacgattaaa catt 1825 2 453 PRT Saccharomyces cerevisiae 2 Met Phe
Asp Leu Lys Thr Ile Leu Asp His Pro Asn Ile Pro Trp Lys 1 5 10 15
Leu Ile Ile Ser Gly Phe Ser Ile Ala Gln Phe Ser Phe Glu Ser Tyr 20
25 30 Leu Thr Tyr Arg Gln Tyr Gln Lys Leu Ser Glu Thr Lys Leu Pro
Pro 35 40 45 Val Leu Glu Asp Glu Ile Asp Asp Glu Thr Phe His Lys
Ser Arg Asn 50 55 60 Tyr Ser Arg Ala Lys Ala Lys Phe Ser Ile Phe
Gly Asp Val Tyr Asn 65 70 75 80 Leu Ala Gln Lys Leu Val Phe Ile Lys
Tyr Asp Leu Phe Pro Lys Ile 85 90 95 Trp His Met Ala Val Ser Leu
Leu Asn Ala Val Leu Pro Val Arg Phe 100 105 110 His Met Val Ser Thr
Val Ala Gln Ser Leu Cys Phe Leu Gly Leu Leu 115 120 125 Ser Ser Leu
Ser Thr Leu Val Asp Leu Pro Leu Ser Tyr Tyr Ser His 130 135 140 Phe
Val Leu Glu Glu Lys Phe Gly Phe Asn Lys Leu Thr Val Gln Leu 145 150
155 160 Trp Ile Thr Asp Met Ile Lys Ser Leu Thr Leu Ala Tyr Ala Ile
Gly 165 170 175 Gly Pro Ile Leu Tyr Leu Phe Leu Lys Ile Phe Asp Lys
Phe Pro Thr 180 185 190 Asp Phe Leu Trp Tyr Ile Met Val Phe Leu Phe
Val Val Gln Ile Leu 195 200 205 Ala Met Thr Ile Ile Pro Val Phe Ile
Met Pro Met Phe Asn Lys Phe 210 215 220 Thr Pro Leu Glu Asp Gly Glu
Leu Lys Lys Ser Ile Glu Ser Leu Ala 225 230 235 240 Asp Arg Val Gly
Phe Pro Leu Asp Lys Ile Phe Val Ile Asp Gly Ser 245 250 255 Lys Arg
Ser Ser His Ser Asn Ala Tyr Phe Thr Gly Leu Pro Phe Thr 260 265 270
Ser Lys Arg Ile Val Leu Phe Asp Thr Leu Val Asn Ser Asn Ser Thr 275
280 285 Asp Glu Ile Thr Ala Val Leu Ala His Glu Ile Gly His Trp Gln
Lys 290 295 300 Asn His Ile Val Asn Met Val Ile Phe Ser Gln Leu His
Thr Phe Leu 305 310 315 320 Ile Phe Ser Leu Phe Thr Ser Ile Tyr Arg
Asn Thr Ser Phe Tyr Asn 325 330 335 Thr Phe Gly Phe Phe Leu Glu Lys
Ser Thr Gly Ser Phe Val Asp Pro 340 345 350 Val Ile Thr Lys Glu Phe
Pro Ile Ile Ile Gly Phe Met Leu Phe Asn 355 360 365 Asp Leu Leu Thr
Pro Leu Glu Cys Ala Met Gln Phe Val Met Ser Leu 370 375 380 Ile Ser
Arg Thr His Glu Tyr Gln Ala Asp Ala Tyr Ala Lys Lys Leu 385 390 395
400 Gly Tyr Lys Gln Asn Leu Cys Arg Ala Leu Ile Asp Leu Gln Ile Lys
405 410 415 Asn Leu Ser Thr Met Asn Val Asp Pro Leu Tyr Ser Ser Tyr
His Tyr 420 425 430 Ser His Pro Thr Leu Ala Glu Arg Ser Thr Ala Leu
Asp Tyr Val Ser 435 440 445 Glu Lys Lys Lys Asn 450 3 2948 DNA
Saccharomyces cerevisiae CDS (1001)..(1945) 3 tgaactgttg atgaacaaag
agaagctgac aagcatcaaa gctttgtacg atgatttcca 60 ttcaaaaatt
tgtgaatatg aaaccaagtt caacaagaat tttcttgaat taaatgagtt 120
atataatatg aataggggag accgtaggcc aaaggaactg aaatttacag attttattac
180 ttcacagctg tttaacgata tcgaaagcat ttgcaacttg aaagttagtg
ttcacaactt 240 atccaacatt tttaaaaaac aggtcagtac cctaaaacaa
cactcaaagc acgcattatc 300 tgaggattca atatcgcaca caggtaacgg
tagttcatcg tcgcccagtt cagcgtcatt 360 aacgccagta acttcttcat
ccaagagtag tttattttta cctagcggta gctcgtctac 420 ttccctgaaa
tttacagacc agattgttca taaatgggtt aggattgctc ctttacagta 480
caaacgagac attaatgtga acttggaatt taataaggac attaaggaaa ctttaattcc
540 aagttttgaa agctgcctat gttgtaggtt ttattgcgtt cgagtaatga
ttaaatttga 600 aaaccatctt ggcgtagcga agattgatat ccctatttct
gttaggcaag tgacaaaata 660 aaaaaacatt agaaaaaatt ctcgttactt
ttcttataga tatagatata tgtatggttt 720 gcttatagat gaaggtattt
atcgcgtcct ttgtattccc tattattaat aaaattcttt 780 taaaatgcat
tttctggtgc tcttttgttg cttctgtatt tttttttttt tggaccactg 840
gatggaaaac ctttgatgat tttattacct ttattttaag ttactaaaat atcgagattt
900 caggaacaaa acatagaatt ttctttgtca agaaaaataa aacgaaataa
attgatgctt 960 tgactactga ctgtctgtca tagagagaac cagaacagca atg cta
caa ttc tca 1015 Met Leu Gln Phe Ser 1 5 aca ttt cta gtg ctc cta
tac atc tcc ata tcc tat gtg cta ccg cta 1063 Thr Phe Leu Val Leu
Leu Tyr Ile Ser Ile Ser Tyr Val Leu Pro Leu 10 15 20 tat gca act
tca caa cca gaa ggg tct aaa cga gat aat cct cga acg 1111 Tyr Ala
Thr Ser Gln Pro Glu Gly Ser Lys Arg Asp Asn Pro Arg Thr 25 30 35
att aaa tct cgc atg caa aaa ctt aca att atg cta att tcc aac ctt
1159 Ile Lys Ser Arg Met Gln Lys Leu Thr Ile Met Leu Ile Ser Asn
Leu 40 45 50 ttt ttg gtg cct ttt tta caa tct caa tta tct agt acc
act tca cat 1207 Phe Leu Val Pro Phe Leu Gln Ser Gln Leu Ser Ser
Thr Thr Ser His 55 60 65 ata agt ttc aag gac gca ttt tta ggc tta
ggt att atc cca ggt tat 1255 Ile Ser Phe Lys Asp Ala Phe Leu Gly
Leu Gly Ile Ile Pro Gly Tyr 70 75 80 85 tac gct gca ttg cca aac cct
tgg caa ttc agc cag ttc gtg aaa gac 1303 Tyr Ala Ala Leu Pro Asn
Pro Trp Gln Phe Ser Gln Phe Val Lys Asp 90 95 100 tta acg aaa tgt
gtt gcg atg tta ttg acc tta tat tgt gga ccc gtt 1351 Leu Thr Lys
Cys Val Ala Met Leu Leu Thr Leu Tyr Cys Gly Pro Val 105 110 115 tta
gat ttt gta tta tat cat tta tta aat cca aag agc tct ata ctt 1399
Leu Asp Phe Val Leu Tyr His Leu Leu Asn Pro Lys Ser Ser Ile Leu 120
125 130 gaa gat ttt tac cat gaa ttc ctg aat att tgg agt ttc agg aat
ttt 1447 Glu Asp Phe Tyr His Glu Phe Leu Asn Ile Trp Ser Phe Arg
Asn Phe 135 140 145 ata ttt gca cca ata act gag gaa ata ttt tac acg
tca atg ctt ttg 1495 Ile Phe Ala Pro Ile Thr Glu Glu Ile Phe Tyr
Thr Ser Met Leu Leu 150 155 160 165 act acg tac tta aac cta ata ccg
cat tcg caa cta agc tat caa cag 1543 Thr Thr Tyr Leu Asn Leu Ile
Pro His Ser Gln Leu Ser Tyr Gln Gln 170 175 180 tta ttt tgg caa cca
tcg ctt ttt ttt gga ctt gcg cac gca cac cat 1591 Leu Phe Trp Gln
Pro Ser Leu Phe Phe Gly Leu Ala His Ala His His 185 190 195 gct tat
gag caa tta cag gaa ggc tcc atg aca act gtt tcc att ctg 1639 Ala
Tyr Glu Gln Leu Gln Glu Gly Ser Met Thr Thr Val Ser Ile Leu 200 205
210 ctg aca aca tgc ttc caa att tta tac aca aca ctt ttt gga ggg tta
1687 Leu Thr Thr Cys Phe Gln Ile Leu Tyr Thr Thr Leu Phe Gly Gly
Leu 215 220 225 acc aag ttt gta ttc gta aga aca ggc ggg aac cta tgg
tgc tgc ata 1735 Thr Lys Phe Val Phe Val Arg Thr Gly Gly Asn Leu
Trp Cys Cys Ile 230 235 240 245 atc ctg cat gcc ctt tgc aat atc atg
ggg ttt cct ggt cct tca aga 1783 Ile Leu His Ala Leu Cys Asn Ile
Met Gly Phe Pro Gly Pro Ser Arg 250 255 260 ttg aat tta cat ttc aca
gta gta gac aag aaa gct gga cgc att tcc 1831 Leu Asn Leu His Phe
Thr Val Val Asp Lys Lys Ala Gly Arg Ile Ser 265 270 275 aaa ttg gtc
tca atc tgg aat aag tgc tac ttc gca ctg ctg gtc ctt 1879 Lys Leu
Val Ser Ile Trp Asn Lys Cys Tyr Phe Ala Leu Leu Val Leu 280 285 290
gga tta ata tcc ctg aag gat acc tta caa act ctg gta gga act cct
1927 Gly Leu Ile Ser Leu Lys Asp Thr Leu Gln Thr Leu Val Gly Thr
Pro 295 300 305 ggt tat aga ata acc ctt tagccttttt tacgtacttg
tataccgttt 1975 Gly Tyr Arg Ile Thr Leu 310 315 aaaatttcct
atgtactata accttttttc actactatta tggaattcta tcgagcgacc 2035
gggcttttgt tacggaagag tgaaaaaatc gagttttggt gttttggtga aagaatttgg
2095 aggactataa agtacctata ctttgtatta cggactcaat aacaagtcgt
tcgtgtcagt 2155 ggtattgaag ttgtcagatc taagagtaga gagaaggtgg
catctaatag gtttcgacgt 2215 ttttcttttt ttaaggtttt tatttggtct
cctagaattt aaggtcttag ttagttttgg 2275 tttgttttgt gggttacata
ttttcaattc aaaggagaat ttagctgtct tttataatgt 2335 ccaatagaga
taacgagagc atgctgcgta ctacatcaag cgataaggcg atcgctagtc 2395
aaagggataa acggaagtct gaagttttga ttgctgcaca gtcccttgac aatgaaatcc
2455 gcagcgtaaa aaacctaaaa agattgtcga ttgggtcaat ggatttactt
attgatccag 2515 aattagatat aaaattcggt ggggaatcta gtgggagacg
atcatggtct ggcacgacat 2575 ccagttctgc gtcaatgcca agtgacacaa
ccaccgttaa taacacacga tatagcgatc 2635 caactccgct agagaacttg
catgggaggg gtaactcagg gatagaatcc tccaataaga 2695 ctaaacaagg
taactactta ggtataaaaa aaggtgttca ctctccatcc aggaaattaa 2755
atgctaacgt attaaagaaa aacttattat gggttcccgc caatcaacac cctaacgtta
2815 agcctgataa tttcctagag cttgtacaag atactttaca aaatatacaa
ctaagcgaca 2875 atggtgaaga taatgatggg aatagcaatg aaaataacga
tattgaggat aatggggagg 2935 ataaagaatc aca 2948 4 315 PRT
Saccharomyces cerevisiae 4 Met Leu Gln Phe Ser Thr Phe Leu Val Leu
Leu Tyr Ile Ser Ile Ser 1 5 10 15 Tyr Val Leu Pro Leu Tyr Ala Thr
Ser Gln Pro Glu Gly Ser Lys Arg 20 25 30 Asp Asn Pro Arg Thr Ile
Lys Ser Arg Met Gln Lys Leu Thr Ile Met 35 40 45 Leu Ile Ser Asn
Leu Phe Leu Val Pro Phe Leu Gln Ser Gln Leu Ser 50 55 60 Ser Thr
Thr Ser His Ile Ser Phe Lys Asp Ala Phe Leu Gly Leu Gly 65 70 75 80
Ile Ile Pro Gly Tyr Tyr Ala Ala Leu Pro Asn Pro Trp Gln Phe Ser 85
90 95 Gln Phe Val Lys Asp Leu Thr Lys Cys Val Ala Met Leu Leu Thr
Leu 100 105 110 Tyr Cys Gly Pro Val Leu Asp Phe Val Leu Tyr His Leu
Leu Asn Pro 115 120 125 Lys Ser Ser Ile Leu Glu Asp Phe Tyr His Glu
Phe Leu Asn Ile Trp 130 135 140 Ser Phe Arg Asn Phe Ile Phe Ala Pro
Ile Thr Glu Glu Ile Phe Tyr 145 150 155 160 Thr Ser Met Leu Leu Thr
Thr Tyr Leu Asn Leu Ile Pro His Ser Gln 165 170 175 Leu Ser Tyr Gln
Gln Leu Phe Trp Gln Pro Ser Leu Phe Phe Gly Leu 180 185 190 Ala His
Ala His His Ala Tyr Glu Gln Leu Gln Glu Gly Ser Met Thr 195 200 205
Thr Val Ser Ile Leu Leu Thr Thr Cys Phe Gln Ile Leu Tyr Thr Thr 210
215 220 Leu Phe Gly Gly Leu Thr Lys Phe Val Phe Val Arg Thr Gly Gly
Asn 225 230 235 240 Leu Trp Cys Cys Ile Ile Leu His Ala Leu Cys Asn
Ile Met Gly Phe 245 250 255 Pro Gly Pro Ser Arg Leu Asn Leu His Phe
Thr Val Val Asp Lys Lys 260 265 270 Ala Gly Arg Ile Ser Lys Leu Val
Ser Ile Trp Asn Lys Cys Tyr Phe 275 280 285 Ala Leu Leu Val Leu Gly
Leu Ile Ser Leu Lys Asp
Thr Leu Gln Thr 290 295 300 Leu Val Gly Thr Pro Gly Tyr Arg Ile Thr
Leu 305 310 315 5 373 DNA mouse 5 tttggagtcg cccattttca ccacattatt
gagcagctgc gcttccgcca gagcagtgtg 60 ggaagtatct tcgtgtctgc
agcgttccag ttctcctaca ccgctgtctt cggtgcttat 120 acagctttcc
tcttcatccg cacaggacac ctgatagggc cggttctctg ccactctttc 180
tgcaactaca tgggcttccc tgcagtgtgt gcagccctgg agcatccaca gaagtggcca
240 ctgctggcag gctatgcctc ggtgtgggac ttttcctgct tctgcttcaa
cccctgacag 300 accccaagct ctatggcagc cttcctcttt gtatgctttt
ggaaagaaca ggggcctcag 360 agaccctact gtg 373 6 362 DNA human
misc_feature (93)..(156) "n" is G, A, C or T 6 cattattagc
cagatgaatt ctttcctgtg ttttttttta tttgctgtat taattggtcg 60
aaaggagctt tttgctgcat ttggttttta tgntagccaa cccactntta ttggactatt
120 gntcatcttc cagtttattt tttcacctta caatgnggtt ctttcttttt
gcctaacagt 180 cctaagccgc agatttgagt ttcaagctga tgcattgcca
agaaacttgg gaaggctaaa 240 gacttatatt ctgctttaat caaacttaac
aaagataact tgggattccc tgtttctgac 300 tggttgttct caatgtggca
ttattctcat cctccactgc tagagagact tcaagctttg 360 aa 362
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