# Patent

## Title
Methods for generating hypermutable yeast

## Abstract

Yeast cells are mutagenized to obtain desirable mutants. Mutagenesis is mediated by a defective mismatch repair system which can be enhanced using conventional exogenously applied mutagens. Yeast cells with the defective mismatch repair system are hypermutable, but after selection of desired mutant yeast strains, they can be rendered genetically stable by restoring the mismatch repair system to proper functionality.

## Background
This application is a continuation of U.S. Ser. No. 10/641,068, filed Aug. 15, 2003, which issued as U.S. Pat. No. 6,921,666 on Jul. 26, 2005, which is a divisional of U.S. Ser. No. 09/788,657, filed Feb. 21, 2001, which issued on Dec. 2, 2003, as U.S. Pat. No. 6,656,736, which claims the benefit of provisional application Ser. No. 60/184,336 filed Feb. 23, 2000. The disclosure of these prior applications is expressly incorporated herein.


FIELD OF THE INVENTION
The invention is related to the area of mismatch repair genes. In particular it is related to the field of in situ mutagenesis of single celled organisms.
BACKGROUND OF THE INVENTION
Within the past four years, the genetic cause of the Hereditary Nonpolyposis Colorectal Cancer Syndrome (HNPCC), also known as Lynch syndrome II, has been ascertained for the majority of kindred's affected with the disease (Liu, B., Parsons, R., Papadopoulos, N., Nicolaides, N. C., Lynch, H. T., Watson, P., Jass, J. R., Dunlop, M., Wyllie, A., Peltomaki, P., de la Chapelle, A., Hamilton, S. R., Vogelstein, B., and Kinzler, K. W. 1996. Analysis of mismatch repair genes in hereditary non-polyposis colorectal cancer patients. Nat. Med. 2:169-174). The molecular basis of HNPCC involves genetic instability resulting from defective mismatch repair (MMR). To date, six genes have been identified in humans that encode for proteins and appear to participate in the MMR process, including the mutS homologs GTBP, hMSH2, and hMSH3 and the mutL homologs hMLH1, hPMS1, and hPMS2 (Bronner, C. E., Baker, S. M., Morrison, P. T., Warren, G., Smith, L. G., Lescoe, M. K., Kane, M., Earabino, C., Lipford, J., Lindblom, A., Tannergard, P., Bollag, R. J., Godwin, A., R., Ward, D. C., Nordenskjold, M., Fishel, R., Kolodner, R., and Liskay, R. M. 1994. Mutation in the DNA mismatch repair gene homologue hMLH1 is associated with hereditary non-polyposis colon cancer. Nature 368:258-261; Fishel, R., Lescoe, M., Rao, M. R. S., Copeland, N. J., Jenkins, N. A., Garber, J., Kane, M., and Kolodner, R. 1993. The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer. Cell 7:1027-1038; Leach, F. S., Nicolaides, N. C, Papadopoulos, N., Liu, B., Jen, J., Parsons, R., Peltomaki, P., Sistonen, P., Aaltonen, L. A., Nystrom-Lahti, M., Guan, X. Y., Zhang, J., Meltzer, P. S., Yu, J. W., Kao, F. T., Chen, D. J., Cerosaletti, K. M., Fournier, R. E. K., Todd, S., Lewis, T., Leach R. J., Naylor, S. L., Weissenbach, J., Mecklin, J. P., Jarvinen, J. A., Petersen, G. M., Hamilton, S. R., Green, J., Jass, J., Watson, P., Lynch, H. T., Trent, J. M., de la Chapelle, A., Kinzler, K. W., and Vogelstein, B. 1993. Mutations of a mutS homolog in hereditary non-polyposis colorectal cancer. Cell 75:1215-1225; Nicolaides, N. C., Papadopoulos, N., Liu, B., Wei, Y. F., Carter, K. C., Ruben, S. M., Rosen, C. A., Haseltine, W. A., Fleischmann, R. D., Fraser, C. M., Adams, M. D., Venter, C. J., Dunlop, M. G., Hamilton, S. R., Petersen, G. M., de la Chapelle, A., Vogelstein, B., and kinzler, K. W. 1994. Mutations of two PMS homologs in hereditary nonpolyposis colon cancer. Nature 371: 75-80; Nicolaides, N. C., Palombo, F., Kinzler, K. W., Vogelstein, B., and Jiricny, J. 1996. Molecular cloning of the N-terminus of GTBP. Genomics 31:395-397; Palombo, F., Hughes, M., Jiricny, J., Truong, O., Hsuan, J. 1994. Mismatch repair and cancer. Nature 36:417; Palombo, F., Gallinari, P., Iaccarino, I., Lettieri, T., Hughes, M. A., Truong, O., Hsuan, J. J., and Jiricny, J. 1995. GTBP, a 160-kilodalton protein essential for mismatch-binding activity in human cells. Science 268:1912-1914; Papadopoulos, N., Nicolaides, N. C., Wei, Y. F., Carter, K. C., Ruben, S. M., Rosen, C. A., Haseltine, W. A., Fleischmann, R. D., Fraser, C. M., Adams, M. D., Venter, C. J., Dunlop, M. G., Hamilton, S. R., Petersen, G. M., de la Chapelle, A.,Vogelstein, B., and Kinzler, K. W. 1994. Mutation of a mutL homolog is associated with hereditary colon cancer. Science 263:1625-1629). Germline mutations in four of these genes (hMSH2, hMLH1, hPMS1, and hPMS2) have been identified in HNPCC kindred's (Bronner, C. E., Baker, S. M., Morrison, P. T., Warren, G., Smith, L. G., Lescoe, M. K., Kane, M., Earabino, C., Lipford, J., Lindblom, A., Tannergard, P., Bollag, R. J., Godwin, A., R., Ward, D. C., Nordenskjold, M., Fishel, R., Kolodner, R., and Liskay, R. M. 1994. Mutation in the DNA mismatch repair gene homologue hMLH1 is associated with hereditary non-polyposis colon cancer. Nature 368:258-261; Leach, F. S., Nicolaides, N. C, Papadopoulos, N., Liu, B., Jen, J., Parsons, R., Peltomaki, P., Sistonen, P., Aaltonen, L. A., Nystrom-Lahti, M., Guan, X. Y., Zhang, J., Meltzer, P. S., Yu, J. W., Kao, F. T., Chen, D. J., Cerosaletti, K. M., Fournier, R. E. K., Todd, S., Lewis, T., Leach R. J., Naylor, S. L., Weissenbach, J., Mecklin, J. P., Jarvinen, J. A., Petersen, G. M., Hamilton, S. R., Green, J., Jass, J., Watson, P., Lynch, H. T., Trent, J. M., de la Chapelle, A., Kinzler, K. W., and Vogelstein, B. 1993. Mutations of a mutS homolog in hereditary non-polyposis colorectal cancer. Cell 75:1215-1225; Liu, B., Parsons, R., Papadopoulos, N., Nicolaides, N. C., Lynch, H. T., Watson, P., Jass, J. R., Dunlop, M., Wyllie, A., Peltomaki, P., de la Chapelle, A., Hamilton, S. R., Vogelstein, B., and Kinzler, K. W. 1996. Analysis of mismatch repair genes in hereditary non-polyposis colorectal cancer patients. Nat. Med. 2:169-174; Nicolaides, N. C., Papadopoulos, N., Liu, B., Wei, Y. F., Carter, K. C., Ruben, S. M., Rosen, C. A., Haseltine, W. A., Fleischmann, R. D., Fraser, C. M., Adams, M. D., Venter, C. J., Dunlop, M. G., Hamilton, S. R., Petersen, G. M., de la Chapelle, A., Vogelstein, B., and kinzler, K. W. 1994. Mutations of two PMS homologs in hereditary nonpolyposis colon cancer. Nature 371: 75-80; Papadopoulos, N., Nicolaides, N. C., Wei, Y. F., Carter, K. C., Ruben, S. M., Rosen, C. A., Haseltine, W. A., Fleischmann, R. D., Fraser, C. M., Adams, M. D., Venter, C. J., Dunlop, M. G., Hamilton, S. R., Petersen, G. M., de la Chapelle, A., Vogelstein, B., and kinzler, K. W. 1994. Mutation of a mutL homolog is associated with hereditary colon cancer. Science 263:1625-1629). Though the mutator defect that arises from the MMR deficiency can affect any DNA sequence, microsatellite sequences are particularly sensitive to MMR abnormalities (Modrich, P. 1994. Mismatch repair, genetic stability, and cancer. Science 266:1959-1960). Microsatellite instability (MI) is therefore a useful indicator of defective MMR. In addition to its occurrence in virtually all tumors arising in HNPCC patients, MI is found in a small fraction of sporadic tumors with distinctive molecular and phenotypic properties (Perucho, M. 1996. Cancer of the microsattelite mutator phenotype. Biol Chem. 377:675-684).
HNPCC is inherited in an autosomal dominant fashion, so that the normal cells of affected family members contain one mutant allele of the relevant MMR gene (inherited from an affected parent) and one wild-type allele (inherited from the unaffected parent). During the early stages of tumor development, however, the wild-type allele is inactivated through a somatic mutation, leaving the cell with no functional MMR gene and resulting in a profound defect in MMR activity. Because a somatic mutation in addition to a germ-line mutation is required to generate defective MMR in the tumor cells, this mechanism is generally referred to as one involving two hits, analogous to the biallelic inactivation of tumor suppressor genes that initiate other hereditary cancers (Leach, F. S., Nicolaides, N. C, Papadopoulos, N., Liu, B., Jen, J., Parsons, R., Peltomaki, P., Sistonen, P., Aaltonen, L. A., Nystrom-Lahti, M., Guan, X. Y., Zhang, J., Meltzer, P. S., Yu, J. W., Kao, F. T., Chen, D. J., Cerosaletti, K. M., Fournier, R. E. K., Todd, S., Lewis, T., Leach R. J., Naylor, S. L., Weissenbach, J., Mecklin, J. P., Jarvinen, J. A., Petersen, G. M., Hamilton, S. R., Green, J., Jass, J., Watson, P., Lynch, H. T., Trent, J. M., de la Chapelle, A., Kinzler, K. W., and Vogelstein, B. 1993. Mutations of a mutS homolog in hereditary non-polyposis colorectal cancer. Cell 75:1215-1225; Liu, B., Parsons, R., Papadopoulos, N., Nicolaides, N. C., Lynch, H. T., Watson, P., Jass, J. R., Dunlop, M., Wyllie, A., Peltomaki, P., de la Chapelle, A., Hamilton, S. R., Vogelstein, B., and Kinzler, K. W. 1996. Analysis of mismatch repair genes in hereditary non-polyposis colorectal cancer patients. Nat. Med. 2:169-174; Parsons, R., Li, G. M., Longley, M. J., Fang, W. H., Papadopolous, N., Jen, J., de la Chapelle, A., Kinzler, K. W., Vogelstein, B., and Modrich, P. 1993. Hypermutability and mismatch repair deficiency in RER+ tumor cells. Cell 75:1227-1236). In line with this two-hit mechanism, the non-neoplastic cells of HNPCC patients generally retain near normal levels of MMR activity due to the presence of the wild-type allele.
The ability to alter the signal transduction pathways by manipulation of a gene products function, either by over-expression of the wild type protein or a fragment thereof, or by introduction of mutations into specific protein domains of the protein, the so-called dominant-negative inhibitory mutant, were described over a decade in the yeast system Saccharomyces cerevisiae by Herskowitz (Nature 329(6136):219-222, 1987). It has been demonstrated that over-expression of wild type gene products can result in a similar, dominant-negative inhibitory phenotype due most likely to the “saturating-out” of a factor, such as a protein, that is present at low levels and necessary for activity; removal of the protein by binding to a high level of its cognate partner results in the same net effect, leading to inactivation of the protein and the associated signal transduction pathway. Recently, work done by Nicolaides et.al. (Nicolaides N C, Littman S J, Modrich P, Kinzler K W, Vogelstein B 1998. A naturally occurring hPMS2 mutation can confer a dominant negative mutator phenotype. Mol Cell Biol 18:1635-1641) has demonstrated the utility of introducing dominant negative inhibitory mismatch repair mutants into mammalian cells to confer global DNA hypermutability. The ability to manipulate the MMR process and therefore increase the mutability of the target host genome at will, in this example a mammalian cell, allows for the generation of innovative cell subtypes or variants of the original wild type cells. These variants can be placed under a specified, desired selective process, the result of which is a novel organism that expresses an altered biological molecule(s) and has a new trait. The concept of creating and introducing dominant negative alleles of a gene, including the MMR alleles, in bacterial cells has been documented to result in genetically altered prokaryotic mismatch repair genes (Aronshtam A, Marinus M G. 1996. Dominant negative mutator mutations in the mutL gene of Escherichia coli. Nucleic Acids Res 24:2498-2504; Wu T H, Marinus M G. 1994. Dominant negative mutator mutations in the mutS gene of Escherichia coli. J Bacteriol 176:5393-400; Brosh R M Jr, Matson S W. 1995. Mutations in motif II of Escherichia coli DNA helicase II render the enzyme nonfunctional in both mismatch repair and excision repair with differential effects on the unwinding reaction. J Bacteriol 177:5612-5621). Furthermore, altered MMR activity has been demonstrated when MMR genes from different species including yeast, mammalian cells, and plants are over-expressed (Fishel, R., Lescoe, M., Rao, M. R. S., Copeland, N. J., Jenkins, N. A., Garber, J., Kane, M., and Kolodner, R. 1993. The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer. Cell 7:1027-1038; Studamire B, Quach T, Alani, E. 1998. Saccharomyces cerevisiae Msh2p and Msh6p ATPase activities are both required during mismatch repair. Mol Cell Biol 18:7590-7601; Alani E, Sokolsky T, Studamire B, Miret J J, Lahue R S. 1997. Genetic and biochemical analysis of Msh2p-Msh6p: role of ATP hydrolysis and Msh2p-Msh6p subunit interactions in mismatch base pair recognition. Mol Cell Biol 17:2436-2447; Lipkin S M, Wang V, Jacoby R, Banerjee-Basu S, Baxevanis A D, Lynch H T, Elliott R M, and Collins F S. 2000. MLH3: a DNA mismatch repair gene associated with mammalian microsatellite instability. Nat. Genet. 24:27-35).
There is a continuing need in the art for methods of genetically manipulating useful strains of yeast to increase their performance characteristics and abilities.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a method for rendering yeast cells hypermutable.
It is another object of the invention to provide hypermutable yeast cells.
It is a further object of the invention to provide a method of mutating a gene of interest in a yeast.
It is yet another object of the present invention to provide a method to produce yeast that are hypermutable.
It is an object of the invention to provide a method to restore normal mismatch repair activity to hypermutable cells following strain selection.
These and other objects of the invention are provided by one or more of the following embodiments. In one embodiment a method is provided for making a hypermutable yeast. A polynucleotide comprising a dominant negative allele of a mismatch repair gene is introduced into a yeast cell. The cell thus becomes hypermutable.
According to another embodiment a homogeneous composition of cultured, hypermutable yeast cells is provided. The yeast cells comprise a dominant negative allele of a mismatch repair gene.
According to still another embodiment of the invention a method is provided for generating a mutation in a gene of interest. A yeast cell culture comprising the gene of interest and a dominant negative allele of a mismatch repair gene is cultivated. The yeast cell is hypermutable. Cells of the culture are tested to determine whether the gene of interest harbors a mutation.
In yet another embodiment of the invention a method is provided for generating a mutation in a gene of interest. A yeast cell comprising the gene of interest and a polynucleotide encoding a dominant negative allele of a mismatch repair gene is grown to create a population of mutated, hypermutable yeast cells. The population of mutated, hypermutable yeast cells is cultivated under trait selection conditions. Yeast cells which grow under trait selection conditions are tested to determine whether the gene of interest harbors a mutation.
Also provided by the present invention is a method for generating enhanced hypermutable yeast. A yeast cell is exposed to a mutagen. The yeast cell is defective in mismatch repair (MMR) due to the presence of a dominant negative allele of at least one MMR gene. An enhanced rate of mutation of the yeast cell is achieved due to the exposure to the mutagen.
According to still another aspect of the invention a method is provided for generating mismatch repair (MMR)-proficient yeast with new output traits. A yeast cell comprising a gene of interest and a polynucleotide encoding a dominant negative allele of a mismatch repair gene is grown to create a population of mutated, hypermutable yeast cells. The population of mutated, hypermutable yeast cells is cultivated under trait selection conditions. The yeast cells which grow under trait selection conditions are tested to determine whether the gene of interest harbors a mutation. Normal mismatch repair activity is restored to the yeast cells.
These and other embodiments of the invention provide the art with methods that can generate enhanced mutability in yeast as well as providing single-celled eukaryotic organisms harboring potentially useful mutations to generate novel output traits for commercial applications.


DETAILED DESCRIPTION OF THE INVENTION
It is a discovery of the present invention that hypermutable yeast can be made by altering the activity of endogenous mismatch repair activity of host cells. Dominant negative alleles of mismatch repair genes, when introduced and expressed in yeast, increase the rate of spontaneous mutations by reducing the effectiveness of endogenous mismatch repair-mediated DNA repair activity, thereby rendering the yeast highly susceptible to genetic alterations, i.e., hypermutable. Hypermutable yeast can then be utilized to screen for mutations in a gene or a set of genes in variant siblings that exhibit an output trait(s) not found in the wild-type cells.
The process of mismatch repair, also called mismatch proofreading, is an evolutionarily highly conserved process that is carried out by protein complexes described in cells as disparate as prokaryotic cells such as bacteria to more complex mammalian cells (Modrich, P. 1994. Mismatch repair, genetic stability, and cancer. Science 266:1959-1960; Parsons, R., Li, G. M., Longley, M., Modrich, P., Liu, B., Berk, T., Hamilton, S. R., Kinzler, K. W., and Vogelstein, B. 1995. Mismatch repair deficiency in phenotypically normal human cells. Science 268:738-740; Perucho, M. 1996. Cancer of the microsattelite mutator phenotype. Biol Chem. 377:675-684). A mismatch repair gene is a gene that encodes one of the proteins of such a mismatch repair complex. Although not wanting to be bound by any particular theory of mechanism of action, a mismatch repair complex is believed to detect distortions of the DNA helix resulting from non-complementary pairing of nucleotide bases. The non-complementary base on the newer DNA strand is excised, and the excised base is replaced with the appropriate base that is complementary to the older DNA strand. In this way, cells eliminate many mutations that occur as a result of mistakes in DNA replication, resulting in genetic stability of the sibling cells derived from the parental cell.
Some wild type alleles as well as dominant negative alleles cause a mismatch repair defective phenotype even in the presence of a wild-type allele in the same cell. An example of a dominant negative allele of a mismatch repair gene is the human gene hPMS2-134, which carries a truncation mutation at codon 134 (Parsons, R., Li, G. M., Longley, M., Modrich, P., Liu, B., Berk, T., Hamilton, S. R., Kinzler, K. W., and Vogelstein, B. 1995. Mismatch repair deficiency in phenotypically normal human cells. Science 268:738-740; Nicolaides N C, Littman S J, Modrich P, Kinzler K W, Vogelstein B 1998. A naturally occurring hPMS2 mutation can confer a dominant negative mutator phenotype. Mol Cell Biol 18:1635-1641). The mutation causes the product of this gene to abnormally terminate at the position of the 134th amino acid, resulting in a shortened polypeptide containing the N-terminal 133 amino acids. Such a mutation causes an increase in the rate of mutations, which accumulate in cells after DNA replication. Expression of a dominant negative allele of a mismatch repair gene results in impairment of mismatch repair activity, even in the presence of the wild-type allele. Any mismatch repair allele, which produces such effect, can be used in this invention, whether it is wild-type or altered, whether it derives from mammalian, yeast, fungal, amphibian, insect, plant, or bacteria. In addition, the use of over-expressed wild type MMR gene alleles from human, mouse, plants, and yeast in bacteria has been shown to cause a dominant negative effect on the bacterial hosts MMR activity (Aronshtam A, Marinus M G. 1996. Dominant negative mutator mutations in the mutL gene of Escherichia coli. Nucleic Acids Res 24:2498-2504; Wu T H, Marinus M G. 1994. Dominant negative mutator mutations in the mutS gene of Escherichia coli. J Bacteriol 176:5393-400; Brosh R M Jr, Matson S W. 1995. Mutations in motif II of Escherichia coli DNA helicase II render the enzyme nonfunctional in both mismatch repair and excision repair with differential effects on the unwinding reaction. J Bacteriol 177:5612-5621; Lipkin S M, Wang V, Jacoby R, Banerjee-Basu S, Baxevanis A D, Lynch H T, Elliott R M, and Collins F S. 2000. MLH3: a DNA mismatch repair gene associated with mammalian microsatellite instability. Nat Genet 24:27-35). This suggests that perturbation of the multi-component MMR protein complex can be accomplished by introduction of MMR components from other species into yeast.
Dominant negative alleles of a mismatch repair gene can be obtained from the cells of humans, animals, yeast, bacteria, plants or other organisms. Screening cells for defective mismatch repair activity can identify such alleles. Mismatch repair genes may be mutant or wild type. Yeast host MMR may be mutated or not. The term yeast used in this application comprises any organism from the eukaryotic kingdom, including but not limited to Saccharomyces sp., Pichia sp., Schizosaccharomyces sp., Kluyveromyces sp., and other fungi (Gellissen, G. and Hollenberg, C P. Gene 190(1):87-97, 1997). These organisms can be exposed to chemical mutagens or radiation, for example, and can be screened for defective mismatch repair. Genomic DNA, cDNA, mRNA, or protein from any cell encoding a mismatch repair protein can be analyzed for variations from the wild-type sequence. Dominant negative alleles of a mismatch repair gene can also be created artificially, for example, by producing variants of the hPMS2-134 allele or other mismatch repair genes (Nicolaides N C, Littman S J, Modrich P, Kinzler K W, Vogelstein B 1998. A naturally occurring hPMS2 mutation can confer a dominant negative mutator phenotype. Mol Cell Biol 18:1635-1641). Various techniques of site-directed mutagenesis can be used. The suitability of such alleles, whether natural or artificial, for use in generating hypermutable yeast can be evaluated by testing the mismatch repair activity (using methods described in Nicolaides N C, Littman S J, Modrich P, Kinzler K W, Vogelstein B 1998. A naturally occurring hPMS2 mutation can confer a dominant negative mutator phenotype. Mol Cell Biol 18:1635-1641) caused by the allele in the presence of one or more wild-type alleles to determine if it is a dominant negative allele.
A yeast that over-expresses a wild type mismatch repair allele or a dominant negative allele of a mismatch repair gene will become hypermutable. This means that the spontaneous mutation rate of such yeast is elevated compared to yeast without such alleles. The degree of elevation of the spontaneous mutation rate can be at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1000-fold that of the normal yeast as measured as a function of yeast doubling/hour.
According to one aspect of the invention, a polynucleotide encoding either a wild type or a dominant negative form of a mismatch repair protein is introduced into yeast. The gene can be any dominant negative allele encoding a protein which is part of a mismatch repair complex, for example, mutS, mutL, mutH, or mutY homologs of the bacterial, yeast, plant or mammalian genes (Modrich, P. 1994. Mismatch repair, genetic stability, and cancer. Science 266:1959-1960; Prolla, T. A, Pang, Q., Alani, E., Kolodner, R. A., and Liskay, R. M. 1994. MLH1, PMS1, and MSH2 Interaction during the initiation of DNA mismatch repair in yeast. Science 264:1091-1093). The dominant negative allele can be naturally occurring or made in the laboratory. The polynucleotide can be in the form of genomic DNA, cDNA, RNA, or a chemically synthesized polynucleotide or polypeptide. The molecule can be introduced into the cell by transformation, electroporation, mating, particle bombardment, or other method described in the literature.
Transformation is used herein as any process whereby a polynucleotide or polypeptide is introduced into a cell. The process of transformation can be carried out in a yeast culture using a suspension of cells. The yeast can be any type classified under the eukayotic kingdom as by international convention.
In general, transformation will be carried out using a suspension of cells but other methods can also be employed as long as a sufficient fraction of the treated cells incorporate the polynucleotide or polypeptide so as to allow transfected cells to be grown and utilized. The protein product of the polynucleotide may be transiently or stably expressed in the cell. Techniques for transformation are well known to those skilled in the art. Available techniques to introduce a polynucleotide or polypeptide into a yeast cell include but are not limited to electroporation, viral transduction, cell fusion, the use of spheroplasts or chemically competent cells (e.g., calcium chloride), and packaging of the polynucleotide together with lipid for fusion with the cells of interest. Once a cell has been transformed with the mismatch repair gene or protein, the cell can be propagated and manipulated in either liquid culture or on a solid agar matrix, such as a petri dish. If the transfected cell is stable, the gene will be expressed at a consistent level for many cell generations, and a stable, hypermutable yeast strain results.
An isolated yeast cell can be obtained from a yeast culture by chemically selecting strains using antibiotic selection of an expression vector. If the yeast cell is derived from a single cell, it is defined as a clone. Techniques for single-cell cloning of microorganisms such as yeast are well known in the art.
A polynucleotide encoding a dominant negative form of a mismatch repair protein can be introduced into the genome of yeast or propagated on an extra-chromosomal plasmid, such as the 2-micron plasmid. Selection of clones harboring a mismatch repair gene expression vector can be accomplished by plating cells on synthetic complete medium lacking the appropriate amino acid or other essential nutrient as described (J. C. Schneider and L. Guarente, Methods in Enzymology 194:373,1991). The yeast can be any species for which suitable techniques are available to produce transgenic microorganisms, such as but not limited to genera including Saccharomyces, Schizosaccharomyces, Pichia, Hansenula, Kluyveromyces and others.
Any method for making transgenic yeast known in the art can be used. According to one process of producing a transgenic microorganism, the polynucleotide is introduced into the yeast by one of the methods well known to those in the art. Next, the yeast culture is grown under conditions that select for cells in which the polynucleotide encoding the mismatch repair gene is either incorporated into the host genome as a stable entity or propagated on a self-replicating extra-chromosomal plasmid, and the protein encoded by the polynucleotide fragment transcribed and subsequently translated into a functional protein within the cell. Once transgenic yeast is engineered to harbor the expression construct, it is then propagated to generate and sustain a culture of transgenic yeast indefinitely.
Once a stable, transgenic yeast cell has been engineered to express a defective mismatch repair (MMR) protein, the yeast can be cultivated to create novel mutations in one or more target gene(s) of interest harbored within the same yeast cell. A gene of interest can be any gene naturally possessed by the yeast or one introduced into the yeast host by standard recombinant DNA techniques. The target gene(s) may be known prior to the selection or unknown. One advantage of employing such transgenic yeast cells to induce mutations in resident or extra-chromosomal genes within the yeast is that it is unnecessary to expose the cells to mutagenic insult, whether it is chemical or radiation, to produce a series of random gene alterations in the target gene(s). This is due to the highly efficient nature and the spectrum of naturally occurring mutations that result as a consequence of the altered mismatch repair process. However, it is possible to increase the spectrum and frequency of mutations by the concomitant use of either chemical and/or radiation together with MMR defective cells. The net effect of the combination treatment is an increase in mutation rate in the genetically altered yeast that are useful for producing new output traits. The rate of the combination treatment is higher than the rate using only the MMR-defective cells or only the mutagen with wild-type MMR cells.
MMR-defective yeast of the invention can be used in genetic screens for the direct selection of variant sub-clones that exhibit new output traits with commercially desirable applications. This permits one to bypass the tedious and time consuming steps of gene identification, isolation and characterization.
Mutations can be detected by analyzing the internally and/or externally mutagenized yeast for alterations in its genotype and/or phenotype. Genes that produce altered phenotypes in MMR-defective microbial cells can be discerned by any of a variety of molecular techniques well known to those in the art. For example, the yeast genome can be isolated and a library of restriction fragments of the yeast genome can be cloned into a plasmid vector. The library can be introduced into a “normal” cell and the cells exhibiting the novel phenotype screened. A plasmid can be isolated from those normal cells that exhibit the novel phenotype and the gene(s) characterized by DNA sequence analysis. Alternatively, differential messenger RNA screen can be employed utilizing driver and tester RNA (derived from wild type and novel mutant, respectively) followed by cloning the differential transcripts and characterizing them by standard molecular biology methods well known to those skilled in the art. Furthermore, if the mutant sought is encoded by an extra-chromosomal plasmid, then following co-expression of the dominant negative MMR gene and the gene of interest, and following phenotypic selection, the plasmid can be isolated from mutant clones and analyzed by DNA sequence analysis using methods well known to those in the art. Phenotypic screening for output traits in MMR-defective mutants can be by biochemical activity and/or a readily observable phenotype of the altered gene product. A mutant phenotype can also be detected by identifying alterations in electrophoretic mobility, DNA binding in the case of transcription factors, spectroscopic properties such as IR, CD, X-ray crystallography or high field NMR analysis, or other physical or structural characteristics of a protein encoded by a mutant gene. It is also possible to screen for altered novel function of a protein in situ, in isolated form, or in model systems. One can screen for alteration of any property of the yeast associated with the function of the gene of interest, whether the gene is known prior to the selection or unknown.
The screening and selection methods discussed are meant to illustrate the potential means of obtaining novel mutants with commercially valuable output traits, but they are not meant to limit the many possible ways in which screening and selection can be carried out by those of skill in the art.
Plasmid expression vectors that harbor a mismatch repair (MMR) gene insert can be used in combination with a number of commercially available regulatory sequences to control both the temporal and quantitative biochemical expression level of the dominant negative MMR protein. The regulatory sequences can be comprised of a promoter, enhancer or promoter/enhancer combination and can be inserted either upstream or downstream of the MMR gene to control the expression level. The regulatory sequences can be any of those well known to those in the art, including but not limited to the AOX1, GAP, GAL1, GAL10, PHO5, and PGK promoters harbored on high or low copy number extra-chromosomal expression vectors or on constructs that are integrated into the genome via homologous recombination. These types of regulatory systems have been disclosed in scientific publications and are familiar to those skilled in the art.
Once a microorganism with a novel, desired output trait of interest is created, the activity of the aberrant MMR activity is desirably attenuated or eliminated by any means known in the art. These include but are not limited to removing an inducer from the culture medium that is responsible for promoter activation, curing a plasmid from a transformed yeast cell, and addition of chemicals, such as 5-fluoro-orotic acid to “loop-out” the gene of interest.
In the case of an inducibly controlled dominant negative MMR allele, expression of the dominant negative MMR gene will be turned on (induced) to generate a population of hypermutable yeast cells with new output traits. Expression of the dominant negative MMR allele can be rapidly turned off to reconstitute a genetically stable strain that displays a new output trait of commercial interest. The resulting yeast strain is now useful as a stable strain that can be applied to various commercial applications, depending upon the selection process placed upon it.
In cases where genetically deficient mismatch repair yeast [strains such as but not limited to: M1 (mutS) and in EC2416 (mutS delta umuDC), and mutL or mutY strains] are used to derive new output traits, transgenic constructs can be used that express wild type mismatch repair genes sufficient to complement the genetic defect and therefore restore mismatch repair activity of the host after trait selection [Grzesiuk, E. et.al. (Mutagenesis 13;127-132, 1998); Bridges, B. A., et.al. (EMBO J. 16:3349-3356, 1997); LeClerc, J. E., Science 15:1208-1211, 1996); Jaworski, A. et.al. (Proc. Natl. Acad. Sci USA 92:11019-11023, 1995)]. The resulting yeast is genetically stable and can be employed for various commercial applications.
The use of over-expression of foreign (exogenous, transgenic) mismatch repair genes from human and yeast such as MSH2, MLH1, MLH3, etc. have been previously demonstrated to produce a dominant negative mutator phenotype in yeast hosts (Shcherbakova, P. V., Hall, M. C., Lewis, M. S., Bennett, S. E., Martin, K. J., Bushel, P. R., Afshari, C. A., and Kunkel, T. A. Mol. Cell Biol. 21(3):940-951; Studamire B, Quach T, Alani, E. 1998. Saccharomyces cerevisiae Msh2p and Msh6p ATPase activities are both required during mismatch repair. Mol Cell Biol 18:7590-7601; Alani E, Sokolsky T, Studamire B, Miret J J, Lahue R S. 1997. Genetic and biochemical analysis of Msh2p-Msh6p: role of ATP hydrolysis and Msh2p-Msh6p subunit interactions in mismatch base pair recognition. Mol Cell Biol 17:2436-2447; Lipkin S M, Wang V, Jacoby R, Banerjee-Basu S, Baxevanis A D, Lynch H T, Elliott R M, and Collins F S. 2000. MLH3: a DNA mismatch repair gene associated with mammalian microsatellite instability. Nat Genet 24:27-35). In addition, the use of yeast strains expressing prokaryotic dominant negative MMR genes as well as hosts that have genomic defects in endogenous MMR proteins have also been previously shown to result in a dominant negative mutator phenotype (Evans, E., Sugawara, N., Haber, J. E., and Alani, E. Mol. Cell. 5(5):789-799, 2000; Aronshtam A, Marinus M G. 1996. Dominant negative mutator mutations in the mutL gene of Escherichia coli. Nucleic Acids Res 24:2498-2504; Wu T H, Marinus M G. 1994. Dominant negative mutator mutations in the mutS gene of Escherichia coli. J Bacteriol 176:5393-400; Brosh R M Jr, Matson S W. 1995. Mutations in motif II of Escherichia coli DNA helicase II render the enzyme nonfunctional in both mismatch repair and excision repair with differential effects on the unwinding reaction. J Bacteriol 177:5612-5621). However, the findings disclosed here teach the use of MMR genes, including the human PMSR2 gene (Nicolaides, N. C., Carter, K. C., Shell, B. K., Papadopoulos, N., Vogelstein, B., and Kinzler, K. W. 1995. Genomic organization of the human PMS2 gene family. Genomics 30:195-206), the related PMS134 truncated MMR gene (Nicolaides N. C., Kinzler, K. W., and Vogelstein, B. 1995. Analysis of the 5′ region of PMS2 reveals heterogenous transcripts and a novel overlapping gene. Genomics 29:329-334), the plant mismatch repair genes (U.S. patent application Ser. No. 09/749,601) and those genes that are homologous to the 134 N-terminal amino acids of the PMS2 gene to create hypermutable yeast.
DNA mutagens can be used in combination with MMR defective yeast hosts to enhance the hypermutable production of genetic alterations. This further reduces MMR activity and is useful for generation of microorganisms with commercially relevant output traits.
The ability to create hypermutable organisms using dominant negative alleles can be used to generate innovative yeast strains that display new output features useful for a variety of applications, including but not limited to the manufacturing industry, for the generation of new biochemicals, for detoxifying noxious chemicals, either by-products of manufacturing processes or those used as catalysts, as well as helping in remediation of toxins present in the environment, including but not limited to polychlorobenzenes (PCBs), heavy metals and other environmental hazards. Novel yeast strains can be selected for enhanced activity to either produce increased quantity or quality of a protein or non-protein therapeutic molecule by means of biotransformation. Biotransformation is the enzymatic conversion of one chemical intermediate to the next intermediate or product in a pathway or scheme by a microbe or an extract derived from the microbe. There are many examples of biotransformation in use for the commercial manufacturing of important biological and chemical products, including penicillin G, erythromycin, and clavulanic acid. Organisms that are efficient at conversion of “raw” materials to advanced intermediates and/or final products also can perform biotransformation (Berry, A. Trends Biotechnol. 14(7):250-256). The ability to control DNA hypermutability in host yeast strains using a dominant negative MMR (as described above) allows for the generation of variant subtypes that can be selected for new phenotypes of commercial interest, including but not limited to organisms that are toxin-resistant, have the capacity to degrade a toxin in situ or the ability to convert a molecule from an intermediate to either an advanced intermediate or a final product. Other applications using dominant negative MMR genes to produce genetic alteration of yeast hosts for new output traits include but are not limited to recombinant production strains that produce higher quantities of a recombinant polypeptide as well as the use of altered endogenous genes that can transform chemical or catalyze manufacturing downstream processes. A regulatable dominant negative MMR phenotype can be used to produce a yeast strain with a commercially beneficial output trait. Using this process, single-celled yeast cells expressing a dominant negative MMR can be directly selected for the phenotype of interest. Once a selected yeast with a specified output trait is isolated, the hypermutable activity of the dominant negative MMR allele can be turned-off by several methods well known to those skilled in the art. For example, if the dominant-negative allele is expressed by an inducible promoter system, the inducer can be removed or depleted. Such systems include but are not limited to promoters such as: lactose inducible_GALi-GAL10 promoter (M. Johnston and R. W. Davis, Mol. Cell Biol. 4:1440, 1984); the phosphate inducible PHO5 promoter (A. Miyanohara, A. Toh-e, C. Nosaki, F. Nosaki, F. Hamada, N. Ohtomo, and K. Matsubara. Proc. Nati. Acad. Sci. U.S.A. 80:1, 1983); the alcohol dehydrogenase I (ADH) and 3-phosphoglycerate kinase (PGK) promoters, that are considered to be constitutive but can be repressed/de-repressed when yeast cells are grown in non-fermentable carbon sources such as but not limited to lactate (G. Ammerer, Methods in Enzymology 194:192, 1991; J. Mellor, M. J. Dobson, N. A. Roberts, M. F. Tuite, J. S. Emtage, S. White, D. A. Lowe, T. Patel, A. J. Kingsman, and S. M. Kingsman, Gene 24:563, 1982); S. Hahn and L. Guarente, Science 240:317, 1988); Alcohol oxidase (AOX) in Pichia pastoris (Tschopp, J F, Brust, P F, Cregg, J M, Stiliman, C A, and Gingeras, T R. Nucleic Acids Res. 15(9):3859-76, 1987; and the thiamine repressible expression promoter nmtl in Schizosaccharomyces pombe (Moreno, M B, Duran, A., and Ribas, J C. Yeast 16(9):861-72, 2000). Yeast cells can be transformed by any means known to those skilled in the art, including chemical transformation with LiCl (Mount, R. C., Jordan, B. E., and Hadfield, C. Methods Mol. Biol. 53:139-145,1996) and electroporation (Thompson, J R, Register, E., Curotto, J., Kurtz, M. and Kelly, R. Yeast 14(6):565-71, 1998). Yeast cells that have been transformed with DNA can be selected for growth by a variety of methods, including but not restricted to selectable markers (URA3; Rose, M., Grisafi, P., and Botstein, D. Gene 29:113,1984; LEU2; A. Andreadis, Y., Hsu, M., Hermodson, G., Kohihaw, and P. Schimmel. J. Biol. Chem. 259:8059,1984; ARG4; G. Tschumper and J. Carbon. Gene 10:157, 1980; and HIS3; K. Struhl, D. T. Stinchcomb, S., Scherer, and R. W. Davis Proc. Natl. Acad. Sci. U.S.A. 76:1035,1979) and drugs that inhibit growth of yeast cells (tunicamycin, TUN; S. Hahn, J., Pinkham, R. Wei, R., Miller, and L. Guarente. Mol. Cell Biol. 8:655,1988). Recombinant DNA can be introduced into yeast as described above and the yeast vectors can be harbored within the yeast cell either extra-chromosomally or integrated into a specific locus. Extra-chromosomal based yeast expression vectors can be either high copy based (such as the 2-.mu.m vector Yepl3; A. B. Rose and J. R. Broach, Methods in Enzymology 185:234,1991), low copy centromeric vectors that contain autonomously replicating sequences (ARS) such as YRp7 (M. Fitzgerald-Hayes, L. Clarke, and J. Carbon, Cell 29:235,1982) and well as integration vectors that permit the gene of interest to be introduced into specified locus within the host genome and propagated in a stable manner (R. J. Rothstein, Methods in Enzymology 101:202, 1991). Ectopic expression of MMR genes in yeast can be attenuated or completely eliminated at will by a variety of methods, including but not limited to removal from the medium of the specific chemical inducer (e.g., deplete galactose that drives expression of the GAL10 promoter in Saccharomyces cerevisiae or methanol that drives expression of the AOX1 promoter in Pichia pastoris), extra-chromosomally replicating plasmids can be “cured” of expression plasmid by growth of cells under non-selective conditions (e.g. YEpl3 harboring cells can be propagated in the presence of leucine) and cells that have genes inserted into the genome can be grown with chemicals that force the inserted locus to “loop-out” (e.g., integrants that have URA3 can be selected for loss of the inserted gene by growth of integrants on 5-fluoro-orotic acid (J. D. Boeke, F. LaCroute and G. R. Fink. Mol. Gen. Genet. 197:345-346,1984). Whether by withdrawal of inducer or treatment of yeast cells with chemicals, removal of MMR expression results in the re-establishment of a genetically stable yeast cell-line. Thereafter, the lack of mutant MMR allows the endogenous, wild type MMR activity in the host cell to function normally to repair DNA. The newly generated mutant yeast strains that exhibit novel, selected output traits are suitable for a wide range of commercial processes or for gene/protein discovery to identify new biomolecules that are involved in generating a particular output trait. While it has been documented that MMR deficiency can lead to as much as a 1000-fold increase in the endogenous DNA mutation rate of a host, there is no assurance that MMR deficiency alone will be sufficient to alter every gene within the DNA of the host bacterium to create altered biochemicals with new activity(s). Therefore, the use of chemical mutagens and their respective analogues such as ethidium bromide, EMS, MNNG, MNu, Tamoxifen, 8-Hydroxyguanine, as well as others such as those taught in: Khromov-Borisov, N. N., et al, (Mutat. Res. 430:55-74, 1999); Ohe, T., et al, (Mutat. Res. 429:189-199, 1999); Hour, T. C. et al, (Food Chem. Toxicol. 37:569-579, 1999); Hrelia, P., et al, (Chem. Biol. Interact. 118:99-111, 1999); Garganta, F., et al, (Environ. Mol. Mutagen. 33:75-85, 1999); Ukawa-Ishikawa S., et al, (Mutat. Res. 412:99-107, 1998); the website having the URL address: www host server, ehs.utah.edu domain name, ohh directory, mutagens subdirectory, etc. can be used to further enhance the spectrum of mutations and increase the likelihood of obtaining alterations in one or more genes that can in turn generate host yeast with a desired new output trait(s). Mismatch repair deficiency leads to hosts with an increased resistance to toxicity by chemicals with DNA damaging activity. This feature allows for the creation of additional genetically diverse hosts when mismatch defective yeast are exposed to such agents, which would be otherwise impossible due to the toxic effects of such chemical mutagens [Colella, G., et al, (Br. J. Cancer 80:338-343, 1999); Moreland, N. J., et al, (Cancer Res. 59:2102-2106, 1999); Humbert, O., et.al. (Carcinogenesis 20:205-214, 1999); Glaab, W. E., et al, (Mutat. Res. 398:197-207, 1998)]. Moreover, mismatch repair is responsible for repairing chemically-induced DNA adducts, therefore blocking this process could theoretically increase the number, types, mutation rate and genomic alterations of a yeast1 [Rasmussen, L. J. et al, (Carcinogenesis 17:2085-2088, 1996); Sledziewska-Gojska, E., et al, (Mutat. Res. 383 :31-37, 1997); and Janion, C. et al, (Mutat. Res. 210:15-22, 1989)]. In addition to the chemicals listed above, other types of DNA mutagens include ionizing radiation and UV-irradiation, which is known to cause DNA mutagenesis in yeast, can also be used to potentially enhance this process (Lee C C, Lin H K, Lin J K. 1994. A reverse mutagenicity assay for alkylating agents based on a point mutation in the beta-lactamase gene at the active site serine codon. Mutagenesis 9:401-405; Vidal A, Abril N, Pueyo C. 1995. DNA repair by Ogt alkyltransferase influences EMS mutational specificity. Carcinogenesis 16:817-821). These agents, which are extremely toxic to host cells and therefore result in a decrease in the actual pool size of altered yeast cells are more tolerated in MMR defective hosts and in turn permit an enriched spectrum and degree of genomic mutagenesis.
The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples that will be provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.
EXAMPLES
Example 1
Generation of Inducible MMR Dominant Negative Allele Vectors and Yeast Cells Harboring the Expression Vectors
Yeast expression constructs were prepared to determine if the human PMS2 related gene (hPMSR2) (Nicolaides et al. Genomics 30(2): 195-206) and the human PMS 134 gene (Nicolaides NC, Littman SJ, Modrich P, Kinzler KW, Vogelstein B 1998. A naturally occurring hPMS2 mutation can confer a dominant negative mutator phenotype. Mol Cell Biol 18:1635-1641) are capable of inactivating the yeast MMR activity and thereby increase the overall frequency of genomic hypermutation, a consequence of which is the generation of variant sib cells with novel output traits following host selection. For these studies, a plasmid encoding the hPMS 134 cDNA was altered by polymerase chain reaction (PCR). The 5′ oligonucleotide has the following structure: 5′-ACG CAT ATG GAG CGA GCT GAG AGC TCG AGT-3′ (SEO ID NO: 1) that includes the NdeI restriction site CAT ATG. The 3 ′ -oligonucleotide has the following structure: 5′ -GAA TTC TTA TCA CGT AGA ATC GAG ACC GAG GAG AGG GTT AGG GAT AGG CTT ACC AGT TCC AAC CTT CGC CGA TGC-3′ (SEG ID NO: 2) that includes an EcoRI site GAA TTC and the 14 amino acid epitope for the V5 antibody. The oligonucleotides were used for PCR under standard conditions that included 25 cycles of PCR (95° C. for 1 minute, 55° C. for 1 minute, 72° C. for 1.5 minutes for 25 cycles followed by 3 minutes at 72° C.). The PCR fragment was purified by gel electrophoresis and cloned into pTA2.1 (Invitrogen) by standard cloning methods (Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, 2001), creating the plasmid pTA2.1-hPMS 134. pTA2.1-hPMS134 was digested with the restriction enzyme EcoRI to release the insert which was cloned into EcoRI restriction site of pPIC3.5K (Invitrogen). The following strategy, similar to that described above to clone human PMS 134, was used to construct an expression vector for the human related gene PMSR2. First, the hPMSR2 fragment was amplified by PCR to introduce two restriction sites, an NdeI restriction site at the 5′- end and an Eco RI site at the 3′-end of the fragment. The 5′-oligonucleotide that was used for PCR has the following structure: 5′-ACG CAT ATG TGT CCT TGG CGG CCT AGA-3′ (SEO ID NO: 3) that includes the NdeI restriction site CAT ATG. The 3′-oligonucleotide used for PCR has the following structure: 5′-GAA TTC TTA TTA CGT AGA ATC GAG ACC GAG GAG AGG GTT AGG GAT AGG CTT ACC CAT GTG TGA TGT TTC AGA GCT-3′ (SEO ID NO: 4) that includes an EcoRI site GAA TTC and the V5 epitope to allow for antibody detection. The plasmid that contained human PMSR3 in pBluescript SK (Nicolaides et al. Genomics 30(2):195-206,1995) was used as the PCR target with the hPMS2-specific oligonucleotides above. Following 25 cycles of PCR (95° C. for 1 minute, 55° C. for 1 minute, 72° C. for 1.5 minutes for 25 cycles followed by 3 minutes at 72° C.). The PCR fragment was purified by gel electrophoresis and cloned into pTA2.1 (Invitrogen) by standard cloning methods (Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition, 2001), creating the plasmid pTA2.1-hR2. pTA2.1-hR2 was next digested with the restriction enzyme EcoRI to release the insert (there are two EcoRI restriction sites in the multiple cloning site of pTA2.1 that flank the insert) and the inserted into the yeast expression vector pPIC3.5K (Invitrogen).
Pichia pastoris yeast cells were transformed with pPIC3.5K vector, pPIC3.5K-pms134, and pPIC3.5K-hR2 as follows. First, 5 ml of YPD (1% yeast extract, 2% bacto-peptone, 1% dextrose) medium was inoculated with a single colony from a YPD plate (same as YPD liquid but add 2% difco-agar to plate) and incubated with shaking overnight at 30° C. The overnight culture was used to inoculate 500 ml of YPD medium (200 ul of overnight culture) and the culture incubated at 30° C. until the optical density at 600 nm reached 1.3 to 1.5. The cells were then spun down (4000×g for 10 minutes), and then washed 2 times in sterile water (one volume each time), then the cells suspended in 20 ml of 1M sorbitol. The sorbitol/cell suspension was spun down (4,000×g for 10 minutes) and suspended in 1 ml of 1M sorbitol. 80 ul of the cell suspension was mixed with 5 to 10 ug of linearized plasmid DNA and placed in a 0.2 cm cuvette, pulsed length 5 to 10 milliseconds at field strength of 7,500V/cm. Next, the cells are diluted in 1 ml of 1M sorbitol and transferred to a 15 ml tube and incubated at 30° C. for 1 to 2 hours without shaking. Next, the cells are spun out (4,000×G for 10 minutes) and suspended in 100 ul of sterile water, and 50 ul/plate spread onto the appropriate selective medium plate. The plates are incubated for 2 to 3 days at 30° C. and colonies patched out onto YPD plates for further testing.
Example 2
Generation of Hypermutable Yeast with Inducible Dominant Negative Alleles of Mismatch Repair Genes
Yeast clones expressing human PMS2 homologue PMS-R2 or empty vector were grown in BMG (100 mM potassium phosphate, pH 6.0, 1.34% YNB (yeast nitrogen base), 4×10−5% biotin, 1% glycerol) liquid culture for 24 hr at 30° C. The next day, cultures were diluted 1:100 in MM medium (1.34% YNB, 4×10−5% biotin, 0.5% methanol) and incubated at 30° C. with shaking. Cells were removed for mutant selection at 24 and 48 hours post methanol induction as described below (see EXAMPLE 3).
Example 3
Dominant Negative MMR Genes can Produce New Genetic Variants and Commercially Viable Output Traits in Yeast
The ability to express MMR genes in yeast, as presented in example 2, demonstrate the ability to generate genetic alterations and new phenotypes in yeast expressing dominant negative MMR genes. In this example we teach the utility of this method to create eukaryotic strains with commercially relevant output traits.
Generation of Uracil Dependent Yeast Strain
One example of utility is the generation of a yeast strain that is mutant for a particular metabolic product, such as an amino acid or nucleotide. Engineering such a yeast strain will allow for recombinant manipulation of the yeast strain for the introduction of genes for scalable process of recombinant manufacturing. In order to demonstrate that MMR can be manipulated in yeast to generate mutants that lack the abilty to produce specific molecular building blocks, the following experiment was performed. Yeast cells that express a methanol inducible human PMS2 homologue, hPMS2-R2 (as described in example 1 above), were grown in BMY medium overnight then diluted 1:100 and transferred to MM medium, which results in activation of the AOX promoter and production of the hPMS2-R2 MMR gene that is resident within the yeast cell. Control cells were treated the same manner; these cells contain the pPIC3.5 vector in yeast and lack an insert. Cells were induced for 24 and 48 hours and then selected for uracil requiring mutations as follows. The cells were plated to 5-FOA medium (Boeke, J. D., LaCroute, F., and Fink, G. R. Mol. Gen. Genet. 197:345-345,1984). The plates are made as follows: (2× concentrate (filter sterilize): yeast nitrogen base 7 grams; 5-fluoro-orotic acid I gram; uracil 50 milligrams; glucose 20 grams; water to 500 ml; Add to 500 ml 4% agar (autoclaved) and pour plates. Cells are plated on 5-FOA plates (0, 24 and 48 hour time points) and incubated at 30° C. for between 3 and 5 days. Data from a typical experiment is shown in Table 1. No uracil requiring clones were observed in the un-induced or induced culture in yeast cells that harbor the “empty” vector whereas those cells that harbor the MMR gene hPMS2-R2 have clones that are capable of growth on the selection medium. Note that the un-induced culture of hPMS2-R2 does not have any colonies that are resistant to 5-FOA, demonstrating that the gene must be induced for the novel phenotype to be generated. It has been demonstrated that the mutagens (such as ethyl methyl sulfonate result in a low number of ura− mutants and that the spontaneous mutation rate for generating this class of mutants is low (Boeke, J. D., LaCroute, F. and Fink, G. R. Mol. Gen. Genet. 197:345-346,1984).







TABLE 1







Generation of uracil requiring mutant Pichia pastoris yeast cells. #


Represents at 24 hour methanol induction and @ a 48 hour induction. For


comparison a wild type yeast cell treated/un-treated is shown (Galli, A.


and Schiestl, R. H. Mutat. Res. 429(1): 13-26, 1999).

















Frequency







(ura−



Strain
Seeded
ura−
URA+
cells)

















Wt
100,000
0
~100,000
0



Empty
100,000
0
~100,000
0



pMORye-1#
100,000
14
~100,000
1/7,142



pMORye2@
100,000
123
~100,000
1/813



Wt
100,000
1-0.1
100,000
1/105-6*



Mutagen
100,000
10
100,000
1/10,000










Generations of Heat-Resistant Producer Strains

One example of commercial utility is the generation of heat-resistant recombinant protein producer strains. In the scalable process of recombinant manufacturing, large-scale fermentation of both prokaryotes and eukaryotes results in the generation of excessive heat within the culture. This heat must be dissipated by physical means such as using cooling jackets that surround the culture while it is actively growing and producing product. Production of a yeast strain that can resist high temperature growth effectively would be advantageous for large-scale recombinant manufacturing processes. To this end, the yeast strain as described in EXAMPLE 2 can be grown in the presence of methanol to induce the dominant negative MMR gene and the cells grown for various times (e.g. 12, 24, 36 and 48 hours) then put on plates and incubated at elevated temperatures to select for mutants that resist high temperature growth (e.g. 37° C. or 42° C.). These strains would be useful for fermentation development and scale-up of processes and should result in a decrease in manufacturing costs due to the need to cool the fermentation less often.
Generation of High Recombinant Protein Producer Strains and Strains with Less Endogenous Pretease Activity
Yeast is a valuable recombinant-manufacturing organism since it is a single celled organism that is inexpensive to grow and easily lends itself to fermentation at scale. Further more, many eukaryotic proteins that are incapable of folding effectively when expressed in Escherichia coli systems fold with the proper conformation in yeast and are structurally identical to their mammalian counterparts. There are several inherent limitations of many proteins that are expressed in yeast including over and/or inappropriate glycosylation of the recombinant protein, proteolysis by endogenous yeast enzymes and insufficient secretion of recombinant protein from the inside of the yeast cell to the medium (which facilitates purification). To generate yeast cells that with this ability to over-secrete proteins, or with less endogenous protease activity and or less hyper-glycosylation activity yeast cells as described in example 1 can be grown with methanol for 12, 24, 36 and 48 hours and yeast cells selected for the ability to over-secrete the protein or interest, under-glycosylate it or a cell with attenuated of no protease activity. Such a strain will be useful for recombinant manufacturing or other commercial purposes and can be combined with the heat resistant strain outlined above. For example, a mutant yeast cell that is resistant to high temperature growth and can secrete large amounts of protein into the medium would result.
Similar results were observed with other dominant negative mutants such as the PMSR2, PMSR3, and the human MLH1 proteins.
Example 4
Mutations Generated in the Host Genome of Yeast by Defective MMR are Genetically Stable
As described in example 3 manipulation of the MMR pathway in yeast results in alterations within the host genome and the ability to select for a novel output traits, for example the ability of a yeast cell to require a specific nutrient. It is important that the mutations introduced by the MMR pathway is genetically stable and passed to daughter cells reproducibly once the wild type MMR pathway is re-established. To determine the genetic stability of mutations introduced into the yeast genome the following experiment was performed. Five independent colonies from pPIC3.5K-hPMS2-R2 that are ura−, five wild type control cells (URA+) and five pPIC3.5K transformed cells (“empty vector”) were grown overnight from an isolated colony in 5 ml of YPD (1% yeast extract, 2% bacto-peptone and 1% dextrose) at 30° C. with shaking. The YPD medium contains all the nutrients necessary for yeast to grow, including uracil. Next, 1 μL of the overnight culture, which was at an optical density (OD) as measured at 600 nM of >3.0, was diluted to an OD600 of 0.01 in YPD and the culture incubated with shaking at 30° C. for an additional 24 hours. This process was repeated 3 more times for a total of 5 overnight incubations. This is the equivalent of greater than 100 generations of doublings (from the initial colony on the plate to the end of the last overnight incubation. Cells (five independent colonies that are ura− and five that were wild type were then plated onto YPD plates at a cell density of 300 to 1,000 cells/plate and incubated for two days at 30° C. The cells from these plates were replica plated to the following plates and scored for growth following three days incubation at 30° C.; Synthetic Complete (SC) SC−ura (1.34% yeast nitrogen base and ammonium sulfate; 4×10−5% biotin; supplemented with all amino acids, NO supplemental uracil; 2% dextrose and 2% agar); SC+URA (same as SC−ura but supplement plate with 50 mg uracil/liter medium), and YPD plates. They were replica plated in the following order—SC−ura, SC complete, YPD. If the novel output trait that is resident within the yeast genome that was generated by expression of the mutant MMR (in this example the human homologue of PMS2, hPMS2-R2) is unstable, the uracil dependent cells should “revert” back a uracil independent phenotype. If the phenotype is stable, growth of the mutant cells under non-selective conditions should result in yeast cells that maintain their viability dependence on exogenous supplementation with uracil. As can be seen in the data presented in Table 2, the uracil dependent phenotype is stable when the yeast cells are grown under non-selective conditions, demonstrating that the MMR-generated phenotype derived from mutation in one of the uracil biosynthetic pathway genes is stable genetically.

















Strain
Seeded
−ura
+URA
YPD






















Wt
650
650
650
650



Empty
560
560
560
560



pMORye-1#
730
0
730
730










These data demonstrate the utility of employing an inducible expression system and a dominant negative MMR gene in a eukaryotic system to generate genetically altered strains. The strain developed in this example, a yeast strain that now requires addition of uracil for growth, is potentially useful as a strain for recombinant manufacturing; by constructing an expression vector that harbors the wild type URA3 gene on either an integration plasmid or an extra-chromosomal vector it is now possible to transform and create novel cells expressing the a protein of interest. It is also possible to modify other resident genes in-yeast cells and select for mutations in genes that that give other useful phenotypes, such as the ability to carry out a novel bio-transformation. Furthermore, it is possible to express a gene extra-chromosomally in a yeast cell that has altered MMR activity as described above and select for mutations in the extra-chromosomal gene. Therefore, in a similar manner to that described above the mutant yeast cell can be put under specific selective pressure and a novel protein with commercially important biochemical attributes selected. These examples are meant only as illustrations and are not meant to limit the scope of the present invention. Finally, as described above once a mutation has been introduced into the gene of interest the MMR activity is attenuated of completely abolished. The result is a yeast cell that harbors a stable mutation in the target gene(s) of interest.
Example 5
Enhanced Generation of MMR-Defective Yeast and Chemical Mutagens for the Generation of New Output Traits
It has been previously documented that MMR deficiency yields to increased mutation frequency and increased resistance to toxic effects of chemical mutagens (CM) and their respective analogues such as but not limited to those as: ethidium bromide, EMS, MNNG, MNU, Tamoxifen, 8-Hydroxyguanine, as well as others listed but not limited to in publications by: Khromov-Borisov, N. N., et al. Mutat. Res. 430:55-74, 1999; Ohe, T., et al. (Mutat. Res. 429:189-199, 1999; Hour, T. C. et al. Food Chem. Toxicol. 37:569-579, 1999; Hrelia, P., et al. Chem. Biol. Interact. 118:99-111, 1999; Garganta, F., et al. Environ. Mol. Mutagen. 33:75-85, 1999; Ukawa-Ishikawa S., et al. Mutat. Res. 412:99-107, 1998; the website having the URL address: www host server, ehs.utah.edu domain name, ohh directory, mutagens subdirectory; Marcelino L A, Andre P C, Khrapko K, Coller H A, Griffith J, Thilly W G. Chemically induced mutations in mitochondrial DNA of human cells: mutational spectrum of N-methyl-N′-nitro-N-nitrosoguanidine. Cancer Res 1998 Jul. 1; 58(13):2857-62; Koi M, Umar A, Chauhan D P, Cherian S P, Carethers J M, Kunkel T A, Boland C R. Human chromosome 3 corrects mismatch repair deficiency and microsatellite instability and reduces N-methyl-N′-nitro-N-nitrosoguanidine tolerance in colon tumor cells with homozygous hMLH1 mutation. Can Res 1994 54:4308-4312,1994. Mismatch repair provokes chromosome aberrations in hamster cells treated with methylating agents or 6-thioguanine, but not with ethylating agents. To demonstrate the ability of CMs to increase the mutation frequency in MMR defective yeast cells, we would predict that exposure of yeast cells to CMs in the presence or absence of methanol (which induces the expression of the resident human homologue to PMS2, hPMS2-R2) will result in an augmentation of mutations within the yeast cell.
Yeast cells that express hPMS2-R2 (induced or un-induced) and empty vector control cells are grown as described in examples 2 and 3) and for 24 hours and diluted into MM medium as described above. Next, the cells in MM are incubated either with or without increasing amounts of ethyl methane sulfonate (EMS) from 0, 1, 10, 50, 100, and 200 μM. 10 μL aliquots of culture (diluted in 300 μl MM) and incubated for 30 minutes, 60 minutes, and 120 minutes followed by plating cells onto 5-FOA plates as described in example 3 above. Mutants are selected and scored as above. We would predict that there will be an increase in the frequency of ura+ mutants in the PMS2-R2 cultures that are induced with methanol as compared to the uninduced parental or wild type strain. In a further extension of this example, human PMS2-R2 harboring cells will be induced for 24 and 48 hours then mutagenized with EMS. This will allow the MMR gene to be fully active and expressed at high levels, thereby resulting in an increase in the number of ura− mutants obtained. We would predict that there will be no change in the number of ura− mutants obtained in the un-induced parental control or the wild type “empty vector” cells.
This example demonstrates the use of employing a regulated dominant negative MMR system plus chemical mutagens to produce enhanced numbers of genetically altered yeast strains that can be selected for new output traits. This method is useful for generating such organisms for commercial applications such as but not limited to recombinant manufacturing, biotransformation, and altered biochemicals with enhanced activities. It is also useful to obtain alterations of protein activity from ectopically expressed proteins harbored on extra-chromosomal expression vectors similar to those described in example 4 above.
Example 6
Alternative Methods to Inhibition of Yeast MMR Activity
The inhibition of MMR activity in a host organism can be achieved by introducing a dominant negative allele as shown in the examples above. This application also teaches us the use of using regulated systems to control MMR in yeast to generate genetic diversity and output traits for commercial applications. Additional methods to regulate the suppression of MMR activity of a host are by using genetic recombination to knock out alleles of a MMR gene within the cell of interest. This can be accomplished by use of homologous recombination that disrupts the endogenous MMR gene; 2) blocking MMR protein dimerization with other subunits (which is required for activity) by the introduction of polypeptides or antibodies into the host via transfection methods routinely used by those skilled in the art (e.g. electroporation); or 3) decreasing the expression of a MMR gene using anti-sense oligonucleotides.
MMR gene knockouts. We intend to generate disrupted targeting vectors of a particular MMR gene and introduce it into the genome of yeast using methods standard in the art. Yeast exhibiting hypermutability will be useful to produce genetically diverse offspring for commercial applications. Yeast will be confirmed to have lost the expression of the MMR gene using standard northern and biochemical techniques (as described in reference 31). MMR gene loci can be knocked out, strains selected for new output traits and MMR restored by introducing a wild type MMR gene to complement the KO locus. Other strategies include using KO vectors that can target a MMR gene locus, select for host output traits and then have the KO vector “spliced” from the genome after strain generation.

Blocking peptides. MMR subunits (MutS and MutL proteins) interact to form active MMR complexes. Peptides are able to specifically inhibit the binding of two proteins by competitive inhibition. Introduction into cells of peptides or antibodies to conserved domains of a particular MMR gene to disrupt activity is straightforward to those skilled in the art. Yeast will be verified for loss of expression of the MMR activity by standard northern and/or biochemical techniques (as described in Nicolaides N C, Littman S J, Modrich P, Kinzler K W, Vogelstein B 1998. A naturally occurring hPMS2 mutation can confer a dominant negative mutatorphenotype. Mol Cell Biol 18:1635-1641). Yeast exhibiting hypermutability will be useful to produce genetically diverse sibs for commercial applications.

Discussion

The results described above will lead to several conclusions. First, expression of dominant negative MMR proteins results in an increase in microsatellite instability and hypermutability in yeast. The hypermutability of the yeast cell is due to the inhibition of the resident, endogenous MMR biochemical activity in these hosts. This method provides a claim for use of MMR genes and their encoded products for the creation of hypermutable yeast to produce new output traits for commercial applications.
EXAMPLES OF MMR GENES AND ENCODED POLYPEPTIDES








Yeast MLH1 cDNA (accession number U07187) (SEQ ID NO: 5)










1
aaataggaat gtgatacctt ctattgcatg caaagatagt gtaggaggcg ctgctattgc



 


61
caaagacttt tgagaccgct tgctgtttca ttatagttga ggagttctcg aagacgagaa


 


121
attagcagtt ttcggtgttt agtaatcgcg ctagcatgct aggacaattt aactgcaaaa


 


181
ttttgatacg atagtgatag taaatggaag gtaaaaataa catagacgta tcaataagca


 


241
atgtctctca gaataaaagc acttgatgca tcagtggtta acaaaattgc tgcaggtgag


 


301
atcataatat cccccgtaaa tgctctcaaa gaaatgatgg agaattccat cgatgcgaat


 


361
gctacaatga ttgatattct agtcaaggaa ggaggaatta aggtacttca aataacagat


 


421
aacggatctg gaattaataa agcagacctg ccaatcttat gtgagcgatt cacgacgtcc


 


481
aaattacaaa aattcgaaga tttgagtcag attcaaacgt atggattccg aggagaagct


 


541
ttagccagta tctcacatgt ggcaagagtc acagtaacga caaaagttaa agaagacaga


 


601
tgtgcatgga gagtttcata tgcagaaggt aagatgttgg aaagccccaa acctgttgct


 


661
ggaaaagacg gtaccacgat cctagttgaa gacctttttt tcaatattcc ttctagatta


 


721
agggccttga ggtcccataa tgatgaatac tctaaaatat tagatgttgt cgggcgatac


 


781
gccattcatt ccaaggacat tggcttttct tgtaaaaagt tcggagactc taattattct


 


841
ttatcagtta aaccttcata tacagtccag gataggatta ggactgtgtt caataaatct


 


901
gtggcttcga atttaattac ttttcatatc agcaaagtag aagatttaaa cctggaaagc


 


961
gttgatggaa aggtgtgtaa tttgaatttc atatccaaaa agtccatttc attaattttt


 


1021
ttcattaata atagactagt gacatgtgat cttctaagaa gagctttgaa cagcgtttac


 


1081
tccaattatc tgccaaaggg cttcagacct tttatttatt tgggaattgt tatagatccg


 


1141
gcggctgttg atgttaacgt tcacccgaca aagagagagg ttcgtttcct gagccaagat


 


1201
gagatgatag agaaaatcgc caatcaattg cacgccgaat tatctgccat tgatacttca


 


1261
cgtactttca aggcttcttc aatttcaaca aacaagccag agtcattgat accatttaat


 


1321
gacaccatag aaagtgatag gaataggaag agtctccgac aagcccaagt ggtagagaat


 


1381
tcatatacga cagccaatag tcaactaagg aaagcgaaaa gacaagagaa taaactagtc


 


1441
agaatagatg cttcacaagc taaaattacg tcatttttat cctcaagtca acagttcaac


 


1501
tttgaaggat cgtctacaaa gcgacaactg agtgaaccca aggtaacaaa tgtaagccac


 


1561
tcccaagagg cagaaaagct gacactaaat gaaagcgaac aaccgcgtga tgccaataca


 


1621
atcaatgata atgacttgaa ggatcaacct aagaagaaac aaaagttggg ggattataaa


 


1681
gttccaagca ttgccgatga cgaaaagaat gcactcccga tttcaaaaga cgggtatatt


 


1741
agagtaccta aggagcgagt taatgttaat cttacgagta tcaagaaatt gcgtgaaaaa


 


1801
gtagatgatt cgatacatcg agaactaaca gacatttttg caaatttgaa ttacgttggg


 


1861
gttgtagatg aggaaagaag attagccgct attcagcatg acttaaagct ttttttaata


 


1921
gattacggat ctgtgtgcta tgagctattc tatcagattg gtttgacaga cttcgcaaac


 


1981
tttggtaaga taaacctaca gagtacaaat gtgtcagatg atatagtttt gtataatctc


 


2041
ctatcagaat ttgacgagtt aaatgacgat gcttccaaag aaaaaataat tagtaaaata


 


2101
tgggacatga gcagtatgct aaatgagtac tattccatag aattggtgaa tgatggtcta


 


2161
gataatgact taaagtctgt gaagctaaaa tctctaccac tacttttaaa aggctacatt


 


2221
ccatctctgg tcaagttacc attttttata tatcgcctgg gtaaagaagt tgattgggag


 


2281
gatgaacaag agtgtctaga tggtatttta agagagattg cattactcta tatacctgat


 


2341
atggttccga aagtcgatac actcgatgca tcgttgtcag aagacgaaaa agcccagttt


 


2401
ataaatagaa aggaacacat atcctcatta ctagaacacg ttctcttccc ttgtatcaaa


 


2461
cgaaggttcc tggcccctag acacattctc aaggatgtcg tggaaatagc caaccttcca


 


2521
gatctataca aagtttttga gaggtgttaa ctttaaaacg ttttggctgt aataccaaag


 


2581
tttttgttta tttcctgagt gtgattgtgt ttcatttgaa agtgtatgcc ctttccttta


 


2641
acgattcatc cgcgagattt caaaggatat gaaatatggt tgcagttagg aaagtatgtc


 


2701
agaaatgtat attcggattg aaactcttct aatagttctg aagtcacttg gttccgtatt


 


2761
gttttcgtcc tcttcctcaa gcaacgattc ttgtctaagc ttattcaacg gtaccaaaga


 


2821
cccgagtcct tttatgagag aaaacatttc atcatttttc aactcaatta tcttaatatc


 


2881
attttgtagt attttgaaaa caggatggta aaacgaatca cctgaatcta gaagctgtac


 


2941
cttgtcccat aaaagtttta atttactgag cctttcggtc aagtaaacta gtttatctag


 


3001
ttttgaaccg aatattgtgg gcagatttgc agtaagttca gttagatcta ctaaaagttg


 


3061
tttgacagca gccgattcca caaaaatttg gtaaaaggag atgaaagaga cctcgcgcgt


 


3121
aatggtttgc atcaccatcg gatgtctgtt gaaaaactca ctttttgcat ggaagttatt


 


3181
aacaataaga ctaatgatta ccttagaata atgtataa


 








Yeast MLH1 protein (accession number U07187) (SEQ ID NO: 15)



MSLRIKALDASVVNKIAAGEIIISPVNALKEMMENSIDANATMI


 


DILVKEGGIKVLQITDNGSGINKADLPILCERFTTSKLQKFEDLSQIQTYGFRGEALA


 


SISHVARVTVTTKVKEDRCAWRVSYAEGKMLESPKPVAGKDGTTILVEDLFFNIPSRL


 


RALRSHNDEYSKILDVVGRYAIHSKDIGFSCKKFGDSNYSLSVKPSYTVQDRIRTVFN


 


KSVASNLITFHISKVEDLNLESVDGKVCNLNFISKKSISLIFFINNRLVTCDLLRRAL


 


NSVYSNYLPKGFRPFIYLGIVIDPAAVDVNVHPTKREVRFLSQDEIIEKIANQLHAEL


 


SAIDTSRTFKASSISTNKPESLIPFNDTIESDRNRKSLRQAQVVENSYTTANSQLRKA


 


KRQENKLVRIDASQAKITSFLSSSQQFNFEGSSTKRQLSEPKVTNVSHSQEAEKLTLN


 


ESEQPRDANTINDNDLKDQPKKKQKLGDYKVPSIADDEKNALPISKDGYIRVPKERVN


 


VNLTSIKKLREKVDDSIHRELTDIFANLNYVGVVDEERRLAAIQHDLKLFLIDYGSVC


 


YELFYQIGLTDFANFGKINLQSTNVSDDIVLYNLLSEFDELNDDASKEKIISKIWDMS


 


SMLNEYYSIELVNDGLDNDLKSVKLKSLPLLLKGYIPSLVKLPFFIYRLGKEVDWEDE


 


QECLDGILREIALLYIPDMVPKVDTLDASLSEDEKAQFINRKEHISSLLEHVLFPCIK


 


RRFLAPRHILKDVVEIANLPDLYKVFERC


 


Mouse PMS2 protein (SEQ ID NO: 16)









MEQTEGVSTE CAKAIKPIDG KSVHQICSGQ VILSLSTAVK ELIENSVDAG ATTIDLRLKD
60



 


YGVDLIEVSD NGCGVEEENF EGLALKHHTS KIQEFADLTQ VETFGFRGEA LSSLCALSDV
120


 


TISTCHGSAS VGTRLVFDHN GKITQKTPYP RPKGTTVSVQ HLFYTLPVRY KEFQRNIKKE
180


 


YSKMVQVLQA YCIISAGVRV SCTNQLGQGK RHAVVCTSGT SGMKENIGSV FGQKQLQSLI
240


 


PFVQLPPSDA VCEEYGLSTS GRHKTFSTFR ASFHSARTAP GGVQQTGSFS SSIRGPVTQQ
300


 


RSLSLSMRFY HMYNRHQYPF VVLNVSVDSE CVDINVTPDK RQILLQEEKL LLAVLKTSLI
360


 


GMFDSDANKL NVNQQPLLDV EGNLVKLHTA ELEKPVPGKQ DNSPSLKSTA DEKRVASISR
420


 


LREAFSLHPT KEIKSRGPET AELTRSFPSE KRGVLSSYPS DVISYRGLRG SQDKLVSPTD
480


 


SPGDCMDREK IEKDSGLSST SAGSEEEFST PEVASSFSSD YNVSSLEDRP SQETINCGDL
540


 


DCRPPGTGQS LKPEDHGYQC KALPLARLSP TNAKRFKTEE RPSNVNISQR LPGPQSTSAA
600


 


EVDVAIKMNK RIVLLEFSLS SLAKRMKQLQ HLKAQNKHEL SYRKFRAKIC PGENQAAEDE
660


 


LRKEISKSMF AEMEILGQFN LGFIVTKLKE DLFLVDQHAA DEKYNFEMLQ QHTVLQAQRL
720


 


ITPQTLNLTA VNEAVLIENL EIFRKNGFDF VIDEDAPVTE RAKLISLPTS KNWTFGPQDI
780


 


DELIFMLSDS PGVMCRPSRV RQMFASRACR KSVMIGTALN ASEMKKLITH MGEMDHPWNC
840


 


PHGRPTMRHV ANLDVISQN
859


 


Mouse PMS2 cDNA (SEQ ID NO: 6)


gaattccggt gaaggtcctg aagaatttcc agattcctga gtatcattgg aggagacaga
60


 


taacctgtcg tcaggtaacg atggtgtata tgcaacagaa atgggtgttc ctggagacgc
120


 


gtcttttccc gagagcggca ccgcaactct cccgcggtga ctgtgactgg aggagtcctg
180


 


catccatgga gcaaaccgaa ggcgtgagta cagaatgtgc taaggccatc aagcctattg
240


 


atgggaagtc agtccatcaa atttgttctg ggcaggtgat actcagttta agcaccgctg
300


 


tgaaggagtt gatagaaaat agtgtagatg ctggtgctac tactattgat ctaaggctta
360


 


aagactatgg ggtggacctc attgaagttt cagacaatgg atgtggggta gaagaagaaa
420


 


actttgaagg tctagctctg aaacatcaca catctaagat tcaagagttt gccgacctca
480


 


cgcaggttga aactttcggc tttcgggggg aagctctgag ctctctgtgt gcactaagtg
540


 


atgtcactat atctacctgc cacgggtctg caagcgttgg gactcgactg gtgtttgacc
600


 


ataatgggaa aatcacccag aaaactccct acccccgacc taaaggaacc acagtcagtg
660


 


tgcagcactt attttataca ctacccgtgc gttacaaaga gtttcagagg aacattaaaa
720


 


aggagtattc caaaatggtg caggtcttac aggcgtactg tatcatctca gcaggcgtcc
780


 


gtgtaagctg cactaatcag ctcggacagg ggaagcggca cgctgtggtg tgcacaagcg
840


 


gcacgtctgg catgaaggaa aatatcgggt ctgtgtttgg ccagaagcag ttgcaaagcc
900


 


tcattccttt tgttcagctg ccccctagtg acgctgtgtg tgaagagtac ggcctgagca
960


 


cttcaggacg ccacaaaacc ttttctacgt ttcgggcttc atttcacagt gcacgcacgg
1020


 


cgccgggagg agtgcaacag acaggcagtt tttcttcatc aatcagaggc cctgtgaccc
1080


 


agcaaaggtc tctaagcttg tcaatgaggt tttatcacat gtataaccgg catcagtacc
1140


 


catttgtcgt ccttaacgtt tccgttgact cagaatgtgt ggatattaat gtaactccag
1200


 


ataaaaggca aattctacta caagaagaga agctattgct ggccgtttta aagacctcct
1260


 


tgataggaat gtttgacagt gatgcaaaca agcttaatgt caaccagcag ccactgctag
1320


 


atgttgaagg taacttagta aagctgcata ctgcagaact agaaaagcct gtgccaggaa
1380


 


agcaagataa ctctccttca ctgaagagca caggagacga gaaaagggta gcatccatct
1440


 


ccaggctgag agaggccttt tctcttcatc ctactaaaga gatcaagtct aggggtccag
1500


 


agactgctga actgacacgg agttttccaa gtgagaaaag gggcgtgtta tcctcttatc
1560


 


cttcagacgt catctcttac agaggcctcc gtggctcgca ggacaaattg gtgagtccca
1620


 


cggacagccc tggtgactgt atggacagag agaaaataga aaaagactca gggctcagca
1680


 


gcacctcagc tggctctgag gaagagttca gcaccccaga agtggccagt agctttagca
1740


 


gtgactataa cgtgagctcc ctagaagaca gaccttctca ggaaaccata aactgtggtg
1800


 


acctggactg ccgtcctcca ggtacaggac agtccttgaa gccagaagac catggatatc
1860


 


aatgcaaagc tctacctcta gctcgtctgt cacccacaaa tgccaagcgc ttcaagacag
1920


 


aggaaagacc ctcaaatgtc aacatttctc aaagattgcc tggtcctcag agcacctcag
1980


 


cagctgaggt cgatgtagcc ataaaaatga ataagagaat cgtgctcctc gagttctctc
2040


 


tgagttctct agctaagcga atgaagcagt tacagcacct aaaggcgcag aacaaacatg
2100


 


aactgagtta cagaaaattt agggccaaga tttgccctgg agaaaaccaa gcagcagaag
2160


 


atgaactcag aaaagagatt agtaaatcga tgtttgcaga gatggagatc ttgggtcagt
2220


 


ttaacctggg atttatagta accaaactga aagaggacct cttcctggtg gaccagcatg
2280


 


ctgcggatga gaagtacaac tttgagatgc tgcagcagca cacggtgctc caggcgcaga
2340


 


ggctcatcac accccagact ctgaacttaa ctgctgtcaa tgaagctgta ctgatagaaa
2400


 


atctggaaat attcagaaag aatggctttg actttgtcat tgatgaggat gctccagtca
2460


 


ctgaaagggc taaattgatt tccttaccaa ctagtaaaaa ctggaccttt ggaccccaag
2520


 


atatagatga actgatcttt atgttaagtg acagccctgg ggtcatgtgc cggccctcac
2580


 


gagtcagaca gatgtttgct tccagagcct gtcggaagtc agtgatgatt ggaacggcgc
2640


 


tcaatgcgag cgagatgaag aagctcatca cccacatggg tgagatggac cacccctgga
2700


 


actgccccca cggcaggcca accatgaggc acgttgccaa tctggatgtc atctctcaga
2760


 


actgacacac cccttgtagc atagagttta ttacagattg ttcggtttgc aaagagaagg
2820


 


ttttaagtaa tctgattatc gttgtacaaa aattagcatg ctgctttaat gtactggatc
2880


 


catttaaaag cagtgttaag gcaggcatga tggagtgttc ctctagctca gctacttggg
2940


 


tgatccggtg ggagctcatg tgagcccagg actttgagac cactccgagc cacattcatg
3000


 


agactcaatt caaggacaaa aaaaaaaaga tatttttgaa gccttttaaa aaaaaa
3056


 


human PMS2 protein (SEQ ID NO: 7)


MKQLPAATVR LLSSSQIITS VVSVVKELIE NSLDAGATSV DVKLENYGFD KIEVRDNGEG
60


 


IKAVDAPVMA MKYYTSKINS HEDLENLTTY GFRGEALGSI CCIAEVLITT RTAADNFSTQ
120


 


YVLDGSGHIL SQKPSHLGQG TTVTALRLFK NLPVRKQFYS TAKKCKDEIK KIQDLLMSFG
180


 


ILKPDLRIVF VHNKAVIWQK SRVSDHKMAL MSVLGTAVMN NMESFQYHSE ESQIYLSGFL
240


 


PKCDADHSFT SLSTPERSFI FINSRPVHQK DILKLIRHHY NLKCLKESTR LYPVFFLKID
300


 


VPTADVDVNL TPDKSQVLLQ NKESVLIALE NLMTTCYGPL PSTNSYENNK TDVSAADIVL
360


 


SKTAETDVLF NKVESSGKNY SNVDTSVIPF QNDMHNDESG KNTDDCLNHQ ISIGDFGYGH
420


 


CSSEISNIDK NTKNAFQDIS MSNVSWENSQ TEYSKTCFIS SVKHTQSENG NKDHIDESGE
480


 


NEEEAGLENS SEISADEWSR GNILKNSVGE NIEPVKILVP EKSLPCKVSN NNYPIPEQMN
540


 


LNEDSCNKKS NVIDNKSGKV TAYDLLSNRV IKKPMSASAL FVQDHRPQFL IENPKTSLED
600


 


ATLQIEELWK TLSEEEKLKY EEKATKDLER YNSQMKRAIE QESQMSLKDG RKKIKPTSAW
660


 


NLAQKHKLKT SLSNQPKLDE LLQSQIEKRR SQNIKMVQIP FSMKNLKINF KKQNKVDLEE
720


 


KDEPCLIHNL RFPDAWLMTS KTEVMLLNPY RVEEALLFKR LLENHKLPAE PLEKPIMLTE
780


 


SLFNGSHYLD VLYKMTADDQ RYSGSTYLSD PRLTANGFKI KLIPGVSITE NYLEIEGMAN
840


 


CLPFYGVADL KEILNAILNR NAKEVYECRP RKVISYLEGE AVRLSRQLPM YLSKEDIQDI
900


 


IYRNKHQFGN EIKECVHGRP FFHHLTYLPE TT
932


 


Human PMS2 cDNA (SEQ ID NO: 17)


cgaggcggat cgggtgttgc atccatggag cgagctgaga gctcgagtac agaacctgct
60


 


aaggccatca aacctattga tcggaagtca gtccatcaga tttgctctgg gcaggtggta
120


 


ctgagtctaa gcactgcggt aaaggagtta gtagaaaaca gtctggatgc tggtgccact
180


 


aatattgatc taaagcttaa ggactatgga gtggatctta ttgaagtttc agacaatgga
240


 


tgtggggtag aagaagaaaa cttcgaaggc ttaactctga aacatcacac atctaagatt
300


 


caagagtttg ccgacctaac tcaggttgaa acttttggct ttcgggggga agctctgagc
360


 


tcactttgtg cactgagcga tgtcaccatt tctacctgcc acgcatcggc gaaggttgga
420


 


actcgactga tgtttgatca caatgggaaa attatccaga aaacccccta cccccgcccc
480


 


agagggacca cagtcagcgt gcagcagtta ttttccacac tacctgtgcg ccataaggaa
540


 


tttcaaagga atattaagaa ggagtatgcc aaaatggtcc aggtcttaca tgcatactgt
600


 


atcatttcag caggcatccg tgtaagttgc accaatcagc ttggacaagg aaaacgacag
660


 


cctgtggtat gcacaggtgg aagccccagc ataaaggaaa atatcggctc tgtgtttggg
720


 


cagaagcagt tgcaaagcct cattcctttt gttcagctgc cccctagtga ctccgtgtgt
780


 


gaagagtacg gtttgagctg ttcggatgct ctgcataatc ttttttacat ctcaggtttc
840


 


atttcacaat gcacgcatgg agttggaagg agttcaacag acagacagtt tttctttatc
900


 


aaccggcggc cttgtgaccc agcaaaggtc tgcagactcg tgaatgaggt ctaccacatg
960


 


tataatcgac accagtatcc atttgttgtt cttaacattt ctgttgattc agaatgcgtt
1020


 


gatatcaatg ttactccaga taaaaggcaa attttgctac aagaggaaaa gcttttgttg
1080


 


gcagttttaa agacctcttt gataggaatg tttgatagtg atgtcaacaa gctaaatgtc
1140


 


agtcagcagc cactgctgga tgttgaaggt aacttaataa aaatgcatgc agcggatttg
1200


 


gaaaagccca tggtagaaaa gcaggatcaa tccccttcat taaggactgg agaagaaaaa
1260


 


aaagacgtgt ccatttccag actgcgagag gccttttctc ttcgtcacac aacagagaac
1320


 


aagcctcaca gcccaaagac tccagaacca agaaggagcc ctctaggaca gaaaaggggt
1380


 


atgctgtctt ctagcacttc aggtgccatc tctgacaaag gcgtcctgag acctcagaaa
1440


 


gaggcagtga gttccagtca cggacccagt gaccgtacgg acagagcgga ggtggagaag
1500


 


gactcggggc acggcagcac ttccgtggat tctgaggggt tcagcatccc agacacgggc
1560


 


agtcactgca gcagcgagta tgcggccagc tccccagggg acaggggctc gcaggaacat
1620


 


gtggactctc aggagaaagc gcctgaaact gacgactctt tttcagatgt ggactgccat
1680


 


tcaaaccagg aagataccgg atgtaaattt cgagttttgc ctcagccaac taatctcgca
1740


 


accccaaaca caaagcgttt taaaaaagaa gaaattcttt ccagttctga catttgtcaa
1800


 


aagttagtaa atactcagga catgtcagcc tctcaggttg atgtagctgt gaaaattaat
1860


 


aagaaagttg tgcccctgga cttttctatg agttctttag ctaaacgaat aaagcagtta
1920


 


catcatgaag cacagcaaag tgaaggggaa cagaattaca ggaagtttag ggcaaagatt
1980


 


tgtcctggag aaaatcaagc agccgaagat gaactaagaa aagagataag taaaacgatg
2040


 


tttgcagaaa tggaaatcat tggtcagttt aacctgggat ttataataac caaactgaat
2100


 


gaggatatct tcatagtgga ccagcatgcc acggacgaga agtataactt cgagatgctg
2160


 


cagcagcaca ccgtgctcca ggggcagagg ctcatagcac ctcagactct caacttaact
2220


 


gctgttaatg aagctgttct gatagaaaat ctggaaatat ttagaaagaa tggctttgat
2280


 


tttgttatcg atgaaaatgc tccagtcact gaaagggcta aactgatttc cttgccaact
2340


 


agtaaaaact ggaccttcgg accccaggac gtcgatgaac tgatcttcat gctgagcgac
2400


 


agccctgggg tcatgtgccg gccttcccga gtcaagcaga tgtttgcctc cagagcctgc
2460


 


cggaagtcgg tgatgattgg gactgctctt aacacaagcg agatgaagaa actgatcacc
2520


 


cacatggggg agatggacca cccctggaac tgtccccatg gaaggccaac catgagacac
2580


 


atcgccaacc tgggtgtcat ttctcagaac tgaccgtagt cactgtatgg aataattggt
2640


 


tttatcgcag atttttatgt tttgaaagac agagtcttca ctaacctttt ttgttttaaa
2700


 


atgaaacctg ctacttaaaa aaaatacaca tcacacccat ttaaaagtga tcttgagaac
2760


 


cttttcaaac c
2771


 


Human PMS1 protein (SEQ ID NO: 18)


MKQLPAATVR LLSSSQIITS VVSVVKELIE NSLDAGATSV DVKLENYGFD KIEVRDNGEG
60


 


IKAVDAPVMA MKYYTSKINS HEDLENLTTY GFRGEALGSI CCIAEVLITT RTAADNFSTQ
120


 


YVLDGSGHIL SQKPSHLGQG TTVTALRLFK NLPVRKQFYS TAKKCKDEIK KIQDLLMSFG
180


 


ILKPDLRIVF VHNKAVIWQK SRVSDHKMAL MSVLGTAVMN NMESFQYHSE ESQIYLSGFL
240


 


PKCDADHSFT SLSTPERSFI FINSRPVHQK DILKLIRHHY NLKCLKESTR LYPVFFLKID
300


 


VPTADVDVNL TPDKSQVLLQ NKESVLIALE NLMTTCYGPL PSTNSYENNK TDVSAADIVL
360


 


SKTAETDVLF NKVESSGKNY SNVDTSVIPF QNDMHNDESG KNTDDCLNHQ ISIGDFGYGH
420


 


CSSEISNIDK NTKNAFQDIS MSNVSWENSQ TEYSKTCFIS SVKHTQSENG NKDHIDESGE
480


 


NEEEAGLENS SEISADEWSR GNILKNSVGE NIEPVKILVP EKSLPCKVSN NNYPIPEQMN
540


 


LNEDSCNKKS NVIDNKSGKV TAYDLLSNRV IKKPMSASAL FVQDHRPQFL IENPKTSLED
600


 


ATLQIEELWK TLSEEEKLKY EEKATKDLER YNSQMKRAIE QESQMSLKDG RKKIKPTSAW
660


 


NLAQKHKLKT SLSNQPKLDE LLQSQIEKRR SQNIKNVQIP FSMKNLKINF KKQNKVDLEE
720


 


KDEPCLIHNL RFPDAWLMTS KTEVMLLNPY RVEEALLFKR LLENHKLPAE PLEKPIMLTE
780


 


SLFNGSHYLD VLYKMTADDQ RYSGSTYLSD PRLTANGFKI KLIPGVSITE NYLEIEGMAN
840


 


CLPFYGVADL KEILNAILNR NAKEVYECRP RKVISYLEGE AVRLSRQLPM YLSKEDIQDI
900


 


IYRMKHQFGN EIKECVHGRP FFHHLTYLPE TT
932


 


Human PMS1 cDNA (SEQ ID NO: 8)


ggcacgagtg gctgcttgcg gctagtggat ggtaattgcc tgcctcgcgc tagcagcaag
60


 


ctgctctgtt aaaagcgaaa atgaaacaat tgcctgcggc aacagttcga ctcctttcaa
120


 


gttctcagat catcacttcg gtggtcagtg ttgtaaaaga gcttattgaa aactccttgg
180


 


atgctggtgc cacaagcgta gatgttaaac tggagaacta tggatttgat aaaattgagg
240


 


tgcgagataa cggggagggt atcaaggctg ttgatgcacc tgtaatggca atgaagtact
300


 


acacctcaaa aataaatagt catgaagatc ttgaaaattt gacaacttac ggttttcgtg
360


 


gagaagcctt ggggtcaatt tgttgtatag ctgaggtttt aattacaaca agaacggctg
420


 


ctgataattt tagcacccag tatgttttag atggcagtgg ccacatactt tctcagaaac
480


 


cttcacatct tggtcaaggt acaactgtaa ctgctttaag attatttaag aatctacctg
540


 


taagaaagca gttttactca actgcaaaaa aatgtaaaga tgaaataaaa aagatccaag
600


 


atctcctcat gagctttggt atccttaaac ctgacttaag gattgtcttt gtacataaca
660


 


aggcagttat ttggcagaaa agcagagtat cagatcacaa gatggctctc atgtcagttc
720


 


tggggactgc tgttatgaac aatatggaat cctttcagta ccactctgaa gaatctcaga
780


 


tttatctcag tggatttctt ccaaagtgtg atgcagacca ctctttcact agtctttcaa
840


 


caccagaaag aagtttcatc ttcataaaca gtcgaccagt acatcaaaaa gatatcttaa
900


 


agttaatccg acatcattac aatctgaaat gcctaaagga atctactcgt ttgtatcctg
960


 


ttttctttct gaaaatcgat gttcctacag ctgatgttga tgtaaattta acaccagata
1020


 


aaagccaagt attattacaa aataaggaat ctgttttaat tgctcttgaa aatctgatga
1080


 


cgacttgtta tggaccatta cctagtacaa attcttatga aaataataaa acagatgttt
1140


 


ccgcagctga catcgttctt agtaaaacag cagaaacaga tgtgcttttt aataaagtgg
1200


 


aatcatctgg aaagaattat tcaaatgttg atacttcagt cattccattc caaaatgata
1260


 


tgcataatga tgaatctgga aaaaacactg atgattgttt aaatcaccag ataagtattg
1320


 


gtgactttgg ttatggtcat tgtagtagtg aaatttctaa cattgataaa aacactaaga
1380


 


atgcatttca ggacatttca atgagtaatg tatcatggga gaactctcag acggaatata
1440


 


gtaaaacttg ttttataagt tccgttaagc acacccagtc agaaaatggc aataaagacc
1500


 


atatagatga gagtggggaa aatgaggaag aagcaggtct tgaaaactct tcggaaattt
1560


 


ctgcagatga gtggagcagg ggaaatatac ttaaaaattc agtgggagag aatattgaac
1620


 


ctgtgaaaat tttagtgcct gaaaaaagtt taccatgtaa agtaagtaat aataattatc
1680


 


caatccctga acaaatgaat cttaatgaag attcatgtaa caaaaaatca aatgtaatag
1740


 


ataataaatc tggaaaagtt acagcttatg atttacttag caatcgagta atcaagaaac
1800


 


ccatgtcagc aagtgctctt tttgttcaag atcatcgtcc tcagtttctc atagaaaatc
1860


 


ctaagactag tttagaggat gcaacactac aaattgaaga actgtggaag acattgagtg
1920


 


aagaggaaaa actgaaatat gaagagaagg ctactaaaga cttggaacga tacaatagtc
1980


 


aaatgaagag agccattgaa caggagtcac aaatgtcact aaaagatggc agaaaaaaga
2040


 


taaaacccac cagcgcatgg aatttggccc agaagcacaa gttaaaaacc tcattatcta
2100


 


atcaaccaaa acttgatgaa ctccttcagt cccaaattga aaaaagaagg agtcaaaata
2160


 


ttaaaatggt acagatcccc ttttctatga aaaacttaaa aataaatttt aagaaacaaa
2220


 


acaaagttga cttagaagag aaggatgaac cttgcttgat ccacaatctc aggtttcctg
2280


 


atgcatggct aatgacatcc aaaacagagg taatgttatt aaatccatat agagtagaag
2340


 


aagccctgct atttaaaaga cttcttgaga atcataaact tcctgcagag ccactggaaa
2400


 


agccaattat gttaacagag agtcttttta atggatctca ttatttagac gttttatata
2460


 


aaatgacagc agatgaccaa agatacagtg gatcaactta cctgtctgat cctcgtctta
2520


 


cagcgaatgg tttcaagata aaattgatac caggagtttc aattactgaa aattacttgg
2580


 


aaatagaagg aatggctaat tgtctcccat tctatggagt agcagattta aaagaaattc
2640


 


ttaatgctat attaaacaga aatgcaaagg aagtttatga atgtagacct cgcaaagtga
2700


 


taagttattt agagggagaa gcagtgcgtc tatccagaca attacccatg tacttatcaa
2760


 


aagaggacat ccaagacatt atctacagaa tgaagcacca gtttggaaat gaaattaaag
2820


 


agtgtgttca tggtcgccca ttttttcatc atttaaccta tcttccagaa actacatgat
2880


 


taaatatgtt taagaagatt agttaccatt gaaattggtt ctgtcataaa acagcatgag
2940


 


tctggtttta aattatcttt gtattatgtg tcacatggtt attttttaaa tgaggattca
3000


 


ctgacttgtt tttatattga aaaaagttcc acgtattgta gaaaacgtaa ataaactaat
3060


 


aac
3063


 


Human MSH2 protein (SEQ ID NO: 19)


MAVQPKETLQ LESAAEVGFV RFFQGMPEKP TTTVRLFDRG DFYTAHGEDA LLAAREVFKT
60


 


QGVIKYMGPA GAKNLQSVVL SKMNFESFVK DLLLVRQYRV EVYKNRAGNK ASKENDWYLA
120


 


YKASPGNLSQ FEDILFGNND MSASIGVVGV KMSAVDGQRQ VGVGYVDSIQ RKLGLCEFPD
180


 


NDQFSNLEAL LIQIGPKECV LPGGETAGDM GKLRQIIQRG GILITERKKA DFSTKDIYQD
240


 


LNRLLKGKKG EQMNSAVLPE MENQVAVSSL SAVIKFLELL SDDSNFGQFE LTTFDFSQYM
300


 


KLDIAAVRAL NLFQGSVEDT TGSQSLAALL NKCKTPQGQR LVNQWIKQPL MDKNRIEERL
360


 


NLVEAFVEDA ELRQTLQEDL LRRFPDLNRL AKKFQRQAAN LQDCYRLYQG INQLPNVIQA
420


 


LEKHEGKHQK LLLAVFVTPL TDLRSDFSKF QEMIETTLDM DQVENHEFLV KPSFDPNLSE
480


 


LREIMNDLEK KMQSTLISAA RDLGLDPGKQ IKLDSSAQFG YYFRVTCKEE KVLRNNKNFS
540


 


TVDIQKNGVK FTNSKLTSLN EEYTKNKTEY EEAQDAIVKE IVNISSGYVE PMQTLNDVLA
600


 


QLDAVVSFAH VSNGAPVPYV RPAILEKGQG RIILKASRHA CVEVQDEIAF IPNDVYFEKD
660


 


KQMFHIITGP NMGGKSTYIR QTGVIVLMAQ IGCFVPCESA EVSIVDCILA RVGAGDSQLK
720


 


GVSTFMAEML ETASILRSAT KDSLIIIDEL GRGTSTYDGF GLAWAISEYI ATKIGAFCMF
780


 


ATHFHELTAL ANQIPTVNNL HVTALTTEET LTMLYQVKKG VCDQSFGIHV AELANFPKHV
840


 


IEGAKQKALE LEEFQYIGES QGYDIMEPAA KKCYLEREQG EKIIQEFLSK VKQMPFTEMS
900


 


EENITIKLKQ LKAEVIAKNN SFVNEIISRI KVTT
934


 


Human MSH2 cDNA (SEQ ID NO: 9)


ggcgggaaac agcttagtgg gtgtggggtc gcgcattttc ttcaaccagg aggtgaggag
60


 


gtttcgacat ggcggtgcag ccgaaggaga cgctgcagtt ggagagcgcg gccgaggtcg
120


 


gcttcgtgcg cttctttcag ggcatgccgg agaagccgac caccacagtg cgccttttcg
180


 


accggggcga cttctatacg gcgcacggcg aggacgcgct gctggccgcc cgggaggtgt
240


 


tcaagaccca gggggtgatc aagtacatgg ggccggcagg agcaaagaat ctgcagagtg
300


 


ttgtgcttag taaaatgaat tttgaatctt ttgtaaaaga tcttcttctg gttcgtcagt
360


 


atagagttga agtttataag aatagagctg gaaataaggc atccaaggag aatgattggt
420


 


atttggcata taaggcttct cctggcaatc tctctcagtt tgaagacatt ctctttggta
480


 


acaatgatat gtcagcttcc attggtgttg tgggtgttaa aatgtccgca gttgatggcc
540


 


agagacaggt tggagttggg tatgtggatt ccatacagag gaaactagga ctgtgtgaat
600


 


tccctgataa tgatcagttc tccaatcttg aggctctcct catccagatt ggaccaaagg
660


 


aatgtgtttt acccggagga gagactgctg gagacatggg gaaactgaga cagataattc
720


 


aaagaggagg aattctgatc acagaaagaa aaaaagctga cttttccaca aaagacattt
780


 


atcaggacct caaccggttg ttgaaaggca aaaagggaga gcagatgaat agtgctgtat
840


 


tgccagaaat ggagaatcag gttgcagttt catcactgtc tgcggtaatc aagtttttag
900


 


aactcttatc agatgattcc aactttggac agtttgaact gactactttt gacttcagcc
960


 


agtatatgaa attggatatt gcagcagtca gagcccttaa cctttttcag ggttctgttg
1020


 


aagataccac tggctctcag tctctggctg ccttgctgaa taagtgtaaa acccctcaag
1080


 


gacaaagact tgttaaccag tggattaagc agcctctcat ggataagaac agaatagagg
1140


 


agagattgaa tttagtggaa gcttttgtag aagatgcaga attgaggcag actttacaag
1200


 


aagatttact tcgtcgattc ccagatctta accgacttgc caagaagttt caaagacaag
1260


 


cagcaaactt acaagattgt taccgactct atcagggtat aaatcaacta cctaatgtta
1320


 


tacaggctct ggaaaaacat gaaggaaaac accagaaatt attgttggca gtttttgtga
1380


 


ctcctcttac tgatcttcgt tctgacttct ccaagtttca ggaaatgata gaaacaactt
1440


 


tagatatgga tcaggtggaa aaccatgaat tccttgtaaa accttcattt gatcctaatc
1500


 


tcagtgaatt aagagaaata atgaatgact tggaaaagaa gatgcagtca acattaataa
1560


 


gtgcagccag agatcttggc ttggaccctg gcaaacagat taaactggat tccagtgcac
1620


 


agtttggata ttactttcgt gtaacctgta aggaagaaaa agtccttcgt aacaataaaa
1680


 


actttagtac tgtagatatc cagaagaatg gtgttaaatt taccaacagc aaattgactt
1740


 


ctttaaatga agagtatacc aaaaataaaa cagaatatga agaagcccag gatgccattg
1800


 


ttaaagaaat tgtcaatatt tcttcaggct atgtagaacc aatgcagaca ctcaatgatg
1860


 


tgttagctca gctagatgct gttgtcagct ttgctcacgt gtcaaatgga gcacctgttc
1920


 


catatgtacg accagccatt ttggagaaag gacaaggaag aattatatta aaagcatcca
1980


 


ggcatgcttg tgttgaagtt caagatgaaa ttgcatttat tcctaatgac gtatactttg
2040


 


aaaaagataa acagatgttc cacatcatta ctggccccaa tatgggaggt aaatcaacat
2100


 


atattggaca aactggggtg atagtactca tggcccaaat tgggtgtttt gtgccatgtg
2160


 


agtcagcaga agtgtccatt gtggactgca tcttagcccg agtaggggct ggtgacagtc
2220


 


aattgaaagg agtctccacg ttcatggctg aaatgttgga aactgcttct atcctcaggt
2280


 


ctgcaaccaa agattcatta ataatcatag atgaattggg aagaggaact tctacctacg
2340


 


atggatttgg gttagcatgg gctatatcag aatacattgc aacaaagatt ggtgcttttt
2400


 


gcatgtttgc aacccatttt catgaactta ctgccttggc caatcagata ccaactgtta
2460


 


ataatctaca tgtcacagca ctcaccactg aagagacctt aactatgctt tatcaggtga
2520


 


agaaaggtgt ctgtgatcaa agttttggga ttcatgttgc agagcttgct aatttcccta
2580


 


agcatgtaat agagtgtgct aaacagaaag ccctggaact tgaggagttt cagtatattg
2640


 


gagaatcgca aggatatgat atcatggaac cagcagcaaa gaagtgctat ctggaaagag
2700


 


agcaaggtga aaaaattatt caggagttcc tgtccaaggt gaaacaaatg ccctttactg
2760


 


aaatgtcaga agaaaacatc acaataaagt taaaacagct aaaagctgaa gtaatagcaa
2820


 


agaataatag ctttgtaaat gaaatcattt cacgaataaa agttactacg tgaaaaatcc
2880


 


cagtaatgga atgaaggtaa tattgataag ctattgtctg taatagtttt atattgtttt
2940


 


atattaaccc tttttccata gtgttaactg tcagtgccca tgggctatca acttaataag
3000


 


atatttagta atattttact ttgaggacat tttcaaagat ttttattttg aaaaatgaga
3060


 


gctgtaactg aggactgttt gcaattgaca taggcaataa taagtgatgt gctgaatttt
3120


 


ataaataaaa tcatgtagtt tgtgg
3145


 


Human MLH1 protein (SEQ ID NO: 20)


MSFVAGVIRR LDETVVNRIA AGEVIQRPAN AIKEMIENCL DAKSTSIQVI VKEGGLKLIQ
60


 


IQDNGTGIRK EDLDIVCERF TTSKLQSFED LASISTYGFR GEALASISHV AHVTITTKTA
120


 


DGKCAYRASY SDGKLKAPPK PGAGNQGTQI TVEDLFYNIA TRRKALKNPS EEYGKILEVV
180


 


GRYSVHNAGI SFSVKKQGET VADVRTLPNA STVDNIRSIF GNAVSRELIE IGCEDKTLAF
240


 


KMNGYISNAN YSVKKCIFLL FINHRLVEST SLRKAIETVY AAYLPKNTHP FLYLSLEISP
300


 


QNVDVNVHPT KHEVHFLHEE SILERVQQHI ESKLLGSNSS RNYFTQTLLP GLAGPSGEMV
360


 


KSTTSLTSSS TSGSSDKVYA HQMVRTDSRE QKLDAFLQPL SKPLSSQPQA IVTEDKTDIS
420


 


SGRARQQDEE MLELPAPAEV AAKNQSLEGD TTKGTSEMSE KRGPTSSNPR KRHREDSDVE
480


 


MVEDDSRKEM TAACTPRRRI INLTSVLSLQ EEINEQGHEV LREMLHNHSF VGCVNPQWAL
540


 


AQHQTKLYLL NTTKLSEELF YQILIYDFAN FGVLRLSEPA PLEDLAMLAL DSPESGWTEE
600


 


DGPKEGLAEY IVEFLKKKAE MLADYFSLEI DEEGNLIGLP LLIDNYVPPL EGLPIFILRL
660


 


ATEVNWDEEK ECFESLSKEC AMFYSIRKQY ISEESTLSGQ QSEVPGSIPN SWKWTVEHIV
720


 


YKALRSHILP PKHFTEDGNI LQLANLPDLY KVFERC
756


 


Human MLH1 cDNA (SEQ ID NO: 10)


cttggctctt ctggcgccaa aatgtcgttc gtggcagggg ttattcggcg gctggacgag
60


 


acagtggtga accgcatcgc ggcgggggaa gttatccagc ggccagctaa tgctatcaaa
120


 


gagatgattg agaactgttt agatgcaaaa tccacaagta ttcaagtgat tgttaaagag
180


 


ggaggcctga agttgattca gatccaagac aatggcaccg ggatcaggaa agaagatctg
240


 


gatattgtat gtgaaaggtt cactactagt aaactgcagt cctttgagga tttagccagt
300


 


atttctacct atggctttcg aggtgaggct ttggccagca taagccatgt ggctcatgtt
360


 


actattacaa cgaaaacagc tgatggaaag tgtgcataca gagcaagtta ctcagatgga
420


 


aaactgaaag cccctcctaa accatgtgct ggcaatcaag ggacccagat cacggtggag
480


 


gacctttttt acaacatagc cacgaggaga aaagctttaa aaaatccaag tgaagaatat
540


 


gggaaaattt tggaagttgt tggcaggtat tcagtacaca atgcaggcat tagtttctca
600


 


gttaaaaaac aaggagagac agtagctgat gttaggacac tacccaatgc ctcaaccgtg
660


 


gacaatattc gctccatctt tggaaatgct gttagtcgag aactgataga aattggatgt
720


 


gaggataaaa ccctagcctt caaaatgaat ggttacatat ccaatgcaaa ctactcagtg
780


 


aagaagtgca tcttcttact cttcatcaac catcgtctgg tagaatcaac ttccttgaga
840


 


aaagccatag aaacagtgta tgcagcctat ttgcccaaaa acacacaccc attcctgtac
900


 


ctcagtttag aaatcagtcc ccagaatgtg gatgttaatg tgcaccccac aaagcatgaa
960


 


gttcacttcc tgcacgagga gagcatcctg gagcgggtgc agcagcacat cgagagcaag
1020


 


ctcctgggct ccaattcctc caggatgtac ttcacccaga ctttgctacc aggacttgct
1080


 


ggcccctctg gggagatggt taaatccaca acaagtctga cctcgtcttc tacttctgga
1140


 


agtagtgata aggtctatgc ccaccagatg gttcgtacag attcccggga acagaagctt
1200


 


gatgcatttc tgcagcctct gagcaaaccc ctgtccagtc agccccaggc cattgtcaca
1260


 


gaggataaga cagatatttc tagtggcagg gctaggcagc aagatgagga gatgcttgaa
1320


 


ctcccagccc ctgctgaagt ggctgccaaa aatcagagct tggaggggga tacaacaaag
1380


 


gggacttcag aaatgtcaga gaagagagga cctacttcca gcaaccccag aaagagacat
1440


 


cgggaagatt ctgatgtgga aatggtggaa gatgattccc gaaaggaaat gactgcagct
1500


 


tgtacccccc ggagaaggat cattaacctc actagtgttt tgagtctcca ggaagaaatt
1560


 


aatgagcagg gacatgaggt tctccgggag atgttgcata accactcctt cgtgggctgt
1620


 


gtgaatcctc agtgggcctt ggcacagcat caaaccaagt tataccttct caacaccacc
1680


 


aagcttagtg aagaactgtt ctaccagata ctcatttatg attttgccaa ttttggtgtt
1740


 


ctcaggttat cggagccagc accgctcttt gaccttgcca tgcttgcctt agatagtcca
1800


 


gagagtggct ggacagagga agatggtccc aaagaaggac ttgctgaata cattgttgag
1860


 


tttctgaaga agaaggctga gatgcttgca gactatttct ctttggaaat tgatgaggaa
1920


 


gggaacctga ttggattacc ccttctgatt gacaactatg tgcccccttt ggagggactg
1980


 


cctatcttca ttcttcgact agccactgag gtgaattggg acgaagaaaa ggaatgtttt
2040


 


gaaagcctca gtaaagaatg cgctatgttc tattccatcc ggaagcagta catatctgag
2100


 


gagtcgaccc tctcaggcca gcagagtgaa gtgcctggct ccattccaaa ctcctggaag
2160


 


tggactgtgg aacacattgt ctataaagcc ttgcgctcac acattctgcc tcctaaacat
2220


 


ttcacagaag atggaaatat cctgcagctt gctaacctgc ctgatctata caaagtcttt
2280


 


gagaggtgtt aaatatggtt atttatgcac tgtgggatgt gttcttcttt ctctgtattc
2340


 


cgatacaaag tgttgtatca aagtgtgata tacaaagtgt accaacataa gtgttggtag
2400


 


cacttaagac ttatacttgc cttctgatag tattccttta tacacagtgg attgattata
2460


 


aataaataga tgtgtcttaa cata
2484


 


hPMS2-134 protein (SEQ ID NO: 21)


MKQLPAATVR LLSSSQIITS VVSVVKELIE NSLDAGATSV DVKLENYGFD KIEVRDNGEG
60


 


IKAVDAPVMA MKYYTSKINS HEDLENLTTY GFRGEALGSI CCIAEVLITT RTAADNFSTQ
120


 


YVLDGSGHIL SQK
133


 


hPMS2-134 cDNA (SEQ ID NO: 11)


cgaggcggat cgggtgttgc atccatggag cgagctgaga gctcgagtac agaacctgct
60


 


aaggccatca aacctattga tcggaagtca gtccatcaga tttgctctgg gcaggtggta
120


 


ctgagtctaa gcactgcggt aaaggagtta gtagaaaaca gtctggatgc tggtgccact
180


 


aatattgatc taaagcttaa ggactatgga gtggatctta ttgaagtttc agacaatgga
240


 


tgtggggtag aagaagaaaa cttcgaaggc ttaactctga aacatcacac atctaagatt
300


 


caagagtttg ccgacctaac tcaggttgaa acttttggct ttcgggggga agctctgagc
360


 


tcactttgtg cactgagcga tgtcaccatt tctacctgcc acgcatcggc gaaggttgga
420


 


acttga
426


 








hMSH6 (human cDNA) ACCESSION  U28946 (SEQ ID NO: 22)



MSRQSTLYSFFPKSPALSDANKASARASREGGRAAAAPGASPSP


 


GGDAAWSEAGPGPRPLARSASPPKAKNLNGGLRRSVAPAAPTSCDFSPGDLVWAKMEG


 


YPWWPCLVYNHPFDGTFIREKGKSVRVHVQFFDDSPTRGWVSKRLLKPYTGSKSKEAQ


 


KGGHFYSAKPEILRAMQRADEALNKDKIKRLELAVCDEPSEPEEEEEMEVGTTYVTDK


 


SEEDNEIESEEEVQPKTQGSRRSSRQIKKRRVISDSESDIGGSDVEFKPDTKEEGSSD


 


EISSGVGDSESEGLNSPVKVARKRKRMVTGNGSLKRKSSRKETPSATKQATSISSETK


 


NTLRAFSAPQNSESQAHVSGGGDDSSRPTVWYHETLEWLKEEKRRDEHRRRPDHPDFD


 


ASTLYVPEDFLNSCTPGMRKWWQIKSQNFDLVICYKVGKFYELYHMDALIGVSELGLV


 


FMKGNWAHSGFPEIAFGRYSDSLVQKGYKVARVEQTETPEMNEARCRKMAHISKYDRV


 


VRREICRIITKGTQTYSVLEGDPSENYSKYLLSLKEKEEDSSGHTRAYGVCFVDTSLG


 


KFFIGQFSDDRHCSRFRTLVAHYPPVQVLFEKGNLSKETKTILKSSLSCSLQEGLIPG


 


SQFWDASKTLRTLLEEEYFREKLSDGIGVMLPQVLKGMTSESDSIGLTPGEKSELALS


 


ALGGCVFYLKKCLIDQELLSMANFEEYIPLDSDTVSTTRSGAIFTKAYQRMVLDAVTL


 


NNLEIFLNGTNGSTEGTLLERVDTCHTPFGKRLLKQWLCAPLCNHYAINDRLDAIEDL


 


MVVPDKISEVVELLKKLPDLERLLSKIHNVGSPLKSQNHPDSRAIMYEETTYSKKKII


 


DFLSALEGFKVMCKIIGIMEEVADGFKSKILKQVISLQTKNPEGRFPDLTVELNRWDT


 


AFDHEKARKTGLITPKAGFDSDYDQALADIRENEQSLLEYLEKQRNRIGCRTIVYWGI


 


GRNRYQLEIPENFTTRNLPEEYELKSTKKGCKRYWTKTIEKKLANLINAEERRDVSLK


 


DCMRRLFYNFDKNYKDWQSAVECIAVLDVLLCLANYSRGGDGPMCRPVILLPEDTPPF


 


LELKGSRHPCITKTFFGDDFIPNDILIGCEEEEQENGKAYCVLVTGPNMGGKSTLMRQ


 


AGLLAVMAQMGCYVPAEVCRLTPIDRVFTRLGASDRIMSGESTFFVELSETASILMHA


 


TAHSLVLVDELGRGTATFDGTAIANAVVKELAETIKCRTLFSTHYHSLVEDYSQNVAV


 


RLGHMACMVENECEDPSQETITFLYKFIKGACPKSYGFNAARLANLPEEVIQKGHRKA


 


REFEKMNQSLRLFREVCLASERSTVDAEAVHKLLTLIKEL”


 


hPMSR2 (human cDNA) ACCESSION  U38964 (SEQ ID NO: 12)









1
ggcgctccta cctgcaagtg gctagtgcca agtgctgggc cgccgctcct gccgtgcatg



 


61
ttggggagcc agtacatgca ggtgggctcc acacggagag gggcgcagac ccggtgacag


 


121
ggctttacct ggtacatcgg catggcgcaa ccaaagcaag agagggtggc gcgtgccaga


 


181
caccaacggt cggaaaccgc cagacaccaa cggtcggaaa ccgccaagac accaacgctc


 


241
ggaaaccgcc agacaccaac gctcggaaac cgccagacac caaggctcgg aatccacgcc


 


301
aggccacgac ggagggcgac tacctccctt ctgaccctgc tgctggcgtt cggaaaaaac


 


361
gcagtccggt gtgctctgat tggtccaggc tctttgacgt cacggactcg acctttgaca


 


421
gagccactag gcgaaaagga gagacgggaa gtattttttc cgccccgccc ggaaagggtg


 


481
gagcacaacg tcgaaagcag ccgttgggag cccaggaggc ggggcgcctg tgggagccgt


 


541
ggagggaact ttcccagtcc ccgaggcgga tccggtgttg catccttgga gcgagctgag


 


601
aactcgagta cagaacctgc taaggccatc aaacctattg atcggaagtc agtccatcag


 


661
atttgctctg ggccggtggt accgagtcta aggccgaatg cggtgaagga gttagtagaa


 


721
aacagtctgg atgctggtgc cactaatgtt gatctaaagc ttaaggacta tggagtggat


 


781
ctcattgaag tttcaggcaa tggatgtggg gtagaagaag aaaacttcga aggctttact


 


841
ctgaaacatc acacatgtaa gattcaagag tttgccgacc taactcaggt ggaaactttt


 


901
ggctttcggg gggaagctct gagctcactt tgtgcactga gtgatgtcac catttctacc


 


961
tgccgtgtat cagcgaaggt tgggactcga ctggtgtttg atcactatgg gaaaatcatc


 


1021
cagaaaaccc cctacccccg ccccagaggg atgacagtca gcgtgaagca gttattttct


 


1081
acgctacctg tgcaccataa agaatttcaa aggaatatta agaagaaacg tgcctgcttc


 


1141
cccttcgcct tctgccgtga ttgtcagttt cctgaggcct ccccagccat gcttcctgta


 


1201
cagcctgtag aactgactcc tagaagtacc ccaccccacc cctgctcctt ggaggacaac


 


1261
gtgatcactg tattcagctc tgtcaagaat ggtccaggtt cttctagatg atctgcacaa


 


1321
atggttcctc tcctccttcc tgatgtctgc cattagcatt ggaataaagt tcctgctgaa


 


1381
aatccaaaaa aaaaaaaaaa aaaaaaaa


 








hPMSR2 (human protein) ACCESSION  U38964 (SEQ ID NO: 23)



MAQPKQERVARARHQRSETARHQRSETAKTPTLGNRQTPTLGNR


 


QTPRLGIHARPRRRATTSLLTLLLAFGKNAVRCALIGPGSLTSRTRPLTEPLGEKERR


 


EVFFPPRPERVEHNVESSRWEPRRRGACGSRGGNFPSPRGGSGVASLERAENSSTEPA


 


KAIKPIDRKSVHQICSGPVVPSLRPNAVKELVENSLDAGATNVDLKLKDYGVDLIEVS


 


GNGCGVEEENFEGFTLKHHTCKIQEFADLTQVETFGFRGEALSSLCALSDVTISTCRV


 


SAKVGTRLVFDHYGKIIQKTPYPRPRGMTVSVKQLFSTLPVHHKEFQRNIKKKRACFP


 


FAFCRDCQFPEASPAMLPVQPVELTPRSTPPHPCSLEDNVITVFSSVKNGPGSSR


 


HPMSR3 (human cDNA) ACCESSION  U38979 (SEQ ID NO: 13)









1
tttttagaaa ctgatgttta ttttccatca accatttttc catgctgctt aagagaatat



 


61
gcaagaacag cttaagacca gtcagtggtt gctcctaccc attcagtggc ctgagcagtg


 


121
gggagctgca gaccagtctt ccgtggcagg ctgagcgctc cagtcttcag tagggaattg


 


181
ctgaataggc acagagggca cctgtacacc ttcagaccag tctgcaacct caggctgagt


 


241
agcagtgaac tcaggagcgg gagcagtcca ttcaccctga aattcctcct tggtcactgc


 


301
cttctcagca gcagcctgct cttctttttc aatctcttca ggatctctgt agaagtacag


 


361
atcaggcatg acctcccatg ggtgttcacg ggaaatggtg ccacgcatgc gcagaacttc


 


421
ccgagccagc atccaccaca ttaaacccac tgagtgagct cccttgttgt tgcatgggat


 


481
ggcaatgtcc acatagcgca gaggagaatc tgtgttacac agcgcaatgg taggtaggtt


 


541
aacataagat gcctccgtga gaggcgaagg ggcggcggga cccgggcctg gcccgtatgt


 


601
gtccttggcg ggctagacta ggccgtcgct gtatggtgag ccccagggag gcggatctgg


 


661
gcccccagaa ggacacccgc ctggatttgc cccgtagccc ggcccgggcc cctcgggagc


 


721
agaacagcct tggtgaggtg gacaggaggg gacctcgcga gcagacgcgc gcgccagcga


 


781
cagcagcccc gccccggcct ctcgggagcc ggggggcaga ggctgcggag ccccaggagg


 


841
gtctatcagc cacagtctct gcatgtttcc aagagcaaca ggaaatgaac acattgcagg


 


901
ggccagtgtc attcaaagat gtggctgtgg atttcaccca ggaggagtgg cggcaactgg


 


961
accctgatga gaagatagca tacggggatg tgatgttgga gaactacagc catctagttt


 


1021
ctgtggggta tgattatcac caagccaaac atcatcatgg agtggaggtg aaggaagtgg


 


1081
agcagggaga ggagccgtgg ataatggaag gtgaatttcc atgtcaacat agtccagaac


 


1141
ctgctaaggc catcaaacct attgatcgga agtcagtcca tcagatttgc tctgggccag


 


1201
tggtactgag tctaagcact gcagtgaagg agttagtaga aaacagtctg gatgctggtg


 


1261
ccactaatat tgatctaaag cttaaggact atggagtgga tctcattgaa gtttcagaca


 


1321
atggatgtgg ggtagaagaa gaaaactttg aaggcttaat ctctttcagc tctgaaacat


 


1381
cacacatgta agattcaaga gtttgccgac ctaactgaag ttgaaacttt cggttttcag


 


1441
ggggaagctc tgagctcact gtgtgcactg agcgatgtca ccatttctac ctgccacgcg


 


1501
ttggtgaagg ttgggactcg actggtgttt gatcacgatg ggaaaatcat ccaggaaacc


 


1561
ccctaccccc accccagagg gaccacagtc agcgtgaagc agttattttc tacgctacct


 


1621
gtgcgccata aggaatttca aaggaatatt aagaagacgt gcctgcttcc ccttcgcctt


 


1681
ctgccgtgat tgtcagtttc ctgaggcctc cccagccatg cttcctgtac agcctgcaga


 


1741
actgtgagtc aattaaacct cttttcttca taaattaaaa aaaaa


 








hPMSR3 (human protein) ACCESSION  U38979 (SEQ ID NO: 24)



MCPWRPRLGRRCMVSPREADLGPQKDTRLDLPRSPARAPREQNS


 


LGEVDRRGPREQTRAPATAAPPRPLGSRGAEAAEPQEGLSATVSACFQEQQEMNTLQG


 


PVSFKDVAVDFTQEEWRQLDPDEKIAYGDVMLENYSHLVSVGYDYHQAKHHHGVEVKE


 


VEQGEEPWIMEGEFPCQHSPEPAKAIKPIDRKSVHQICSGPVVLSLSTAVKELVENSL


 


DAGATNIDLKLKDYGVDLIEVSDNGCGVEEENFEGLISFSSETSHM”


 


hPMSL9 (human cDNA) ACCESSION  NM_005395 (SEQ ID NO: 14)









1
atgtgtcctt ggcggcctag actaggccgt cgctgtatgg tgagccccag ggaggcggat



 


61
ctgggccccc agaaggacac ccgcctggat ttgccccgta gcccggcccg ggcccctcgg


 


121
gagcagaaca gccttggtga ggtggacagg aggggacctc gcgagcagac gcgcgcgcca


 


181
gcgacagcag ccccgccccg gcctctcggg agccgggggg cagaggctgc ggagccccag


 


241
gagggtctat cagccacagt ctctgcatgt ttccaagagc aacaggaaat gaacacattg


 


301
caggggccag tgtcattcaa agatgtggct gtggatttca cccaggagga gtggcggcaa


 


361
ctggaccctg atgagaagat agcatacggg gatgtgatgt tggagaacta cagccatcta


 


421
gtttctgtgg ggtatgatta tcaccaagcc aaacatcatc atggagtgga ggtgaaggaa


 


481
gtggagcagg gagaggagcc gtggataatg gaaggtgaat ttccatgtca acatagtcca


 


541
gaacctgcta aggccatcaa acctattgat cggaagtcag tccatcagat ttgctctggg


 


601
ccagtggtac tgagtctaag cactgcagtg aaggagttag tagaaaacag tctggatgct


 


661
ggtgccacta atattgatct aaagcttaag gactatggag tggatctcat tgaagtttca


 


721
gacaatggat gtggggtaga agaagaaaac tttgaaggct taatctcttt cagctctgaa


 


781
acatgacaca tgtaa


 








hPMSL9 (human protein) ACCESSION  NM_005395 (SEQ ID NO: 25)



MCPWRPRLGRRCMVSPREADLGPQKDTRLDLPRSPARAPREQNS


 


LGEVDRRGPREQTRAPATAAPPRPLGSRGAEAAEPQEGLSATVSACFQEQQEMNTLQG


 


PVSFKDVAVDFTQEEWRQLDPDEKIAYGDVMLENYSHLVSVGYDYHQAKHHHGVEVKE


 


VEQGEEPWIMEGEFPCQHSPEPAKAIKPIDRKSVHQICSGPVVLSLSTAVKELVENSL


 


DAGATNIDLKLKDYGVDLIEVSDNGCGVEEENFEGLISFSSETSHM”


















 


 


<160> NUMBER OF SEQ ID NOS: 25 


 


<210> SEQ ID NO 1 


<211> LENGTH: 30 


<212> TYPE: DNA 


<213> ORGANISM: Artificial Sequence 


<220> FEATURE: 


<223> OTHER INFORMATION: PCR primer 


 


<400> SEQUENCE: 1 


 


acgcatatgg agcgagctga gagctcgagt                                      30 


 


 


<210> SEQ ID NO 2 


<211> LENGTH: 75 


<212> TYPE: DNA 


<213> ORGANISM: Artificial Sequence 


<220> FEATURE: 


<223> OTHER INFORMATION: PCR primer 


 


<400> SEQUENCE: 2 


 


gaattcttat cacgtagaat cgagaccgag gagagggtta gggataggct taccagttcc     60 


 


aaccttcgcc gatgc                                                      75 


 


 


<210> SEQ ID NO 3 


<211> LENGTH: 27 


<212> TYPE: DNA 


<213> ORGANISM: Artificial Sequence 


<220> FEATURE: 


<223> OTHER INFORMATION: PCR primer 


 


<400> SEQUENCE: 3 


 


acgcatatgt gtccttggcg gcctaga                                         27 


 


 


<210> SEQ ID NO 4 


<211> LENGTH: 75 


<212> TYPE: DNA 


<213> ORGANISM: Artificial Sequence 


<220> FEATURE: 


<223> OTHER INFORMATION: PCR primer 


 


<400> SEQUENCE: 4 


 


gaattcttat tacgtagaat cgagaccgag gagagggtta gggataggct tacccatgtg     60 


 


tgatgtttca gagct                                                      75 


 


 


<210> SEQ ID NO 5 


<211> LENGTH: 3218 


<212> TYPE: DNA 


<213> ORGANISM: Saccharomyces cerevisiae 


 


<400> SEQUENCE: 5 


 


aaataggaat gtgatacctt ctattgcatg caaagatagt gtaggaggcg ctgctattgc     60 


 


caaagacttt tgagaccgct tgctgtttca ttatagttga ggagttctcg aagacgagaa    120 


 


attagcagtt ttcggtgttt agtaatcgcg ctagcatgct aggacaattt aactgcaaaa    180 


 


ttttgatacg atagtgatag taaatggaag gtaaaaataa catagaccta tcaataagca    240 


 


atgtctctca gaataaaagc acttgatgca tcagtggtta acaaaattgc tgcaggtgag    300 


 


atcataatat cccccgtaaa tgctctcaaa gaaatgatgg agaattccat cgatgcgaat    360 


 


gctacaatga ttgatattct agtcaaggaa ggaggaatta aggtacttca aataacagat    420 


 


aacggatctg gaattaataa agcagacctg ccaatcttat gtgagcgatt cacgacgtcc    480 


 


aaattacaaa aattcgaaga tttgagtcag attcaaacgt atggattccg aggagaagct    540 


 


ttagccagta tctcacatgt ggcaagagtc acagtaacga caaaagttaa agaagacaga    600 


 


tgtgcatgga gagtttcata tgcagaaggt aagatgttgg aaagccccaa acctgttgct    660 


 


ggaaaagacg gtaccacgat cctagttgaa gacctttttt tcaatattcc ttctagatta    720 


 


agggccttga ggtcccataa tgatgaatac tctaaaatat tagatgttgt cgggcgatac    780 


 


gccattcatt ccaaggacat tggcttttct tgtaaaaagt tcggagactc taattattct    840 


 


ttatcagtta aaccttcata tacagtccag gataggatta ggactgtgtt caataaatct    900 


 


gtggcttcga atttaattac ttttcatatc agcaaagtag aagatttaaa cctggaaagc    960 


 


gttgatggaa aggtgtgtaa tttgaatttc atatccaaaa agtccatttc attaattttt   1020 


 


ttcattaata atagactagt gacatgtgat cttctaagaa gagctttgaa cagcgtttac   1080 


 


tccaattatc tgccaaaggg cttcagacct tttatttatt tgggaattgt tatagatccg   1140 


 


gcggctgttg atgttaacgt tcacccgaca aagagagagg ttcgtttcct gagccaagat   1200 


 


gagatcatag agaaaatcgc caatcaattg cacgccgaat tatctgccat tgatacttca   1260 


 


cgtactttca aggcttcttc aatttcaaca aacaagccag agtcattgat accatttaat   1320 


 


gacaccatag aaagtgatag gaataggaag agtctccgac aagcccaagt ggtagagaat   1380 


 


tcatatacga cagccaatag tcaactaagg aaagcgaaaa gacaagagaa taaactagtc   1440 


 


agaatagatg cttcacaagc taaaattacg tcatttttat cctcaagtca acagttcaac   1500 


 


tttgaaggat cgtctacaaa gcgacaactg agtgaaccca aggtaacaaa tgtaagccac   1560 


 


tcccaagagg cagaaaagct gacactaaat gaaagcgaac aaccgcgtga tgccaataca   1620 


 


atcaatgata atgacttgaa ggatcaacct aagaagaaac aaaagttggg ggattataaa   1680 


 


gttccaagca ttgccgatga cgaaaagaat gcactcccga tttcaaaaga cgggtatatt   1740 


 


agagtaccta aggagcgagt taatgttaat cttacgagta tcaagaaatt gcgtgaaaaa   1800 


 


gtagatgatt cgatacatcg agaactaaca gacatttttg caaatttgaa ttacgttggg   1860 


 


gttgtagatg aggaaagaag attagccgct attcagcatg acttaaagct ttttttaata   1920 


 


gattacggat ctgtgtgcta tgagctattc tatcagattg gtttgacaga cttcgcaaac   1980 


 


tttggtaaga taaacctaca gagtacaaat gtgtcagatg atatagtttt gtataatctc   2040 


 


ctatcagaat ttgacgagtt aaatgacgat gcttccaaag aaaaaataat tagtaaaata   2100 


 


tgggacatga gcagtatgct aaatgagtac tattccatag aattggtgaa tgatggtcta   2160 


 


gataatgact taaagtctgt gaagctaaaa tctctaccac tacttttaaa aggctacatt   2220 


 


ccatctctgg tcaagttacc attttttata tatcgcctgg gtaaagaagt tgattgggag   2280 


 


gatgaacaag agtgtctaga tggtatttta agagagattg cattactcta tatacctgat   2340 


 


atggttccga aagtcgatac actcgatgca tcgttgtcag aagacgaaaa agcccagttt   2400 


 


ataaatagaa aggaacacat atcctcatta ctagaacacg ttctcttccc ttgtatcaaa   2460 


 


cgaaggttcc tggcccctag acacattctc aaggatgtcg tggaaatagc caaccttcca   2520 


 


gatctataca aagtttttga gaggtgttaa ctttaaaacg ttttggctgt aataccaaag   2580 


 


tttttgttta tttcctgagt gtgattgtgt ttcatttgaa agtgtatgcc ctttccttta   2640 


 


acgattcatc cgcgagattt caaaggatat gaaatatggt tgcagttagg aaagtatgtc   2700 


 


agaaatgtat attcggattg aaactcttct aatagttctg aagtcacttg gttccgtatt   2760 


 


gttttcgtcc tcttcctcaa gcaacgattc ttgtctaagc ttattcaacg gtaccaaaga   2820 


 


cccgagtcct tttatgagag aaaacatttc atcatttttc aactcaatta tcttaatatc   2880 


 


attttgtagt attttgaaaa caggatggta aaacgaatca cctgaatcta gaagctgtac   2940 


 


cttgtcccat aaaagtttta atttactgag cctttcggtc aagtaaacta gtttatctag   3000 


 


ttttgaaccg aatattgtgg gcagatttgc agtaagttca gttagatcta ctaaaagttg   3060 


 


tttgacagca gccgattcca caaaaatttg gtaaaaggag atgaaagaga cctcgcgcgt   3120 


 


aatggtttgc atcaccatcg gatgtctgtt gaaaaactca ctttttgcat ggaagttatt   3180 


 


aacaataaga ctaatgatta ccttagaata atgtataa                           3218 


 


 


<210> SEQ ID NO 6 


<211> LENGTH: 3056 


<212> TYPE: DNA 


<213> ORGANISM: Mus musculus 


 


<400> SEQUENCE: 6 


 


gaattccggt gaaggtcctg aagaatttcc agattcctga gtatcattgg aggagacaga     60 


 


taacctgtcg tcaggtaacg atggtgtata tgcaacagaa atgggtgttc ctggagacgc    120 


 


gtcttttccc gagagcggca ccgcaactct cccgcggtga ctgtgactgg aggagtcctg    180 


 


catccatgga gcaaaccgaa ggcgtgagta cagaatgtgc taaggccatc aagcctattg    240 


 


atgggaagtc agtccatcaa atttgttctg ggcaggtgat actcagttta agcaccgctg    300 


 


tgaaggagtt gatagaaaat agtgtagatg ctggtgctac tactattgat ctaaggctta    360 


 


aagactatgg ggtggacctc attgaagttt cagacaatgg atgtggggta gaagaagaaa    420 


 


actttgaagg tctagctctg aaacatcaca catctaagat tcaagagttt gccgacctca    480 


 


cgcaggttga aactttcggc tttcgggggg aagctctgag ctctctgtgt gcactaagtg    540 


 


atgtcactat atctacctgc cacgggtctg caagcgttgg gactcgactg gtgtttgacc    600 


 


ataatgggaa aatcacccag aaaactccct acccccgacc taaaggaacc acagtcagtg    660 


 


tgcagcactt attttataca ctacccgtgc gttacaaaga gtttcagagg aacattaaaa    720 


 


aggagtattc caaaatggtg caggtcttac aggcgtactg tatcatctca gcaggcgtcc    780 


 


gtgtaagctg cactaatcag ctcggacagg ggaagcggca cgctgtggtg tgcacaagcg    840 


 


gcacgtctgg catgaaggaa aatatcgggt ctgtgtttgg ccagaagcag ttgcaaagcc    900 


 


tcattccttt tgttcagctg ccccctagtg acgctgtgtg tgaagagtac ggcctgagca    960 


 


cttcaggacg ccacaaaacc ttttctacgt ttcgggcttc atttcacagt gcacgcacgg   1020 


 


cgccgggagg agtgcaacag acaggcagtt tttcttcatc aatcagaggc cctgtgaccc   1080 


 


agcaaaggtc tctaagcttg tcaatgaggt tttatcacat gtataaccgg catcagtacc   1140 


 


catttgtcgt ccttaacgtt tccgttgact cagaatgtgt ggatattaat gtaactccag   1200 


 


ataaaaggca aattctacta caagaagaga agctattgct ggccgtttta aagacctcct   1260 


 


tgataggaat gtttgacagt gatgcaaaca agcttaatgt caaccagcag ccactgctag   1320 


 


atgttgaagg taacttagta aagctgcata ctgcagaact agaaaagcct gtgccaggaa   1380 


 


agcaagataa ctctccttca ctgaagagca cagcagacga gaaaagggta gcatccatct   1440 


 


ccaggctgag agaggccttt tctcttcatc ctactaaaga gatcaagtct aggggtccag   1500 


 


agactgctga actgacacgg agttttccaa gtgagaaaag gggcgtgtta tcctcttatc   1560 


 


cttcagacgt catctcttac agaggcctcc gtggctcgca ggacaaattg gtgagtccca   1620 


 


cggacagccc tggtgactgt atggacagag agaaaataga aaaagactca gggctcagca   1680 


 


gcacctcagc tggctctgag gaagagttca gcaccccaga agtggccagt agctttagca   1740 


 


gtgactataa cgtgagctcc ctagaagaca gaccttctca ggaaaccata aactgtggtg   1800 


 


acctggactg ccgtcctcca ggtacaggac agtccttgaa gccagaagac catggatatc   1860 


 


aatgcaaagc tctacctcta gctcgtctgt cacccacaaa tgccaagcgc ttcaagacag   1920 


 


aggaaagacc ctcaaatgtc aacatttctc aaagattgcc tggtcctcag agcacctcag   1980 


 


cagctgaggt cgatgtagcc ataaaaatga ataagagaat cgtgctcctc gagttctctc   2040 


 


tgagttctct agctaagcga atgaagcagt tacagcacct aaaggcgcag aacaaacatg   2100 


 


aactgagtta cagaaaattt agggccaaga tttgccctgg agaaaaccaa gcagcagaag   2160 


 


atgaactcag aaaagagatt agtaaatcga tgtttgcaga gatggagatc ttgggtcagt   2220 


 


ttaacctggg atttatagta accaaactga aagaggacct cttcctggtg gaccagcatg   2280 


 


ctgcggatga gaagtacaac tttgagatgc tgcagcagca cacggtgctc caggcgcaga   2340 


 


ggctcatcac accccagact ctgaacttaa ctgctgtcaa tgaagctgta ctgatagaaa   2400 


 


atctggaaat attcagaaag aatggctttg actttgtcat tgatgaggat gctccagtca   2460 


 


ctgaaagggc taaattgatt tccttaccaa ctagtaaaaa ctggaccttt ggaccccaag   2520 


 


atatagatga actgatcttt atgttaagtg acagccctgg ggtcatgtgc cggccctcac   2580 


 


gagtcagaca gatgtttgct tccagagcct gtcggaagtc agtgatgatt ggaacggcgc   2640 


 


tcaatgcgag cgagatgaag aagctcatca cccacatggg tgagatggac cacccctgga   2700 


 


actgccccca cggcaggcca accatgaggc acgttgccaa tctggatgtc atctctcaga   2760 


 


actgacacac cccttgtagc atagagttta ttacagattg ttcggtttgc aaagagaagg   2820 


 


ttttaagtaa tctgattatc gttgtacaaa aattagcatg ctgctttaat gtactggatc   2880 


 


catttaaaag cagtgttaag gcaggcatga tggagtgttc ctctagctca gctacttggg   2940 


 


tgatccggtg ggagctcatg tgagcccagg actttgagac cactccgagc cacattcatg   3000 


 


agactcaatt caaggacaaa aaaaaaaaga tatttttgaa gccttttaaa aaaaaa       3056 


 


 


<210> SEQ ID NO 7 


<211> LENGTH: 2771 


<212> TYPE: DNA 


<213> ORGANISM: Homo sapiens 


 


<400> SEQUENCE: 7 


 


cgaggcggat cgggtgttgc atccatggag cgagctgaga gctcgagtac agaacctgct     60 


 


aaggccatca aacctattga tcggaagtca gtccatcaga tttgctctgg gcaggtggta    120 


 


ctgagtctaa gcactgcggt aaaggagtta gtagaaaaca gtctggatgc tggtgccact    180 


 


aatattgatc taaagcttaa ggactatgga gtggatctta ttgaagtttc agacaatgga    240 


 


tgtggggtag aagaagaaaa cttcgaaggc ttaactctga aacatcacac atctaagatt    300 


 


caagagtttg ccgacctaac tcaggttgaa acttttggct ttcgggggga agctctgagc    360 


 


tcactttgtg cactgagcga tgtcaccatt tctacctgcc acgcatcggc gaaggttgga    420 


 


actcgactga tgtttgatca caatgggaaa attatccaga aaacccccta cccccgcccc    480 


 


agagggacca cagtcagcgt gcagcagtta ttttccacac tacctgtgcg ccataaggaa    540 


 


tttcaaagga atattaagaa ggagtatgcc aaaatggtcc aggtcttaca tgcatactgt    600 


 


atcatttcag caggcatccg tgtaagttgc accaatcagc ttggacaagg aaaacgacag    660 


 


cctgtggtat gcacaggtgg aagccccagc ataaaggaaa atatcggctc tgtgtttggg    720 


 


cagaagcagt tgcaaagcct cattcctttt gttcagctgc cccctagtga ctccgtgtgt    780 


 


gaagagtacg gtttgagctg ttcggatgct ctgcataatc ttttttacat ctcaggtttc    840 


 


atttcacaat gcacgcatgg agttggaagg agttcaacag acagacagtt tttctttatc    900 


 


aaccggcggc cttgtgaccc agcaaaggtc tgcagactcg tgaatgaggt ctaccacatg    960 


 


tataatcgac accagtatcc atttgttgtt cttaacattt ctgttgattc agaatgcgtt   1020 


 


gatatcaatg ttactccaga taaaaggcaa attttgctac aagaggaaaa gcttttgttg   1080 


 


gcagttttaa agacctcttt gataggaatg tttgatagtg atgtcaacaa gctaaatgtc   1140 


 


agtcagcagc cactgctgga tgttgaaggt aacttaataa aaatgcatgc agcggatttg   1200 


 


gaaaagccca tggtagaaaa gcaggatcaa tccccttcat taaggactgg agaagaaaaa   1260 


 


aaagacgtgt ccatttccag actgcgagag gccttttctc ttcgtcacac aacagagaac   1320 


 


aagcctcaca gcccaaagac tccagaacca agaaggagcc ctctaggaca gaaaaggggt   1380 


 


atgctgtctt ctagcacttc aggtgccatc tctgacaaag gcgtcctgag acctcagaaa   1440 


 


gaggcagtga gttccagtca cggacccagt gaccctacgg acagagcgga ggtggagaag   1500 


 


gactcggggc acggcagcac ttccgtggat tctgaggggt tcagcatccc agacacgggc   1560 


 


agtcactgca gcagcgagta tgcggccagc tccccagggg acaggggctc gcaggaacat   1620 


 


gtggactctc aggagaaagc gcctgaaact gacgactctt tttcagatgt ggactgccat   1680 


 


tcaaaccagg aagataccgg atgtaaattt cgagttttgc ctcagccaac taatctcgca   1740 


 


accccaaaca caaagcgttt taaaaaagaa gaaattcttt ccagttctga catttgtcaa   1800 


 


aagttagtaa atactcagga catgtcagcc tctcaggttg atgtagctgt gaaaattaat   1860 


 


aagaaagttg tgcccctgga cttttctatg agttctttag ctaaacgaat aaagcagtta   1920 


 


catcatgaag cacagcaaag tgaaggggaa cagaattaca ggaagtttag ggcaaagatt   1980 


 


tgtcctggag aaaatcaagc agccgaagat gaactaagaa aagagataag taaaacgatg   2040 


 


tttgcagaaa tggaaatcat tggtcagttt aacctgggat ttataataac caaactgaat   2100 


 


gaggatatct tcatagtgga ccagcatgcc acggacgaga agtataactt cgagatgctg   2160 


 


cagcagcaca ccgtgctcca ggggcagagg ctcatagcac ctcagactct caacttaact   2220 


 


gctgttaatg aagctgttct gatagaaaat ctggaaatat ttagaaagaa tggctttgat   2280 


 


tttgttatcg atgaaaatgc tccagtcact gaaagggcta aactgatttc cttgccaact   2340 


 


agtaaaaact ggaccttcgg accccaggac gtcgatgaac tgatcttcat gctgagcgac   2400 


 


agccctgggg tcatgtgccg gccttcccga gtcaagcaga tgtttgcctc cagagcctgc   2460 


 


cggaagtcgg tgatgattgg gactgctctt aacacaagcg agatgaagaa actgatcacc   2520 


 


cacatggggg agatggacca cccctggaac tgtccccatg gaaggccaac catgagacac   2580 


 


atcgccaacc tgggtgtcat ttctcagaac tgaccgtagt cactgtatgg aataattggt   2640 


 


tttatcgcag atttttatgt tttgaaagac agagtcttca ctaacctttt ttgttttaaa   2700 


 


atgaaacctg ctacttaaaa aaaatacaca tcacacccat ttaaaagtga tcttgagaac   2760 


 


cttttcaaac c                                                        2771 


 


 


<210> SEQ ID NO 8 


<211> LENGTH: 3063 


<212> TYPE: DNA 


<213> ORGANISM: Homo sapiens 


 


<400> SEQUENCE: 8 


 


ggcacgagtg gctgcttgcg gctagtggat ggtaattgcc tgcctcgcgc tagcagcaag     60 


 


ctgctctgtt aaaagcgaaa atgaaacaat tgcctgcggc aacagttcga ctcctttcaa    120 


 


gttctcagat catcacttcg gtggtcagtg ttgtaaaaga gcttattgaa aactccttgg    180 


 


atgctggtgc cacaagcgta gatgttaaac tggagaacta tggatttgat aaaattgagg    240 


 


tgcgagataa cggggagggt atcaaggctg ttgatgcacc tgtaatggca atgaagtact    300 


 


acacctcaaa aataaatagt catgaagatc ttgaaaattt gacaacttac ggttttcgtg    360 


 


gagaagcctt ggggtcaatt tgttgtatag ctgaggtttt aattacaaca agaacggctg    420 


 


ctgataattt tagcacccag tatgttttag atggcagtgg ccacatactt tctcagaaac    480 


 


cttcacatct tggtcaaggt acaactgtaa ctgctttaag attatttaag aatctacctg    540 


 


taagaaagca gttttactca actgcaaaaa aatgtaaaga tgaaataaaa aagatccaag    600 


 


atctcctcat gagctttggt atccttaaac ctgacttaag gattgtcttt gtacataaca    660 


 


aggcagttat ttggcagaaa agcagagtat cagatcacaa gatggctctc atgtcagttc    720 


 


tggggactgc tgttatgaac aatatggaat cctttcagta ccactctgaa gaatctcaga    780 


 


tttatctcag tggatttctt ccaaagtgtg atgcagacca ctctttcact agtctttcaa    840 


 


caccagaaag aagtttcatc ttcataaaca gtcgaccagt acatcaaaaa gatatcttaa    900 


 


agttaatccg acatcattac aatctgaaat gcctaaagga atctactcgt ttgtatcctg    960 


 


ttttctttct gaaaatcgat gttcctacag ctgatgttga tgtaaattta acaccagata   1020 


 


aaagccaagt attattacaa aataaggaat ctgttttaat tgctcttgaa aatctgatga   1080 


 


cgacttgtta tggaccatta cctagtacaa attcttatga aaataataaa acagatgttt   1140 


 


ccgcagctga catcgttctt agtaaaacag cagaaacaga tgtgcttttt aataaagtgg   1200 


 


aatcatctgg aaagaattat tcaaatgttg atacttcagt cattccattc caaaatgata   1260 


 


tgcataatga tgaatctgga aaaaacactg atgattgttt aaatcaccag ataagtattg   1320 


 


gtgactttgg ttatggtcat tgtagtagtg aaatttctaa cattgataaa aacactaaga   1380 


 


atgcatttca ggacatttca atgagtaatg tatcatggga gaactctcag acggaatata   1440 


 


gtaaaacttg ttttataagt tccgttaagc acacccagtc agaaaatggc aataaagacc   1500 


 


atatagatga gagtggggaa aatgaggaag aagcaggtct tgaaaactct tcggaaattt   1560 


 


ctgcagatga gtggagcagg ggaaatatac ttaaaaattc agtgggagag aatattgaac   1620 


 


ctgtgaaaat tttagtgcct gaaaaaagtt taccatgtaa agtaagtaat aataattatc   1680 


 


caatccctga acaaatgaat cttaatgaag attcatgtaa caaaaaatca aatgtaatag   1740 


 


ataataaatc tggaaaagtt acagcttatg atttacttag caatcgagta atcaagaaac   1800 


 


ccatgtcagc aagtgctctt tttgttcaag atcatcgtcc tcagtttctc atagaaaatc   1860 


 


ctaagactag tttagaggat gcaacactac aaattgaaga actgtggaag acattgagtg   1920 


 


aagaggaaaa actgaaatat gaagagaagg ctactaaaga cttggaacga tacaatagtc   1980 


 


aaatgaagag agccattgaa caggagtcac aaatgtcact aaaagatggc agaaaaaaga   2040 


 


taaaacccac cagcgcatgg aatttggccc agaagcacaa gttaaaaacc tcattatcta   2100 


 


atcaaccaaa acttgatgaa ctccttcagt cccaaattga aaaaagaagg agtcaaaata   2160 


 


ttaaaatggt acagatcccc ttttctatga aaaacttaaa aataaatttt aagaaacaaa   2220 


 


acaaagttga cttagaagag aaggatgaac cttgcttgat ccacaatctc aggtttcctg   2280 


 


atgcatggct aatgacatcc aaaacagagg taatgttatt aaatccatat agagtagaag   2340 


 


aagccctgct atttaaaaga cttcttgaga atcataaact tcctgcagag ccactggaaa   2400 


 


agccaattat gttaacagag agtcttttta atggatctca ttatttagac gttttatata   2460 


 


aaatgacagc agatgaccaa agatacagtg gatcaactta cctgtctgat cctcgtctta   2520 


 


cagcgaatgg tttcaagata aaattgatac caggagtttc aattactgaa aattacttgg   2580 


 


aaatagaagg aatggctaat tgtctcccat tctatggagt agcagattta aaagaaattc   2640 


 


ttaatgctat attaaacaga aatgcaaagg aagtttatga atgtagacct cgcaaagtga   2700 


 


taagttattt agagggagaa gcagtgcgtc tatccagaca attacccatg tacttatcaa   2760 


 


aagaggacat ccaagacatt atctacagaa tgaagcacca gtttggaaat gaaattaaag   2820 


 


agtgtgttca tggtcgccca ttttttcatc atttaaccta tcttccagaa actacatgat   2880 


 


taaatatgtt taagaagatt agttaccatt gaaattggtt ctgtcataaa acagcatgag   2940 


 


tctggtttta aattatcttt gtattatgtg tcacatggtt attttttaaa tgaggattca   3000 


 


ctgacttgtt tttatattga aaaaagttcc acgtattgta gaaaacgtaa ataaactaat   3060 


 


aac                                                                 3063 


 


 


<210> SEQ ID NO 9 


<211> LENGTH: 3145 


<212> TYPE: DNA 


<213> ORGANISM: Homo sapiens 


 


<400> SEQUENCE: 9 


 


ggcgggaaac agcttagtgg gtgtggggtc gcgcattttc ttcaaccagg aggtgaggag     60 


 


gtttcgacat ggcggtgcag ccgaaggaga cgctgcagtt ggagagcgcg gccgaggtcg    120 


 


gcttcgtgcg cttctttcag ggcatgccgg agaagccgac caccacagtg cgccttttcg    180 


 


accggggcga cttctatacg gcgcacggcg aggacgcgct gctggccgcc cgggaggtgt    240 


 


tcaagaccca gggggtgatc aagtacatgg ggccggcagg agcaaagaat ctgcagagtg    300 


 


ttgtgcttag taaaatgaat tttgaatctt ttgtaaaaga tcttcttctg gttcgtcagt    360 


 


atagagttga agtttataag aatagagctg gaaataaggc atccaaggag aatgattggt    420 


 


atttggcata taaggcttct cctggcaatc tctctcagtt tgaagacatt ctctttggta    480 


 


acaatgatat gtcagcttcc attggtgttg tgggtgttaa aatgtccgca gttgatggcc    540 


 


agagacaggt tggagttggg tatgtggatt ccatacagag gaaactagga ctgtgtgaat    600 


 


tccctgataa tgatcagttc tccaatcttg aggctctcct catccagatt ggaccaaagg    660 


 


aatgtgtttt acccggagga gagactgctg gagacatggg gaaactgaga cagataattc    720 


 


aaagaggagg aattctgatc acagaaagaa aaaaagctga cttttccaca aaagacattt    780 


 


atcaggacct caaccggttg ttgaaaggca aaaagggaga gcagatgaat agtgctgtat    840 


 


tgccagaaat ggagaatcag gttgcagttt catcactgtc tgcggtaatc aagtttttag    900 


 


aactcttatc agatgattcc aactttggac agtttgaact gactactttt gacttcagcc    960 


 


agtatatgaa attggatatt gcagcagtca gagcccttaa cctttttcag ggttctgttg   1020 


 


aagataccac tggctctcag tctctggctg ccttgctgaa taagtgtaaa acccctcaag   1080 


 


gacaaagact tgttaaccag tggattaagc agcctctcat ggataagaac agaatagagg   1140 


 


agagattgaa tttagtggaa gcttttgtag aagatgcaga attgaggcag actttacaag   1200 


 


aagatttact tcgtcgattc ccagatctta accgacttgc caagaagttt caaagacaag   1260 


 


cagcaaactt acaagattgt taccgactct atcagggtat aaatcaacta cctaatgtta   1320 


 


tacaggctct ggaaaaacat gaaggaaaac accagaaatt attgttggca gtttttgtga   1380 


 


ctcctcttac tgatcttcgt tctgacttct ccaagtttca ggaaatgata gaaacaactt   1440 


 


tagatatgga tcaggtggaa aaccatgaat tccttgtaaa accttcattt gatcctaatc   1500 


 


tcagtgaatt aagagaaata atgaatgact tggaaaagaa gatgcagtca acattaataa   1560 


 


gtgcagccag agatcttggc ttggaccctg gcaaacagat taaactggat tccagtgcac   1620 


 


agtttggata ttactttcgt gtaacctgta aggaagaaaa agtccttcgt aacaataaaa   1680 


 


actttagtac tgtagatatc cagaagaatg gtgttaaatt taccaacagc aaattgactt   1740 


 


ctttaaatga agagtatacc aaaaataaaa cagaatatga agaagcccag gatgccattg   1800 


 


ttaaagaaat tgtcaatatt tcttcaggct atgtagaacc aatgcagaca ctcaatgatg   1860 


 


tgttagctca gctagatgct gttgtcagct ttgctcacgt gtcaaatgga gcacctgttc   1920 


 


catatgtacg accagccatt ttggagaaag gacaaggaag aattatatta aaagcatcca   1980 


 


ggcatgcttg tgttgaagtt caagatgaaa ttgcatttat tcctaatgac gtatactttg   2040 


 


aaaaagataa acagatgttc cacatcatta ctggccccaa tatgggaggt aaatcaacat   2100 


 


atattcgaca aactggggtg atagtactca tggcccaaat tgggtgtttt gtgccatgtg   2160 


 


agtcagcaga agtgtccatt gtggactgca tcttagcccg agtaggggct ggtgacagtc   2220 


 


aattgaaagg agtctccacg ttcatggctg aaatgttgga aactgcttct atcctcaggt   2280 


 


ctgcaaccaa agattcatta ataatcatag atgaattggg aagaggaact tctacctacg   2340 


 


atggatttgg gttagcatgg gctatatcag aatacattgc aacaaagatt ggtgcttttt   2400 


 


gcatgtttgc aacccatttt catgaactta ctgccttggc caatcagata ccaactgtta   2460 


 


ataatctaca tgtcacagca ctcaccactg aagagacctt aactatgctt tatcaggtga   2520 


 


agaaaggtgt ctgtgatcaa agttttggga ttcatgttgc agagcttgct aatttcccta   2580 


 


agcatgtaat agagtgtgct aaacagaaag ccctggaact tgaggagttt cagtatattg   2640 


 


gagaatcgca aggatatgat atcatggaac cagcagcaaa gaagtgctat ctggaaagag   2700 


 


agcaaggtga aaaaattatt caggagttcc tgtccaaggt gaaacaaatg ccctttactg   2760 


 


aaatgtcaga agaaaacatc acaataaagt taaaacagct aaaagctgaa gtaatagcaa   2820 


 


agaataatag ctttgtaaat gaaatcattt cacgaataaa agttactacg tgaaaaatcc   2880 


 


cagtaatgga atgaaggtaa tattgataag ctattgtctg taatagtttt atattgtttt   2940 


 


atattaaccc tttttccata gtgttaactg tcagtgccca tgggctatca acttaataag   3000 


 


atatttagta atattttact ttgaggacat tttcaaagat ttttattttg aaaaatgaga   3060 


 


gctgtaactg aggactgttt gcaattgaca taggcaataa taagtgatgt gctgaatttt   3120 


 


ataaataaaa tcatgtagtt tgtgg                                         3145 


 


 


<210> SEQ ID NO 10 


<211> LENGTH: 2484 


<212> TYPE: DNA 


<213> ORGANISM: Homo sapiens 


 


<400> SEQUENCE: 10 


 


cttggctctt ctggcgccaa aatgtcgttc gtggcagggg ttattcggcg gctggacgag     60 


 


acagtggtga accgcatcgc ggcgggggaa gttatccagc ggccagctaa tgctatcaaa    120 


 


gagatgattg agaactgttt agatgcaaaa tccacaagta ttcaagtgat tgttaaagag    180 


 


ggaggcctga agttgattca gatccaagac aatggcaccg ggatcaggaa agaagatctg    240 


 


gatattgtat gtgaaaggtt cactactagt aaactgcagt cctttgagga tttagccagt    300 


 


atttctacct atggctttcg aggtgaggct ttggccagca taagccatgt ggctcatgtt    360 


 


actattacaa cgaaaacagc tgatggaaag tgtgcataca gagcaagtta ctcagatgga    420 


 


aaactgaaag cccctcctaa accatgtgct ggcaatcaag ggacccagat cacggtggag    480 


 


gacctttttt acaacatagc cacgaggaga aaagctttaa aaaatccaag tgaagaatat    540 


 


gggaaaattt tggaagttgt tggcaggtat tcagtacaca atgcaggcat tagtttctca    600 


 


gttaaaaaac aaggagagac agtagctgat gttaggacac tacccaatgc ctcaaccgtg    660 


 


gacaatattc gctccatctt tggaaatgct gttagtcgag aactgataga aattggatgt    720 


 


gaggataaaa ccctagcctt caaaatgaat ggttacatat ccaatgcaaa ctactcagtg    780 


 


aagaagtgca tcttcttact cttcatcaac catcgtctgg tagaatcaac ttccttgaga    840 


 


aaagccatag aaacagtgta tgcagcctat ttgcccaaaa acacacaccc attcctgtac    900 


 


ctcagtttag aaatcagtcc ccagaatgtg gatgttaatg tgcaccccac aaagcatgaa    960 


 


gttcacttcc tgcacgagga gagcatcctg gagcgggtgc agcagcacat cgagagcaag   1020 


 


ctcctgggct ccaattcctc caggatgtac ttcacccaga ctttgctacc aggacttgct   1080 


 


ggcccctctg gggagatggt taaatccaca acaagtctga cctcgtcttc tacttctgga   1140 


 


agtagtgata aggtctatgc ccaccagatg gttcgtacag attcccggga acagaagctt   1200 


 


gatgcatttc tgcagcctct gagcaaaccc ctgtccagtc agccccaggc cattgtcaca   1260 


 


gaggataaga cagatatttc tagtggcagg gctaggcagc aagatgagga gatgcttgaa   1320 


 


ctcccagccc ctgctgaagt ggctgccaaa aatcagagct tggaggggga tacaacaaag   1380 


 


gggacttcag aaatgtcaga gaagagagga cctacttcca gcaaccccag aaagagacat   1440 


 


cgggaagatt ctgatgtgga aatggtggaa gatgattccc gaaaggaaat gactgcagct   1500 


 


tgtacccccc ggagaaggat cattaacctc actagtgttt tgagtctcca ggaagaaatt   1560 


 


aatgagcagg gacatgaggt tctccgggag atgttgcata accactcctt cgtgggctgt   1620 


 


gtgaatcctc agtgggcctt ggcacagcat caaaccaagt tataccttct caacaccacc   1680 


 


aagcttagtg aagaactgtt ctaccagata ctcatttatg attttgccaa ttttggtgtt   1740 


 


ctcaggttat cggagccagc accgctcttt gaccttgcca tgcttgcctt agatagtcca   1800 


 


gagagtggct ggacagagga agatggtccc aaagaaggac ttgctgaata cattgttgag   1860 


 


tttctgaaga agaaggctga gatgcttgca gactatttct ctttggaaat tgatgaggaa   1920 


 


gggaacctga ttggattacc ccttctgatt gacaactatg tgcccccttt ggagggactg   1980 


 


cctatcttca ttcttcgact agccactgag gtgaattggg acgaagaaaa ggaatgtttt   2040 


 


gaaagcctca gtaaagaatg cgctatgttc tattccatcc ggaagcagta catatctgag   2100 


 


gagtcgaccc tctcaggcca gcagagtgaa gtgcctggct ccattccaaa ctcctggaag   2160 


 


tggactgtgg aacacattgt ctataaagcc ttgcgctcac acattctgcc tcctaaacat   2220 


 


ttcacagaag atggaaatat cctgcagctt gctaacctgc ctgatctata caaagtcttt   2280 


 


gagaggtgtt aaatatggtt atttatgcac tgtgggatgt gttcttcttt ctctgtattc   2340 


 


cgatacaaag tgttgtatca aagtgtgata tacaaagtgt accaacataa gtgttggtag   2400 


 


cacttaagac ttatacttgc cttctgatag tattccttta tacacagtgg attgattata   2460 


 


aataaataga tgtgtcttaa cata                                          2484 


 


 


<210> SEQ ID NO 11 


<211> LENGTH: 426 


<212> TYPE: DNA 


<213> ORGANISM: Homo sapiens 


 


<400> SEQUENCE: 11 


 


cgaggcggat cgggtgttgc atccatggag cgagctgaga gctcgagtac agaacctgct     60 


 


aaggccatca aacctattga tcggaagtca gtccatcaga tttgctctgg gcaggtggta    120 


 


ctgagtctaa gcactgcggt aaaggagtta gtagaaaaca gtctggatgc tggtgccact    180 


 


aatattgatc taaagcttaa ggactatgga gtggatctta ttgaagtttc agacaatgga    240 


 


tgtggggtag aagaagaaaa cttcgaaggc ttaactctga aacatcacac atctaagatt    300 


 


caagagtttg ccgacctaac tcaggttgaa acttttggct ttcgggggga agctctgagc    360 


 


tcactttgtg cactgagcga tgtcaccatt tctacctgcc acgcatcggc gaaggttgga    420 


 


acttga                                                               426 


 


 


<210> SEQ ID NO 12 


<211> LENGTH: 1408 


<212> TYPE: DNA 


<213> ORGANISM: Homo sapiens 


 


<400> SEQUENCE: 12 


 


ggcgctccta cctgcaagtg gctagtgcca agtgctgggc cgccgctcct gccgtgcatg     60 


 


ttggggagcc agtacatgca ggtgggctcc acacggagag gggcgcagac ccggtgacag    120 


 


ggctttacct ggtacatcgg catggcgcaa ccaaagcaag agagggtggc gcgtgccaga    180 


 


caccaacggt cggaaaccgc cagacaccaa cggtcggaaa ccgccaagac accaacgctc    240 


 


ggaaaccgcc agacaccaac gctcggaaac cgccagacac caaggctcgg aatccacgcc    300 


 


aggccacgac ggagggcgac tacctccctt ctgaccctgc tgctggcgtt cggaaaaaac    360 


 


gcagtccggt gtgctctgat tggtccaggc tctttgacgt cacggactcg acctttgaca    420 


 


gagccactag gcgaaaagga gagacgggaa gtattttttc cgccccgccc ggaaagggtg    480 


 


gagcacaacg tcgaaagcag ccgttgggag cccaggaggc ggggcgcctg tgggagccgt    540 


 


ggagggaact ttcccagtcc ccgaggcgga tccggtgttg catccttgga gcgagctgag    600 


 


aactcgagta cagaacctgc taaggccatc aaacctattg atcggaagtc agtccatcag    660 


 


atttgctctg ggccggtggt accgagtcta aggccgaatg cggtgaagga gttagtagaa    720 


 


aacagtctgg atgctggtgc cactaatgtt gatctaaagc ttaaggacta tggagtggat    780 


 


ctcattgaag tttcaggcaa tggatgtggg gtagaagaag aaaacttcga aggctttact    840 


 


ctgaaacatc acacatgtaa gattcaagag tttgccgacc taactcaggt ggaaactttt    900 


 


ggctttcggg gggaagctct gagctcactt tgtgcactga gtgatgtcac catttctacc    960 


 


tgccgtgtat cagcgaaggt tgggactcga ctggtgtttg atcactatgg gaaaatcatc   1020 


 


cagaaaaccc cctacccccg ccccagaggg atgacagtca gcgtgaagca gttattttct   1080 


 


acgctacctg tgcaccataa agaatttcaa aggaatatta agaagaaacg tgcctgcttc   1140 


 


cccttcgcct tctgccgtga ttgtcagttt cctgaggcct ccccagccat gcttcctgta   1200 


 


cagcctgtag aactgactcc tagaagtacc ccaccccacc cctgctcctt ggaggacaac   1260 


 


gtgatcactg tattcagctc tgtcaagaat ggtccaggtt cttctagatg atctgcacaa   1320 


 


atggttcctc tcctccttcc tgatgtctgc cattagcatt ggaataaagt tcctgctgaa   1380 


 


aatccaaaaa aaaaaaaaaa aaaaaaaa                                      1408 


 


 


<210> SEQ ID NO 13 


<211> LENGTH: 1785 


<212> TYPE: DNA 


<213> ORGANISM: Homo sapiens 


 


<400> SEQUENCE: 13 


 


tttttagaaa ctgatgttta ttttccatca accatttttc catgctgctt aagagaatat     60 


 


gcaagaacag cttaagacca gtcagtggtt gctcctaccc attcagtggc ctgagcagtg    120 


 


gggagctgca gaccagtctt ccgtggcagg ctgagcgctc cagtcttcag tagggaattg    180 


 


ctgaataggc acagagggca cctgtacacc ttcagaccag tctgcaacct caggctgagt    240 


 


agcagtgaac tcaggagcgg gagcagtcca ttcaccctga aattcctcct tggtcactgc    300 


 


cttctcagca gcagcctgct cttctttttc aatctcttca ggatctctgt agaagtacag    360 


 


atcaggcatg acctcccatg ggtgttcacg ggaaatggtg ccacgcatgc gcagaacttc    420 


 


ccgagccagc atccaccaca ttaaacccac tgagtgagct cccttgttgt tgcatgggat    480 


 


ggcaatgtcc acatagcgca gaggagaatc tgtgttacac agcgcaatgg taggtaggtt    540 


 


aacataagat gcctccgtga gaggcgaagg ggcggcggga cccgggcctg gcccgtatgt    600 


 


gtccttggcg gcctagacta ggccgtcgct gtatggtgag ccccagggag gcggatctgg    660 


 


gcccccagaa ggacacccgc ctggatttgc cccgtagccc ggcccgggcc cctcgggagc    720 


 


agaacagcct tggtgaggtg gacaggaggg gacctcgcga gcagacgcgc gcgccagcga    780 


 


cagcagcccc gccccggcct ctcgggagcc ggggggcaga ggctgcggag ccccaggagg    840 


 


gtctatcagc cacagtctct gcatgtttcc aagagcaaca ggaaatgaac acattgcagg    900 


 


ggccagtgtc attcaaagat gtggctgtgg atttcaccca ggaggagtgg cggcaactgg    960 


 


accctgatga gaagatagca tacggggatg tgatgttgga gaactacagc catctagttt   1020 


 


ctgtggggta tgattatcac caagccaaac atcatcatgg agtggaggtg aaggaagtgg   1080 


 


agcagggaga ggagccgtgg ataatggaag gtgaatttcc atgtcaacat agtccagaac   1140 


 


ctgctaaggc catcaaacct attgatcgga agtcagtcca tcagatttgc tctgggccag   1200 


 


tggtactgag tctaagcact gcagtgaagg agttagtaga aaacagtctg gatgctggtg   1260 


 


ccactaatat tgatctaaag cttaaggact atggagtgga tctcattgaa gtttcagaca   1320 


 


atggatgtgg ggtagaagaa gaaaactttg aaggcttaat ctctttcagc tctgaaacat   1380 


 


cacacatgta agattcaaga gtttgccgac ctaactgaag ttgaaacttt cggttttcag   1440 


 


ggggaagctc tgagctcact gtgtgcactg agcgatgtca ccatttctac ctgccacgcg   1500 


 


ttggtgaagg ttgggactcg actggtgttt gatcacgatg ggaaaatcat ccaggaaacc   1560 


 


ccctaccccc accccagagg gaccacagtc agcgtgaagc agttattttc tacgctacct   1620 


 


gtgcgccata aggaatttca aaggaatatt aagaagacgt gcctgcttcc ccttcgcctt   1680 


 


ctgccgtgat tgtcagtttc ctgaggcctc cccagccatg cttcctgtac agcctgcaga   1740 


 


actgtgagtc aattaaacct cttttcttca taaattaaaa aaaaa                   1785 


 


 


<210> SEQ ID NO 14 


<211> LENGTH: 795 


<212> TYPE: DNA 


<213> ORGANISM: Homo sapiens 


 


<400> SEQUENCE: 14 


 


atgtgtcctt ggcggcctag actaggccgt cgctgtatgg tgagccccag ggaggcggat     60 


 


ctgggccccc agaaggacac ccgcctggat ttgccccgta gcccggcccg ggcccctcgg    120 


 


gagcagaaca gccttggtga ggtggacagg aggggacctc gcgagcagac gcgcgcgcca    180 


 


gcgacagcag ccccgccccg gcctctcggg agccgggggg cagaggctgc ggagccccag    240 


 


gagggtctat cagccacagt ctctgcatgt ttccaagagc aacaggaaat gaacacattg    300 


 


caggggccag tgtcattcaa agatgtggct gtggatttca cccaggagga gtggcggcaa    360 


 


ctggaccctg atgagaagat agcatacggg gatgtgatgt tggagaacta cagccatcta    420 


 


gtttctgtgg ggtatgatta tcaccaagcc aaacatcatc atggagtgga ggtgaaggaa    480 


 


gtggagcagg gagaggagcc gtggataatg gaaggtgaat ttccatgtca acatagtcca    540 


 


gaacctgcta aggccatcaa acctattgat cggaagtcag tccatcagat ttgctctggg    600 


 


ccagtggtac tgagtctaag cactgcagtg aaggagttag tagaaaacag tctggatgct    660 


 


ggtgccacta atattgatct aaagcttaag gactatggag tggatctcat tgaagtttca    720 


 


gacaatggat gtggggtaga agaagaaaac tttgaaggct taatctcttt cagctctgaa    780 


 


acatcacaca tgtaa                                                     795 


 


 


<210> SEQ ID NO 15 


<211> LENGTH: 769 


<212> TYPE: PRT 


<213> ORGANISM: Saccharomyces cerevisiae 


 


<400> SEQUENCE: 15 


 


Met Ser Leu Arg Ile Lys Ala Leu Asp Ala Ser Val Val Asn Lys Ile 


 1               5                  10                  15 


 


Ala Ala Gly Glu Ile Ile Ile Ser Pro Val Asn Ala Leu Lys Glu Met 


            20                  25                  30 


 


Met Glu Asn Ser Ile Asp Ala Asn Ala Thr Met Ile Asp Ile Leu Val 


        35                  40                  45 


 


Lys Glu Gly Gly Ile Lys Val Leu Gln Ile Thr Asp Asn Gly Ser Gly 


    50                  55                  60 


 


Ile Asn Lys Ala Asp Leu Pro Ile Leu Cys Glu Arg Phe Thr Thr Ser 


65                  70                  75                  80 


 


Lys Leu Gln Lys Phe Glu Asp Leu Ser Gln Ile Gln Thr Tyr Gly Phe 


                85                  90                  95 


 


Arg Gly Glu Ala Leu Ala Ser Ile Ser His Val Ala Arg Val Thr Val 


            100                 105                 110 


 


Thr Thr Lys Val Lys Glu Asp Arg Cys Ala Trp Arg Val Ser Tyr Ala 


        115                 120                 125 


 


Glu Gly Lys Met Leu Glu Ser Pro Lys Pro Val Ala Gly Lys Asp Gly 


    130                 135                 140 


 


Thr Thr Ile Leu Val Glu Asp Leu Phe Phe Asn Ile Pro Ser Arg Leu 


145                 150                 155                 160 


 


Arg Ala Leu Arg Ser His Asn Asp Glu Tyr Ser Lys Ile Leu Asp Val 


                165                 170                 175 


 


Val Gly Arg Tyr Ala Ile His Ser Lys Asp Ile Gly Phe Ser Cys Lys 


            180                 185                 190 


 


Lys Phe Gly Asp Ser Asn Tyr Ser Leu Ser Val Lys Pro Ser Tyr Thr 


        195                 200                 205 


 


Val Gln Asp Arg Ile Arg Thr Val Phe Asn Lys Ser Val Ala Ser Asn 


    210                 215                 220 


 


Leu Ile Thr Phe His Ile Ser Lys Val Glu Asp Leu Asn Leu Glu Ser 


225                 230                 235                 240 


 


Val Asp Gly Lys Val Cys Asn Leu Asn Phe Ile Ser Lys Lys Ser Ile 


                245                 250                 255 


 


Ser Leu Ile Phe Phe Ile Asn Asn Arg Leu Val Thr Cys Asp Leu Leu 


            260                 265                 270 


 


Arg Arg Ala Leu Asn Ser Val Tyr Ser Asn Tyr Leu Pro Lys Gly Phe 


        275                 280                 285 


 


Arg Pro Phe Ile Tyr Leu Gly Ile Val Ile Asp Pro Ala Ala Val Asp 


    290                 295                 300 


 


Val Asn Val His Pro Thr Lys Arg Glu Val Arg Phe Leu Ser Gln Asp 


305                 310                 315                 320 


 


Glu Ile Ile Glu Lys Ile Ala Asn Gln Leu His Ala Glu Leu Ser Ala 


                325                 330                 335 


 


Ile Asp Thr Ser Arg Thr Phe Lys Ala Ser Ser Ile Ser Thr Asn Lys 


            340                 345                 350 


 


Pro Glu Ser Leu Ile Pro Phe Asn Asp Thr Ile Glu Ser Asp Arg Asn 


        355                 360                 365 


 


Arg Lys Ser Leu Arg Gln Ala Gln Val Val Glu Asn Ser Tyr Thr Thr 


    370                 375                 380 


 


Ala Asn Ser Gln Leu Arg Lys Ala Lys Arg Gln Glu Asn Lys Leu Val 


385                 390                 395                 400 


 


Arg Ile Asp Ala Ser Gln Ala Lys Ile Thr Ser Phe Leu Ser Ser Ser 


                405                 410                 415 


 


Gln Gln Phe Asn Phe Glu Gly Ser Ser Thr Lys Arg Gln Leu Ser Glu 


            420                 425                 430 


 


Pro Lys Val Thr Asn Val Ser His Ser Gln Glu Ala Glu Lys Leu Thr 


        435                 440                 445 


 


Leu Asn Glu Ser Glu Gln Pro Arg Asp Ala Asn Thr Ile Asn Asp Asn 


    450                 455                 460 


 


Asp Leu Lys Asp Gln Pro Lys Lys Lys Gln Lys Leu Gly Asp Tyr Lys 


465                 470                 475                 480 


 


Val Pro Ser Ile Ala Asp Asp Glu Lys Asn Ala Leu Pro Ile Ser Lys 


                485                 490                 495 


 


Asp Gly Tyr Ile Arg Val Pro Lys Glu Arg Val Asn Val Asn Leu Thr 


            500                 505                 510 


 


Ser Ile Lys Lys Leu Arg Glu Lys Val Asp Asp Ser Ile His Arg Glu 


        515                 520                 525 


 


Leu Thr Asp Ile Phe Ala Asn Leu Asn Tyr Val Gly Val Val Asp Glu 


    530                 535                 540 


 


Glu Arg Arg Leu Ala Ala Ile Gln His Asp Leu Lys Leu Phe Leu Ile 


545                 550                 555                 560 


 


Asp Tyr Gly Ser Val Cys Tyr Glu Leu Phe Tyr Gln Ile Gly Leu Thr 


                565                 570                 575 


 


Asp Phe Ala Asn Phe Gly Lys Ile Asn Leu Gln Ser Thr Asn Val Ser 


            580                 585                 590 


 


Asp Asp Ile Val Leu Tyr Asn Leu Leu Ser Glu Phe Asp Glu Leu Asn 


        595                 600                 605 


 


Asp Asp Ala Ser Lys Glu Lys Ile Ile Ser Lys Ile Trp Asp Met Ser 


    610                 615                 620 


 


Ser Met Leu Asn Glu Tyr Tyr Ser Ile Glu Leu Val Asn Asp Gly Leu 


625                 630                 635                 640 


 


Asp Asn Asp Leu Lys Ser Val Lys Leu Lys Ser Leu Pro Leu Leu Leu 


                645                 650                 655 


 


Lys Gly Tyr Ile Pro Ser Leu Val Lys Leu Pro Phe Phe Ile Tyr Arg 


            660                 665                 670 


 


Leu Gly Lys Glu Val Asp Trp Glu Asp Glu Gln Glu Cys Leu Asp Gly 


        675                 680                 685 


 


Ile Leu Arg Glu Ile Ala Leu Leu Tyr Ile Pro Asp Met Val Pro Lys 


    690                 695                 700 


 


Val Asp Thr Leu Asp Ala Ser Leu Ser Glu Asp Glu Lys Ala Gln Phe 


705                 710                 715                 720 


 


Ile Asn Arg Lys Glu His Ile Ser Ser Leu Leu Glu His Val Leu Phe 


                725                 730                 735 


 


Pro Cys Ile Lys Arg Arg Phe Leu Ala Pro Arg His Ile Leu Lys Asp 


            740                 745                 750 


 


Val Val Glu Ile Ala Asn Leu Pro Asp Leu Tyr Lys Val Phe Glu Arg 


        755                 760                 765 


 


Cys 


 


 


<210> SEQ ID NO 16 


<211> LENGTH: 859 


<212> TYPE: PRT 


<213> ORGANISM: Mus musculus 


 


<400> SEQUENCE: 16 


 


Met Glu Gln Thr Glu Gly Val Ser Thr Glu Cys Ala Lys Ala Ile Lys 


 1               5                  10                  15 


 


Pro Ile Asp Gly Lys Ser Val His Gln Ile Cys Ser Gly Gln Val Ile 


            20                  25                  30 


 


Leu Ser Leu Ser Thr Ala Val Lys Glu Leu Ile Glu Asn Ser Val Asp 


        35                  40                  45 


 


Ala Gly Ala Thr Thr Ile Asp Leu Arg Leu Lys Asp Tyr Gly Val Asp 


    50                  55                  60 


 


Leu Ile Glu Val Ser Asp Asn Gly Cys Gly Val Glu Glu Glu Asn Phe 


65                  70                  75                  80 


 


Glu Gly Leu Ala Leu Lys His His Thr Ser Lys Ile Gln Glu Phe Ala 


                85                  90                  95 


 


Asp Leu Thr Gln Val Glu Thr Phe Gly Phe Arg Gly Glu Ala Leu Ser 


            100                 105                 110 


 


Ser Leu Cys Ala Leu Ser Asp Val Thr Ile Ser Thr Cys His Gly Ser 


        115                 120                 125 


 


Ala Ser Val Gly Thr Arg Leu Val Phe Asp His Asn Gly Lys Ile Thr 


    130                 135                 140 


 


Gln Lys Thr Pro Tyr Pro Arg Pro Lys Gly Thr Thr Val Ser Val Gln 


145                 150                 155                 160 


 


His Leu Phe Tyr Thr Leu Pro Val Arg Tyr Lys Glu Phe Gln Arg Asn 


                165                 170                 175 


 


Ile Lys Lys Glu Tyr Ser Lys Met Val Gln Val Leu Gln Ala Tyr Cys 


            180                 185                 190 


 


Ile Ile Ser Ala Gly Val Arg Val Ser Cys Thr Asn Gln Leu Gly Gln 


        195                 200                 205 


 


Gly Lys Arg His Ala Val Val Cys Thr Ser Gly Thr Ser Gly Met Lys 


    210                 215                 220 


 


Glu Asn Ile Gly Ser Val Phe Gly Gln Lys Gln Leu Gln Ser Leu Ile 


225                 230                 235                 240 


 


Pro Phe Val Gln Leu Pro Pro Ser Asp Ala Val Cys Glu Glu Tyr Gly 


                245                 250                 255 


 


Leu Ser Thr Ser Gly Arg His Lys Thr Phe Ser Thr Phe Arg Ala Ser 


            260                 265                 270 


 


Phe His Ser Ala Arg Thr Ala Pro Gly Gly Val Gln Gln Thr Gly Ser 


        275                 280                 285 


 


Phe Ser Ser Ser Ile Arg Gly Pro Val Thr Gln Gln Arg Ser Leu Ser 


    290                 295                 300 


 


Leu Ser Met Arg Phe Tyr His Met Tyr Asn Arg His Gln Tyr Pro Phe 


305                 310                 315                 320 


 


Val Val Leu Asn Val Ser Val Asp Ser Glu Cys Val Asp Ile Asn Val 


                325                 330                 335 


 


Thr Pro Asp Lys Arg Gln Ile Leu Leu Gln Glu Glu Lys Leu Leu Leu 


            340                 345                 350 


 


Ala Val Leu Lys Thr Ser Leu Ile Gly Met Phe Asp Ser Asp Ala Asn 


        355                 360                 365 


 


Lys Leu Asn Val Asn Gln Gln Pro Leu Leu Asp Val Glu Gly Asn Leu 


    370                 375                 380 


 


Val Lys Leu His Thr Ala Glu Leu Glu Lys Pro Val Pro Gly Lys Gln 


385                 390                 395                 400 


 


Asp Asn Ser Pro Ser Leu Lys Ser Thr Ala Asp Glu Lys Arg Val Ala 


                405                 410                 415 


 


Ser Ile Ser Arg Leu Arg Glu Ala Phe Ser Leu His Pro Thr Lys Glu 


            420                 425                 430 


 


Ile Lys Ser Arg Gly Pro Glu Thr Ala Glu Leu Thr Arg Ser Phe Pro 


        435                 440                 445 


 


Ser Glu Lys Arg Gly Val Leu Ser Ser Tyr Pro Ser Asp Val Ile Ser 


    450                 455                 460 


 


Tyr Arg Gly Leu Arg Gly Ser Gln Asp Lys Leu Val Ser Pro Thr Asp 


465                 470                 475                 480 


 


Ser Pro Gly Asp Cys Met Asp Arg Glu Lys Ile Glu Lys Asp Ser Gly 


                485                 490                 495 


 


Leu Ser Ser Thr Ser Ala Gly Ser Glu Glu Glu Phe Ser Thr Pro Glu 


            500                 505                 510 


 


Val Ala Ser Ser Phe Ser Ser Asp Tyr Asn Val Ser Ser Leu Glu Asp 


        515                 520                 525 


 


Arg Pro Ser Gln Glu Thr Ile Asn Cys Gly Asp Leu Asp Cys Arg Pro 


    530                 535                 540 


 


Pro Gly Thr Gly Gln Ser Leu Lys Pro Glu Asp His Gly Tyr Gln Cys 


545                 550                 555                 560 


 


Lys Ala Leu Pro Leu Ala Arg Leu Ser Pro Thr Asn Ala Lys Arg Phe 


                565                 570                 575 


 


Lys Thr Glu Glu Arg Pro Ser Asn Val Asn Ile Ser Gln Arg Leu Pro 


            580                 585                 590 


 


Gly Pro Gln Ser Thr Ser Ala Ala Glu Val Asp Val Ala Ile Lys Met 


        595                 600                 605 


 


Asn Lys Arg Ile Val Leu Leu Glu Phe Ser Leu Ser Ser Leu Ala Lys 


    610                 615                 620 


 


Arg Met Lys Gln Leu Gln His Leu Lys Ala Gln Asn Lys His Glu Leu 


625                 630                 635                 640 


 


Ser Tyr Arg Lys Phe Arg Ala Lys Ile Cys Pro Gly Glu Asn Gln Ala 


                645                 650                 655 


 


Ala Glu Asp Glu Leu Arg Lys Glu Ile Ser Lys Ser Met Phe Ala Glu 


            660                 665                 670 


 


Met Glu Ile Leu Gly Gln Phe Asn Leu Gly Phe Ile Val Thr Lys Leu 


        675                 680                 685 


 


Lys Glu Asp Leu Phe Leu Val Asp Gln His Ala Ala Asp Glu Lys Tyr 


    690                 695                 700 


 


Asn Phe Glu Met Leu Gln Gln His Thr Val Leu Gln Ala Gln Arg Leu 


705                 710                 715                 720 


 


Ile Thr Pro Gln Thr Leu Asn Leu Thr Ala Val Asn Glu Ala Val Leu 


                725                 730                 735 


 


Ile Glu Asn Leu Glu Ile Phe Arg Lys Asn Gly Phe Asp Phe Val Ile 


            740                 745                 750 


 


Asp Glu Asp Ala Pro Val Thr Glu Arg Ala Lys Leu Ile Ser Leu Pro 


        755                 760                 765 


 


Thr Ser Lys Asn Trp Thr Phe Gly Pro Gln Asp Ile Asp Glu Leu Ile 


    770                 775                 780 


 


Phe Met Leu Ser Asp Ser Pro Gly Val Met Cys Arg Pro Ser Arg Val 


785                 790                 795                 800 


 


Arg Gln Met Phe Ala Ser Arg Ala Cys Arg Lys Ser Val Met Ile Gly 


                805                 810                 815 


 


Thr Ala Leu Asn Ala Ser Glu Met Lys Lys Leu Ile Thr His Met Gly 


            820                 825                 830 


 


Glu Met Asp His Pro Trp Asn Cys Pro His Gly Arg Pro Thr Met Arg 


        835                 840                 845 


 


His Val Ala Asn Leu Asp Val Ile Ser Gln Asn 


    850                 855 


 


 


<210> SEQ ID NO 17 


<211> LENGTH: 932 


<212> TYPE: PRT 


<213> ORGANISM: Homo sapiens 


 


<400> SEQUENCE: 17 


 


Met Lys Gln Leu Pro Ala Ala Thr Val Arg Leu Leu Ser Ser Ser Gln 


 1               5                  10                  15 


 


Ile Ile Thr Ser Val Val Ser Val Val Lys Glu Leu Ile Glu Asn Ser 


            20                  25                  30 


 


Leu Asp Ala Gly Ala Thr Ser Val Asp Val Lys Leu Glu Asn Tyr Gly 


        35                  40                  45 


 


Phe Asp Lys Ile Glu Val Arg Asp Asn Gly Glu Gly Ile Lys Ala Val 


    50                  55                  60 


 


Asp Ala Pro Val Met Ala Met Lys Tyr Tyr Thr Ser Lys Ile Asn Ser 


65                  70                  75                  80 


 


His Glu Asp Leu Glu Asn Leu Thr Thr Tyr Gly Phe Arg Gly Glu Ala 


                85                  90                  95 


 


Leu Gly Ser Ile Cys Cys Ile Ala Glu Val Leu Ile Thr Thr Arg Thr 


            100                 105                 110 


 


Ala Ala Asp Asn Phe Ser Thr Gln Tyr Val Leu Asp Gly Ser Gly His 


        115                 120                 125 


 


Ile Leu Ser Gln Lys Pro Ser His Leu Gly Gln Gly Thr Thr Val Thr 


    130                 135                 140 


 


Ala Leu Arg Leu Phe Lys Asn Leu Pro Val Arg Lys Gln Phe Tyr Ser 


145                 150                 155                 160 


 


Thr Ala Lys Lys Cys Lys Asp Glu Ile Lys Lys Ile Gln Asp Leu Leu 


                165                 170                 175 


 


Met Ser Phe Gly Ile Leu Lys Pro Asp Leu Arg Ile Val Phe Val His 


            180                 185                 190 


 


Asn Lys Ala Val Ile Trp Gln Lys Ser Arg Val Ser Asp His Lys Met 


        195                 200                 205 


 


Ala Leu Met Ser Val Leu Gly Thr Ala Val Met Asn Asn Met Glu Ser 


    210                 215                 220 


 


Phe Gln Tyr His Ser Glu Glu Ser Gln Ile Tyr Leu Ser Gly Phe Leu 


225                 230                 235                 240 


 


Pro Lys Cys Asp Ala Asp His Ser Phe Thr Ser Leu Ser Thr Pro Glu 


                245                 250                 255 


 


Arg Ser Phe Ile Phe Ile Asn Ser Arg Pro Val His Gln Lys Asp Ile 


            260                 265                 270 


 


Leu Lys Leu Ile Arg His His Tyr Asn Leu Lys Cys Leu Lys Glu Ser 


        275                 280                 285 


 


Thr Arg Leu Tyr Pro Val Phe Phe Leu Lys Ile Asp Val Pro Thr Ala 


    290                 295                 300 


 


Asp Val Asp Val Asn Leu Thr Pro Asp Lys Ser Gln Val Leu Leu Gln 


305                 310                 315                 320 


 


Asn Lys Glu Ser Val Leu Ile Ala Leu Glu Asn Leu Met Thr Thr Cys 


                325                 330                 335 


 


Tyr Gly Pro Leu Pro Ser Thr Asn Ser Tyr Glu Asn Asn Lys Thr Asp 


            340                 345                 350 


 


Val Ser Ala Ala Asp Ile Val Leu Ser Lys Thr Ala Glu Thr Asp Val 


        355                 360                 365 


 


Leu Phe Asn Lys Val Glu Ser Ser Gly Lys Asn Tyr Ser Asn Val Asp 


    370                 375                 380 


 


Thr Ser Val Ile Pro Phe Gln Asn Asp Met His Asn Asp Glu Ser Gly 


385                 390                 395                 400 


 


Lys Asn Thr Asp Asp Cys Leu Asn His Gln Ile Ser Ile Gly Asp Phe 


                405                 410                 415 


 


Gly Tyr Gly His Cys Ser Ser Glu Ile Ser Asn Ile Asp Lys Asn Thr 


            420                 425                 430 


 


Lys Asn Ala Phe Gln Asp Ile Ser Met Ser Asn Val Ser Trp Glu Asn 


        435                 440                 445 


 


Ser Gln Thr Glu Tyr Ser Lys Thr Cys Phe Ile Ser Ser Val Lys His 


    450                 455                 460 


 


Thr Gln Ser Glu Asn Gly Asn Lys Asp His Ile Asp Glu Ser Gly Glu 


465                 470                 475                 480 


 


Asn Glu Glu Glu Ala Gly Leu Glu Asn Ser Ser Glu Ile Ser Ala Asp 


                485                 490                 495 


 


Glu Trp Ser Arg Gly Asn Ile Leu Lys Asn Ser Val Gly Glu Asn Ile 


            500                 505                 510 


 


Glu Pro Val Lys Ile Leu Val Pro Glu Lys Ser Leu Pro Cys Lys Val 


        515                 520                 525 


 


Ser Asn Asn Asn Tyr Pro Ile Pro Glu Gln Met Asn Leu Asn Glu Asp 


    530                 535                 540 


 


Ser Cys Asn Lys Lys Ser Asn Val Ile Asp Asn Lys Ser Gly Lys Val 


545                 550                 555                 560 


 


Thr Ala Tyr Asp Leu Leu Ser Asn Arg Val Ile Lys Lys Pro Met Ser 


                565                 570                 575 


 


Ala Ser Ala Leu Phe Val Gln Asp His Arg Pro Gln Phe Leu Ile Glu 


            580                 585                 590 


 


Asn Pro Lys Thr Ser Leu Glu Asp Ala Thr Leu Gln Ile Glu Glu Leu 


        595                 600                 605 


 


Trp Lys Thr Leu Ser Glu Glu Glu Lys Leu Lys Tyr Glu Glu Lys Ala 


    610                 615                 620 


 


Thr Lys Asp Leu Glu Arg Tyr Asn Ser Gln Met Lys Arg Ala Ile Glu 


625                 630                 635                 640 


 


Gln Glu Ser Gln Met Ser Leu Lys Asp Gly Arg Lys Lys Ile Lys Pro 


                645                 650                 655 


 


Thr Ser Ala Trp Asn Leu Ala Gln Lys His Lys Leu Lys Thr Ser Leu 


            660                 665                 670 


 


Ser Asn Gln Pro Lys Leu Asp Glu Leu Leu Gln Ser Gln Ile Glu Lys 


        675                 680                 685 


 


Arg Arg Ser Gln Asn Ile Lys Met Val Gln Ile Pro Phe Ser Met Lys 


    690                 695                 700 


 


Asn Leu Lys Ile Asn Phe Lys Lys Gln Asn Lys Val Asp Leu Glu Glu 


705                 710                 715                 720 


 


Lys Asp Glu Pro Cys Leu Ile His Asn Leu Arg Phe Pro Asp Ala Trp 


                725                 730                 735 


 


Leu Met Thr Ser Lys Thr Glu Val Met Leu Leu Asn Pro Tyr Arg Val 


            740                 745                 750 


 


Glu Glu Ala Leu Leu Phe Lys Arg Leu Leu Glu Asn His Lys Leu Pro 


        755                 760                 765 


 


Ala Glu Pro Leu Glu Lys Pro Ile Met Leu Thr Glu Ser Leu Phe Asn 


    770                 775                 780 


 


Gly Ser His Tyr Leu Asp Val Leu Tyr Lys Met Thr Ala Asp Asp Gln 


785                 790                 795                 800 


 


Arg Tyr Ser Gly Ser Thr Tyr Leu Ser Asp Pro Arg Leu Thr Ala Asn 


                805                 810                 815 


 


Gly Phe Lys Ile Lys Leu Ile Pro Gly Val Ser Ile Thr Glu Asn Tyr 


            820                 825                 830 


 


Leu Glu Ile Glu Gly Met Ala Asn Cys Leu Pro Phe Tyr Gly Val Ala 


        835                 840                 845 


 


Asp Leu Lys Glu Ile Leu Asn Ala Ile Leu Asn Arg Asn Ala Lys Glu 


    850                 855                 860 


 


Val Tyr Glu Cys Arg Pro Arg Lys Val Ile Ser Tyr Leu Glu Gly Glu 


865                 870                 875                 880 


 


Ala Val Arg Leu Ser Arg Gln Leu Pro Met Tyr Leu Ser Lys Glu Asp 


                885                 890                 895 


 


Ile Gln Asp Ile Ile Tyr Arg Met Lys His Gln Phe Gly Asn Glu Ile 


            900                 905                 910 


 


Lys Glu Cys Val His Gly Arg Pro Phe Phe His His Leu Thr Tyr Leu 


        915                 920                 925 


 


Pro Glu Thr Thr 


    930 


 


 


<210> SEQ ID NO 18 


<211> LENGTH: 932 


<212> TYPE: PRT 


<213> ORGANISM: Homo sapiens 


 


<400> SEQUENCE: 18 


 


Met Lys Gln Leu Pro Ala Ala Thr Val Arg Leu Leu Ser Ser Ser Gln 


 1               5                  10                  15 


 


Ile Ile Thr Ser Val Val Ser Val Val Lys Glu Leu Ile Glu Asn Ser 


            20                  25                  30 


 


Leu Asp Ala Gly Ala Thr Ser Val Asp Val Lys Leu Glu Asn Tyr Gly 


        35                  40                  45 


 


Phe Asp Lys Ile Glu Val Arg Asp Asn Gly Glu Gly Ile Lys Ala Val 


    50                  55                  60 


 


Asp Ala Pro Val Met Ala Met Lys Tyr Tyr Thr Ser Lys Ile Asn Ser 


65                  70                  75                  80 


 


His Glu Asp Leu Glu Asn Leu Thr Thr Tyr Gly Phe Arg Gly Glu Ala 


                85                  90                  95 


 


Leu Gly Ser Ile Cys Cys Ile Ala Glu Val Leu Ile Thr Thr Arg Thr 


            100                 105                 110 


 


Ala Ala Asp Asn Phe Ser Thr Gln Tyr Val Leu Asp Gly Ser Gly His 


        115                 120                 125 


 


Ile Leu Ser Gln Lys Pro Ser His Leu Gly Gln Gly Thr Thr Val Thr 


    130                 135                 140 


 


Ala Leu Arg Leu Phe Lys Asn Leu Pro Val Arg Lys Gln Phe Tyr Ser 


145                 150                 155                 160 


 


Thr Ala Lys Lys Cys Lys Asp Glu Ile Lys Lys Ile Gln Asp Leu Leu 


                165                 170                 175 


 


Met Ser Phe Gly Ile Leu Lys Pro Asp Leu Arg Ile Val Phe Val His 


            180                 185                 190 


 


Asn Lys Ala Val Ile Trp Gln Lys Ser Arg Val Ser Asp His Lys Met 


        195                 200                 205 


 


Ala Leu Met Ser Val Leu Gly Thr Ala Val Met Asn Asn Met Glu Ser 


    210                 215                 220 


 


Phe Gln Tyr His Ser Glu Glu Ser Gln Ile Tyr Leu Ser Gly Phe Leu 


225                 230                 235                 240 


 


Pro Lys Cys Asp Ala Asp His Ser Phe Thr Ser Leu Ser Thr Pro Glu 


                245                 250                 255 


 


Arg Ser Phe Ile Phe Ile Asn Ser Arg Pro Val His Gln Lys Asp Ile 


            260                 265                 270 


 


Leu Lys Leu Ile Arg His His Tyr Asn Leu Lys Cys Leu Lys Glu Ser 


        275                 280                 285 


 


Thr Arg Leu Tyr Pro Val Phe Phe Leu Lys Ile Asp Val Pro Thr Ala 


    290                 295                 300 


 


Asp Val Asp Val Asn Leu Thr Pro Asp Lys Ser Gln Val Leu Leu Gln 


305                 310                 315                 320 


 


Asn Lys Glu Ser Val Leu Ile Ala Leu Glu Asn Leu Met Thr Thr Cys 


                325                 330                 335 


 


Tyr Gly Pro Leu Pro Ser Thr Asn Ser Tyr Glu Asn Asn Lys Thr Asp 


            340                 345                 350 


 


Val Ser Ala Ala Asp Ile Val Leu Ser Lys Thr Ala Glu Thr Asp Val 


        355                 360                 365 


 


Leu Phe Asn Lys Val Glu Ser Ser Gly Lys Asn Tyr Ser Asn Val Asp 


    370                 375                 380 


 


Thr Ser Val Ile Pro Phe Gln Asn Asp Met His Asn Asp Glu Ser Gly 


385                 390                 395                 400 


 


Lys Asn Thr Asp Asp Cys Leu Asn His Gln Ile Ser Ile Gly Asp Phe 


                405                 410                 415 


 


Gly Tyr Gly His Cys Ser Ser Glu Ile Ser Asn Ile Asp Lys Asn Thr 


            420                 425                 430 


 


Lys Asn Ala Phe Gln Asp Ile Ser Met Ser Asn Val Ser Trp Glu Asn 


        435                 440                 445 


 


Ser Gln Thr Glu Tyr Ser Lys Thr Cys Phe Ile Ser Ser Val Lys His 


    450                 455                 460 


 


Thr Gln Ser Glu Asn Gly Asn Lys Asp His Ile Asp Glu Ser Gly Glu 


465                 470                 475                 480 


 


Asn Glu Glu Glu Ala Gly Leu Glu Asn Ser Ser Glu Ile Ser Ala Asp 


                485                 490                 495 


 


Glu Trp Ser Arg Gly Asn Ile Leu Lys Asn Ser Val Gly Glu Asn Ile 


            500                 505                 510 


 


Glu Pro Val Lys Ile Leu Val Pro Glu Lys Ser Leu Pro Cys Lys Val 


        515                 520                 525 


 


Ser Asn Asn Asn Tyr Pro Ile Pro Glu Gln Met Asn Leu Asn Glu Asp 


    530                 535                 540 


 


Ser Cys Asn Lys Lys Ser Asn Val Ile Asp Asn Lys Ser Gly Lys Val 


545                 550                 555                 560 


 


Thr Ala Tyr Asp Leu Leu Ser Asn Arg Val Ile Lys Lys Pro Met Ser 


                565                 570                 575 


 


Ala Ser Ala Leu Phe Val Gln Asp His Arg Pro Gln Phe Leu Ile Glu 


            580                 585                 590 


 


Asn Pro Lys Thr Ser Leu Glu Asp Ala Thr Leu Gln Ile Glu Glu Leu 


        595                 600                 605 


 


Trp Lys Thr Leu Ser Glu Glu Glu Lys Leu Lys Tyr Glu Glu Lys Ala 


    610                 615                 620 


 


Thr Lys Asp Leu Glu Arg Tyr Asn Ser Gln Met Lys Arg Ala Ile Glu 


625                 630                 635                 640 


 


Gln Glu Ser Gln Met Ser Leu Lys Asp Gly Arg Lys Lys Ile Lys Pro 


                645                 650                 655 


 


Thr Ser Ala Trp Asn Leu Ala Gln Lys His Lys Leu Lys Thr Ser Leu 


            660                 665                 670 


 


Ser Asn Gln Pro Lys Leu Asp Glu Leu Leu Gln Ser Gln Ile Glu Lys 


        675                 680                 685 


 


Arg Arg Ser Gln Asn Ile Lys Met Val Gln Ile Pro Phe Ser Met Lys 


    690                 695                 700 


 


Asn Leu Lys Ile Asn Phe Lys Lys Gln Asn Lys Val Asp Leu Glu Glu 


705                 710                 715                 720 


 


Lys Asp Glu Pro Cys Leu Ile His Asn Leu Arg Phe Pro Asp Ala Trp 


                725                 730                 735 


 


Leu Met Thr Ser Lys Thr Glu Val Met Leu Leu Asn Pro Tyr Arg Val 


            740                 745                 750 


 


Glu Glu Ala Leu Leu Phe Lys Arg Leu Leu Glu Asn His Lys Leu Pro 


        755                 760                 765 


 


Ala Glu Pro Leu Glu Lys Pro Ile Met Leu Thr Glu Ser Leu Phe Asn 


    770                 775                 780 


 


Gly Ser His Tyr Leu Asp Val Leu Tyr Lys Met Thr Ala Asp Asp Gln 


785                 790                 795                 800 


 


Arg Tyr Ser Gly Ser Thr Tyr Leu Ser Asp Pro Arg Leu Thr Ala Asn 


                805                 810                 815 


 


Gly Phe Lys Ile Lys Leu Ile Pro Gly Val Ser Ile Thr Glu Asn Tyr 


            820                 825                 830 


 


Leu Glu Ile Glu Gly Met Ala Asn Cys Leu Pro Phe Tyr Gly Val Ala 


        835                 840                 845 


 


Asp Leu Lys Glu Ile Leu Asn Ala Ile Leu Asn Arg Asn Ala Lys Glu 


    850                 855                 860 


 


Val Tyr Glu Cys Arg Pro Arg Lys Val Ile Ser Tyr Leu Glu Gly Glu 


865                 870                 875                 880 


 


Ala Val Arg Leu Ser Arg Gln Leu Pro Met Tyr Leu Ser Lys Glu Asp 


                885                 890                 895 


 


Ile Gln Asp Ile Ile Tyr Arg Met Lys His Gln Phe Gly Asn Glu Ile 


            900                 905                 910 


 


Lys Glu Cys Val His Gly Arg Pro Phe Phe His His Leu Thr Tyr Leu 


        915                 920                 925 


 


Pro Glu Thr Thr 


    930 


 


 


<210> SEQ ID NO 19 


<211> LENGTH: 934 


<212> TYPE: PRT 


<213> ORGANISM: Homo sapiens 


 


<400> SEQUENCE: 19 


 


Met Ala Val Gln Pro Lys Glu Thr Leu Gln Leu Glu Ser Ala Ala Glu 


 1               5                  10                  15 


 


Val Gly Phe Val Arg Phe Phe Gln Gly Met Pro Glu Lys Pro Thr Thr 


            20                  25                  30 


 


Thr Val Arg Leu Phe Asp Arg Gly Asp Phe Tyr Thr Ala His Gly Glu 


        35                  40                  45 


 


Asp Ala Leu Leu Ala Ala Arg Glu Val Phe Lys Thr Gln Gly Val Ile 


    50                  55                  60 


 


Lys Tyr Met Gly Pro Ala Gly Ala Lys Asn Leu Gln Ser Val Val Leu 


65                  70                  75                  80 


 


Ser Lys Met Asn Phe Glu Ser Phe Val Lys Asp Leu Leu Leu Val Arg 


                85                  90                  95 


 


Gln Tyr Arg Val Glu Val Tyr Lys Asn Arg Ala Gly Asn Lys Ala Ser 


            100                 105                 110 


 


Lys Glu Asn Asp Trp Tyr Leu Ala Tyr Lys Ala Ser Pro Gly Asn Leu 


        115                 120                 125 


 


Ser Gln Phe Glu Asp Ile Leu Phe Gly Asn Asn Asp Met Ser Ala Ser 


    130                 135                 140 


 


Ile Gly Val Val Gly Val Lys Met Ser Ala Val Asp Gly Gln Arg Gln 


145                 150                 155                 160 


 


Val Gly Val Gly Tyr Val Asp Ser Ile Gln Arg Lys Leu Gly Leu Cys 


                165                 170                 175 


 


Glu Phe Pro Asp Asn Asp Gln Phe Ser Asn Leu Glu Ala Leu Leu Ile 


            180                 185                 190 


 


Gln Ile Gly Pro Lys Glu Cys Val Leu Pro Gly Gly Glu Thr Ala Gly 


        195                 200                 205 


 


Asp Met Gly Lys Leu Arg Gln Ile Ile Gln Arg Gly Gly Ile Leu Ile 


    210                 215                 220 


 


Thr Glu Arg Lys Lys Ala Asp Phe Ser Thr Lys Asp Ile Tyr Gln Asp 


225                 230                 235                 240 


 


Leu Asn Arg Leu Leu Lys Gly Lys Lys Gly Glu Gln Met Asn Ser Ala 


                245                 250                 255 


 


Val Leu Pro Glu Met Glu Asn Gln Val Ala Val Ser Ser Leu Ser Ala 


            260                 265                 270 


 


Val Ile Lys Phe Leu Glu Leu Leu Ser Asp Asp Ser Asn Phe Gly Gln 


        275                 280                 285 


 


Phe Glu Leu Thr Thr Phe Asp Phe Ser Gln Tyr Met Lys Leu Asp Ile 


    290                 295                 300 


 


Ala Ala Val Arg Ala Leu Asn Leu Phe Gln Gly Ser Val Glu Asp Thr 


305                 310                 315                 320 


 


Thr Gly Ser Gln Ser Leu Ala Ala Leu Leu Asn Lys Cys Lys Thr Pro 


                325                 330                 335 


 


Gln Gly Gln Arg Leu Val Asn Gln Trp Ile Lys Gln Pro Leu Met Asp 


            340                 345                 350 


 


Lys Asn Arg Ile Glu Glu Arg Leu Asn Leu Val Glu Ala Phe Val Glu 


        355                 360                 365 


 


Asp Ala Glu Leu Arg Gln Thr Leu Gln Glu Asp Leu Leu Arg Arg Phe 


    370                 375                 380 


 


Pro Asp Leu Asn Arg Leu Ala Lys Lys Phe Gln Arg Gln Ala Ala Asn 


385                 390                 395                 400 


 


Leu Gln Asp Cys Tyr Arg Leu Tyr Gln Gly Ile Asn Gln Leu Pro Asn 


                405                 410                 415 


 


Val Ile Gln Ala Leu Glu Lys His Glu Gly Lys His Gln Lys Leu Leu 


            420                 425                 430 


 


Leu Ala Val Phe Val Thr Pro Leu Thr Asp Leu Arg Ser Asp Phe Ser 


        435                 440                 445 


 


Lys Phe Gln Glu Met Ile Glu Thr Thr Leu Asp Met Asp Gln Val Glu 


    450                 455                 460 


 


Asn His Glu Phe Leu Val Lys Pro Ser Phe Asp Pro Asn Leu Ser Glu 


465                 470                 475                 480 


 


Leu Arg Glu Ile Met Asn Asp Leu Glu Lys Lys Met Gln Ser Thr Leu 


                485                 490                 495 


 


Ile Ser Ala Ala Arg Asp Leu Gly Leu Asp Pro Gly Lys Gln Ile Lys 


            500                 505                 510 


 


Leu Asp Ser Ser Ala Gln Phe Gly Tyr Tyr Phe Arg Val Thr Cys Lys 


        515                 520                 525 


 


Glu Glu Lys Val Leu Arg Asn Asn Lys Asn Phe Ser Thr Val Asp Ile 


    530                 535                 540 


 


Gln Lys Asn Gly Val Lys Phe Thr Asn Ser Lys Leu Thr Ser Leu Asn 


545                 550                 555                 560 


 


Glu Glu Tyr Thr Lys Asn Lys Thr Glu Tyr Glu Glu Ala Gln Asp Ala 


                565                 570                 575 


 


Ile Val Lys Glu Ile Val Asn Ile Ser Ser Gly Tyr Val Glu Pro Met 


            580                 585                 590 


 


Gln Thr Leu Asn Asp Val Leu Ala Gln Leu Asp Ala Val Val Ser Phe 


        595                 600                 605 


 


Ala His Val Ser Asn Gly Ala Pro Val Pro Tyr Val Arg Pro Ala Ile 


    610                 615                 620 


 


Leu Glu Lys Gly Gln Gly Arg Ile Ile Leu Lys Ala Ser Arg His Ala 


625                 630                 635                 640 


 


Cys Val Glu Val Gln Asp Glu Ile Ala Phe Ile Pro Asn Asp Val Tyr 


                645                 650                 655 


 


Phe Glu Lys Asp Lys Gln Met Phe His Ile Ile Thr Gly Pro Asn Met 


            660                 665                 670 


 


Gly Gly Lys Ser Thr Tyr Ile Arg Gln Thr Gly Val Ile Val Leu Met 


        675                 680                 685 


 


Ala Gln Ile Gly Cys Phe Val Pro Cys Glu Ser Ala Glu Val Ser Ile 


    690                 695                 700 


 


Val Asp Cys Ile Leu Ala Arg Val Gly Ala Gly Asp Ser Gln Leu Lys 


705                 710                 715                 720 


 


Gly Val Ser Thr Phe Met Ala Glu Met Leu Glu Thr Ala Ser Ile Leu 


                725                 730                 735 


 


Arg Ser Ala Thr Lys Asp Ser Leu Ile Ile Ile Asp Glu Leu Gly Arg 


            740                 745                 750 


 


Gly Thr Ser Thr Tyr Asp Gly Phe Gly Leu Ala Trp Ala Ile Ser Glu 


        755                 760                 765 


 


Tyr Ile Ala Thr Lys Ile Gly Ala Phe Cys Met Phe Ala Thr His Phe 


    770                 775                 780 


 


His Glu Leu Thr Ala Leu Ala Asn Gln Ile Pro Thr Val Asn Asn Leu 


785                 790                 795                 800 


 


His Val Thr Ala Leu Thr Thr Glu Glu Thr Leu Thr Met Leu Tyr Gln 


                805                 810                 815 


 


Val Lys Lys Gly Val Cys Asp Gln Ser Phe Gly Ile His Val Ala Glu 


            820                 825                 830 


 


Leu Ala Asn Phe Pro Lys His Val Ile Glu Cys Ala Lys Gln Lys Ala 


        835                 840                 845 


 


Leu Glu Leu Glu Glu Phe Gln Tyr Ile Gly Glu Ser Gln Gly Tyr Asp 


    850                 855                 860 


 


Ile Met Glu Pro Ala Ala Lys Lys Cys Tyr Leu Glu Arg Glu Gln Gly 


865                 870                 875                 880 


 


Glu Lys Ile Ile Gln Glu Phe Leu Ser Lys Val Lys Gln Met Pro Phe 


                885                 890                 895 


 


Thr Glu Met Ser Glu Glu Asn Ile Thr Ile Lys Leu Lys Gln Leu Lys 


            900                 905                 910 


 


Ala Glu Val Ile Ala Lys Asn Asn Ser Phe Val Asn Glu Ile Ile Ser 


        915                 920                 925 


 


Arg Ile Lys Val Thr Thr 


    930 


 


 


<210> SEQ ID NO 20 


<211> LENGTH: 756 


<212> TYPE: PRT 


<213> ORGANISM: Homo sapiens 


 


<400> SEQUENCE: 20 


 


Met Ser Phe Val Ala Gly Val Ile Arg Arg Leu Asp Glu Thr Val Val 


 1               5                  10                  15 


 


Asn Arg Ile Ala Ala Gly Glu Val Ile Gln Arg Pro Ala Asn Ala Ile 


            20                  25                  30 


 


Lys Glu Met Ile Glu Asn Cys Leu Asp Ala Lys Ser Thr Ser Ile Gln 


        35                  40                  45 


 


Val Ile Val Lys Glu Gly Gly Leu Lys Leu Ile Gln Ile Gln Asp Asn 


    50                  55                  60 


 


Gly Thr Gly Ile Arg Lys Glu Asp Leu Asp Ile Val Cys Glu Arg Phe 


65                  70                  75                  80 


 


Thr Thr Ser Lys Leu Gln Ser Phe Glu Asp Leu Ala Ser Ile Ser Thr 


                85                  90                  95 


 


Tyr Gly Phe Arg Gly Glu Ala Leu Ala Ser Ile Ser His Val Ala His 


            100                 105                 110 


 


Val Thr Ile Thr Thr Lys Thr Ala Asp Gly Lys Cys Ala Tyr Arg Ala 


        115                 120                 125 


 


Ser Tyr Ser Asp Gly Lys Leu Lys Ala Pro Pro Lys Pro Cys Ala Gly 


    130                 135                 140 


 


Asn Gln Gly Thr Gln Ile Thr Val Glu Asp Leu Phe Tyr Asn Ile Ala 


145                 150                 155                 160 


 


Thr Arg Arg Lys Ala Leu Lys Asn Pro Ser Glu Glu Tyr Gly Lys Ile 


                165                 170                 175 


 


Leu Glu Val Val Gly Arg Tyr Ser Val His Asn Ala Gly Ile Ser Phe 


            180                 185                 190 


 


Ser Val Lys Lys Gln Gly Glu Thr Val Ala Asp Val Arg Thr Leu Pro 


        195                 200                 205 


 


Asn Ala Ser Thr Val Asp Asn Ile Arg Ser Ile Phe Gly Asn Ala Val 


    210                 215                 220 


 


Ser Arg Glu Leu Ile Glu Ile Gly Cys Glu Asp Lys Thr Leu Ala Phe 


225                 230                 235                 240 


 


Lys Met Asn Gly Tyr Ile Ser Asn Ala Asn Tyr Ser Val Lys Lys Cys 


                245                 250                 255 


 


Ile Phe Leu Leu Phe Ile Asn His Arg Leu Val Glu Ser Thr Ser Leu 


            260                 265                 270 


 


Arg Lys Ala Ile Glu Thr Val Tyr Ala Ala Tyr Leu Pro Lys Asn Thr 


        275                 280                 285 


 


His Pro Phe Leu Tyr Leu Ser Leu Glu Ile Ser Pro Gln Asn Val Asp 


    290                 295                 300 


 


Val Asn Val His Pro Thr Lys His Glu Val His Phe Leu His Glu Glu 


305                 310                 315                 320 


 


Ser Ile Leu Glu Arg Val Gln Gln His Ile Glu Ser Lys Leu Leu Gly 


                325                 330                 335 


 


Ser Asn Ser Ser Arg Met Tyr Phe Thr Gln Thr Leu Leu Pro Gly Leu 


            340                 345                 350 


 


Ala Gly Pro Ser Gly Glu Met Val Lys Ser Thr Thr Ser Leu Thr Ser 


        355                 360                 365 


 


Ser Ser Thr Ser Gly Ser Ser Asp Lys Val Tyr Ala His Gln Met Val 


    370                 375                 380 


 


Arg Thr Asp Ser Arg Glu Gln Lys Leu Asp Ala Phe Leu Gln Pro Leu 


385                 390                 395                 400 


 


Ser Lys Pro Leu Ser Ser Gln Pro Gln Ala Ile Val Thr Glu Asp Lys 


                405                 410                 415 


 


Thr Asp Ile Ser Ser Gly Arg Ala Arg Gln Gln Asp Glu Glu Met Leu 


            420                 425                 430 


 


Glu Leu Pro Ala Pro Ala Glu Val Ala Ala Lys Asn Gln Ser Leu Glu 


        435                 440                 445 


 


Gly Asp Thr Thr Lys Gly Thr Ser Glu Met Ser Glu Lys Arg Gly Pro 


    450                 455                 460 


 


Thr Ser Ser Asn Pro Arg Lys Arg His Arg Glu Asp Ser Asp Val Glu 


465                 470                 475                 480 


 


Met Val Glu Asp Asp Ser Arg Lys Glu Met Thr Ala Ala Cys Thr Pro 


                485                 490                 495 


 


Arg Arg Arg Ile Ile Asn Leu Thr Ser Val Leu Ser Leu Gln Glu Glu 


            500                 505                 510 


 


Ile Asn Glu Gln Gly His Glu Val Leu Arg Glu Met Leu His Asn His 


        515                 520                 525 


 


Ser Phe Val Gly Cys Val Asn Pro Gln Trp Ala Leu Ala Gln His Gln 


    530                 535                 540 


 


Thr Lys Leu Tyr Leu Leu Asn Thr Thr Lys Leu Ser Glu Glu Leu Phe 


545                 550                 555                 560 


 


Tyr Gln Ile Leu Ile Tyr Asp Phe Ala Asn Phe Gly Val Leu Arg Leu 


                565                 570                 575 


 


Ser Glu Pro Ala Pro Leu Phe Asp Leu Ala Met Leu Ala Leu Asp Ser 


            580                 585                 590 


 


Pro Glu Ser Gly Trp Thr Glu Glu Asp Gly Pro Lys Glu Gly Leu Ala 


        595                 600                 605 


 


Glu Tyr Ile Val Glu Phe Leu Lys Lys Lys Ala Glu Met Leu Ala Asp 


    610                 615                 620 


 


Tyr Phe Ser Leu Glu Ile Asp Glu Glu Gly Asn Leu Ile Gly Leu Pro 


625                 630                 635                 640 


 


Leu Leu Ile Asp Asn Tyr Val Pro Pro Leu Glu Gly Leu Pro Ile Phe 


                645                 650                 655 


 


Ile Leu Arg Leu Ala Thr Glu Val Asn Trp Asp Glu Glu Lys Glu Cys 


            660                 665                 670 


 


Phe Glu Ser Leu Ser Lys Glu Cys Ala Met Phe Tyr Ser Ile Arg Lys 


        675                 680                 685 


 


Gln Tyr Ile Ser Glu Glu Ser Thr Leu Ser Gly Gln Gln Ser Glu Val 


    690                 695                 700 


 


Pro Gly Ser Ile Pro Asn Ser Trp Lys Trp Thr Val Glu His Ile Val 


705                 710                 715                 720 


 


Tyr Lys Ala Leu Arg Ser His Ile Leu Pro Pro Lys His Phe Thr Glu 


                725                 730                 735 


 


Asp Gly Asn Ile Leu Gln Leu Ala Asn Leu Pro Asp Leu Tyr Lys Val 


            740                 745                 750 


 


Phe Glu Arg Cys 


        755 


 


 


<210> SEQ ID NO 21 


<211> LENGTH: 133 


<212> TYPE: PRT 


<213> ORGANISM: Homo sapiens 


 


<400> SEQUENCE: 21 


 


Met Lys Gln Leu Pro Ala Ala Thr Val Arg Leu Leu Ser Ser Ser Gln 


 1               5                  10                  15 


 


Ile Ile Thr Ser Val Val Ser Val Val Lys Glu Leu Ile Glu Asn Ser 


            20                  25                  30 


 


Leu Asp Ala Gly Ala Thr Ser Val Asp Val Lys Leu Glu Asn Tyr Gly 


        35                  40                  45 


 


Phe Asp Lys Ile Glu Val Arg Asp Asn Gly Glu Gly Ile Lys Ala Val 


    50                  55                  60 


 


Asp Ala Pro Val Met Ala Met Lys Tyr Tyr Thr Ser Lys Ile Asn Ser 


65                  70                  75                  80 


 


His Glu Asp Leu Glu Asn Leu Thr Thr Tyr Gly Phe Arg Gly Glu Ala 


                85                  90                  95 


 


Leu Gly Ser Ile Cys Cys Ile Ala Glu Val Leu Ile Thr Thr Arg Thr 


            100                 105                 110 


 


Ala Ala Asp Asn Phe Ser Thr Gln Tyr Val Leu Asp Gly Ser Gly His 


        115                 120                 125 


 


Ile Leu Ser Gln Lys 


    130 


 


 


<210> SEQ ID NO 22 


<211> LENGTH: 1360 


<212> TYPE: PRT 


<213> ORGANISM: Homo sapiens 


 


<400> SEQUENCE: 22 


 


Met Ser Arg Gln Ser Thr Leu Tyr Ser Phe Phe Pro Lys Ser Pro Ala 


 1               5                  10                  15 


 


Leu Ser Asp Ala Asn Lys Ala Ser Ala Arg Ala Ser Arg Glu Gly Gly 


            20                  25                  30 


 


Arg Ala Ala Ala Ala Pro Gly Ala Ser Pro Ser Pro Gly Gly Asp Ala 


        35                  40                  45 


 


Ala Trp Ser Glu Ala Gly Pro Gly Pro Arg Pro Leu Ala Arg Ser Ala 


    50                  55                  60 


 


Ser Pro Pro Lys Ala Lys Asn Leu Asn Gly Gly Leu Arg Arg Ser Val 


65                  70                  75                  80 


 


Ala Pro Ala Ala Pro Thr Ser Cys Asp Phe Ser Pro Gly Asp Leu Val 


                85                  90                  95 


 


Trp Ala Lys Met Glu Gly Tyr Pro Trp Trp Pro Cys Leu Val Tyr Asn 


            100                 105                 110 


 


His Pro Phe Asp Gly Thr Phe Ile Arg Glu Lys Gly Lys Ser Val Arg 


        115                 120                 125 


 


Val His Val Gln Phe Phe Asp Asp Ser Pro Thr Arg Gly Trp Val Ser 


    130                 135                 140 


 


Lys Arg Leu Leu Lys Pro Tyr Thr Gly Ser Lys Ser Lys Glu Ala Gln 


145                 150                 155                 160 


 


Lys Gly Gly His Phe Tyr Ser Ala Lys Pro Glu Ile Leu Arg Ala Met 


                165                 170                 175 


 


Gln Arg Ala Asp Glu Ala Leu Asn Lys Asp Lys Ile Lys Arg Leu Glu 


            180                 185                 190 


 


Leu Ala Val Cys Asp Glu Pro Ser Glu Pro Glu Glu Glu Glu Glu Met 


        195                 200                 205 


 


Glu Val Gly Thr Thr Tyr Val Thr Asp Lys Ser Glu Glu Asp Asn Glu 


    210                 215                 220 


 


Ile Glu Ser Glu Glu Glu Val Gln Pro Lys Thr Gln Gly Ser Arg Arg 


225                 230                 235                 240 


 


Ser Ser Arg Gln Ile Lys Lys Arg Arg Val Ile Ser Asp Ser Glu Ser 


                245                 250                 255 


 


Asp Ile Gly Gly Ser Asp Val Glu Phe Lys Pro Asp Thr Lys Glu Glu 


            260                 265                 270 


 


Gly Ser Ser Asp Glu Ile Ser Ser Gly Val Gly Asp Ser Glu Ser Glu 


        275                 280                 285 


 


Gly Leu Asn Ser Pro Val Lys Val Ala Arg Lys Arg Lys Arg Met Val 


    290                 295                 300 


 


Thr Gly Asn Gly Ser Leu Lys Arg Lys Ser Ser Arg Lys Glu Thr Pro 


305                 310                 315                 320 


 


Ser Ala Thr Lys Gln Ala Thr Ser Ile Ser Ser Glu Thr Lys Asn Thr 


                325                 330                 335 


 


Leu Arg Ala Phe Ser Ala Pro Gln Asn Ser Glu Ser Gln Ala His Val 


            340                 345                 350 


 


Ser Gly Gly Gly Asp Asp Ser Ser Arg Pro Thr Val Trp Tyr His Glu 


        355                 360                 365 


 


Thr Leu Glu Trp Leu Lys Glu Glu Lys Arg Arg Asp Glu His Arg Arg 


    370                 375                 380 


 


Arg Pro Asp His Pro Asp Phe Asp Ala Ser Thr Leu Tyr Val Pro Glu 


385                 390                 395                 400 


 


Asp Phe Leu Asn Ser Cys Thr Pro Gly Met Arg Lys Trp Trp Gln Ile 


                405                 410                 415 


 


Lys Ser Gln Asn Phe Asp Leu Val Ile Cys Tyr Lys Val Gly Lys Phe 


            420                 425                 430 


 


Tyr Glu Leu Tyr His Met Asp Ala Leu Ile Gly Val Ser Glu Leu Gly 


        435                 440                 445 


 


Leu Val Phe Met Lys Gly Asn Trp Ala His Ser Gly Phe Pro Glu Ile 


    450                 455                 460 


 


Ala Phe Gly Arg Tyr Ser Asp Ser Leu Val Gln Lys Gly Tyr Lys Val 


465                 470                 475                 480 


 


Ala Arg Val Glu Gln Thr Glu Thr Pro Glu Met Met Glu Ala Arg Cys 


                485                 490                 495 


 


Arg Lys Met Ala His Ile Ser Lys Tyr Asp Arg Val Val Arg Arg Glu 


            500                 505                 510 


 


Ile Cys Arg Ile Ile Thr Lys Gly Thr Gln Thr Tyr Ser Val Leu Glu 


        515                 520                 525 


 


Gly Asp Pro Ser Glu Asn Tyr Ser Lys Tyr Leu Leu Ser Leu Lys Glu 


    530                 535                 540 


 


Lys Glu Glu Asp Ser Ser Gly His Thr Arg Ala Tyr Gly Val Cys Phe 


545                 550                 555                 560 


 


Val Asp Thr Ser Leu Gly Lys Phe Phe Ile Gly Gln Phe Ser Asp Asp 


                565                 570                 575 


 


Arg His Cys Ser Arg Phe Arg Thr Leu Val Ala His Tyr Pro Pro Val 


            580                 585                 590 


 


Gln Val Leu Phe Glu Lys Gly Asn Leu Ser Lys Glu Thr Lys Thr Ile 


        595                 600                 605 


 


Leu Lys Ser Ser Leu Ser Cys Ser Leu Gln Glu Gly Leu Ile Pro Gly 


    610                 615                 620 


 


Ser Gln Phe Trp Asp Ala Ser Lys Thr Leu Arg Thr Leu Leu Glu Glu 


625                 630                 635                 640 


 


Glu Tyr Phe Arg Glu Lys Leu Ser Asp Gly Ile Gly Val Met Leu Pro 


                645                 650                 655 


 


Gln Val Leu Lys Gly Met Thr Ser Glu Ser Asp Ser Ile Gly Leu Thr 


            660                 665                 670 


 


Pro Gly Glu Lys Ser Glu Leu Ala Leu Ser Ala Leu Gly Gly Cys Val 


        675                 680                 685 


 


Phe Tyr Leu Lys Lys Cys Leu Ile Asp Gln Glu Leu Leu Ser Met Ala 


    690                 695                 700 


 


Asn Phe Glu Glu Tyr Ile Pro Leu Asp Ser Asp Thr Val Ser Thr Thr 


705                 710                 715                 720 


 


Arg Ser Gly Ala Ile Phe Thr Lys Ala Tyr Gln Arg Met Val Leu Asp 


                725                 730                 735 


 


Ala Val Thr Leu Asn Asn Leu Glu Ile Phe Leu Asn Gly Thr Asn Gly 


            740                 745                 750 


 


Ser Thr Glu Gly Thr Leu Leu Glu Arg Val Asp Thr Cys His Thr Pro 


        755                 760                 765 


 


Phe Gly Lys Arg Leu Leu Lys Gln Trp Leu Cys Ala Pro Leu Cys Asn 


    770                 775                 780 


 


His Tyr Ala Ile Asn Asp Arg Leu Asp Ala Ile Glu Asp Leu Met Val 


785                 790                 795                 800 


 


Val Pro Asp Lys Ile Ser Glu Val Val Glu Leu Leu Lys Lys Leu Pro 


                805                 810                 815 


 


Asp Leu Glu Arg Leu Leu Ser Lys Ile His Asn Val Gly Ser Pro Leu 


            820                 825                 830 


 


Lys Ser Gln Asn His Pro Asp Ser Arg Ala Ile Met Tyr Glu Glu Thr 


        835                 840                 845 


 


Thr Tyr Ser Lys Lys Lys Ile Ile Asp Phe Leu Ser Ala Leu Glu Gly 


    850                 855                 860 


 


Phe Lys Val Met Cys Lys Ile Ile Gly Ile Met Glu Glu Val Ala Asp 


865                 870                 875                 880 


 


Gly Phe Lys Ser Lys Ile Leu Lys Gln Val Ile Ser Leu Gln Thr Lys 


                885                 890                 895 


 


Asn Pro Glu Gly Arg Phe Pro Asp Leu Thr Val Glu Leu Asn Arg Trp 


            900                 905                 910 


 


Asp Thr Ala Phe Asp His Glu Lys Ala Arg Lys Thr Gly Leu Ile Thr 


        915                 920                 925 


 


Pro Lys Ala Gly Phe Asp Ser Asp Tyr Asp Gln Ala Leu Ala Asp Ile 


    930                 935                 940 


 


Arg Glu Asn Glu Gln Ser Leu Leu Glu Tyr Leu Glu Lys Gln Arg Asn 


945                 950                 955                 960 


 


Arg Ile Gly Cys Arg Thr Ile Val Tyr Trp Gly Ile Gly Arg Asn Arg 


                965                 970                 975 


 


Tyr Gln Leu Glu Ile Pro Glu Asn Phe Thr Thr Arg Asn Leu Pro Glu 


            980                 985                 990 


 


Glu Tyr Glu Leu Lys Ser Thr Lys Lys Gly Cys Lys Arg Tyr Trp Thr 


        995                 1000                1005 


 


Lys Thr Ile Glu Lys Lys Leu Ala Asn Leu Ile Asn Ala Glu Glu Arg 


    1010                1015                1020 


 


Arg Asp Val Ser Leu Lys Asp Cys Met Arg Arg Leu Phe Tyr Asn Phe 


1025                1030                1035                1040 


 


Asp Lys Asn Tyr Lys Asp Trp Gln Ser Ala Val Glu Cys Ile Ala Val 


                1045                1050                1055 


 


Leu Asp Val Leu Leu Cys Leu Ala Asn Tyr Ser Arg Gly Gly Asp Gly 


            1060                1065                1070 


 


Pro Met Cys Arg Pro Val Ile Leu Leu Pro Glu Asp Thr Pro Pro Phe 


        1075                1080                1085 


 


Leu Glu Leu Lys Gly Ser Arg His Pro Cys Ile Thr Lys Thr Phe Phe 


    1090                1095                1100 


 


Gly Asp Asp Phe Ile Pro Asn Asp Ile Leu Ile Gly Cys Glu Glu Glu 


1105                1110                1115                1120 


 


Glu Gln Glu Asn Gly Lys Ala Tyr Cys Val Leu Val Thr Gly Pro Asn 


                1125                1130                1135 


 


Met Gly Gly Lys Ser Thr Leu Met Arg Gln Ala Gly Leu Leu Ala Val 


            1140                1145                1150 


 


Met Ala Gln Met Gly Cys Tyr Val Pro Ala Glu Val Cys Arg Leu Thr 


        1155                1160                1165 


 


Pro Ile Asp Arg Val Phe Thr Arg Leu Gly Ala Ser Asp Arg Ile Met 


    1170                1175                1180 


 


Ser Gly Glu Ser Thr Phe Phe Val Glu Leu Ser Glu Thr Ala Ser Ile 


1185                1190                1195                1200 


 


Leu Met His Ala Thr Ala His Ser Leu Val Leu Val Asp Glu Leu Gly 


                1205                1210                1215 


 


Arg Gly Thr Ala Thr Phe Asp Gly Thr Ala Ile Ala Asn Ala Val Val 


            1220                1225                1230 


 


Lys Glu Leu Ala Glu Thr Ile Lys Cys Arg Thr Leu Phe Ser Thr His 


        1235                1240                1245 


 


Tyr His Ser Leu Val Glu Asp Tyr Ser Gln Asn Val Ala Val Arg Leu 


    1250                1255                1260 


 


Gly His Met Ala Cys Met Val Glu Asn Glu Cys Glu Asp Pro Ser Gln 


1265                1270                1275                1280 


 


Glu Thr Ile Thr Phe Leu Tyr Lys Phe Ile Lys Gly Ala Cys Pro Lys 


                1285                1290                1295 


 


Ser Tyr Gly Phe Asn Ala Ala Arg Leu Ala Asn Leu Pro Glu Glu Val 


            1300                1305                1310 


 


Ile Gln Lys Gly His Arg Lys Ala Arg Glu Phe Glu Lys Met Asn Gln 


        1315                1320                1325 


 


Ser Leu Arg Leu Phe Arg Glu Val Cys Leu Ala Ser Glu Arg Ser Thr 


    1330                1335                1340 


 


Val Asp Ala Glu Ala Val His Lys Leu Leu Thr Leu Ile Lys Glu Leu 


1345                1350                1355                1360 


 


 


<210> SEQ ID NO 23 


<211> LENGTH: 389 


<212> TYPE: PRT 


<213> ORGANISM: Homo sapiens 


 


<400> SEQUENCE: 23 


 


Met Ala Gln Pro Lys Gln Glu Arg Val Ala Arg Ala Arg His Gln Arg 


 1               5                  10                  15 


 


Ser Glu Thr Ala Arg His Gln Arg Ser Glu Thr Ala Lys Thr Pro Thr 


            20                  25                  30 


 


Leu Gly Asn Arg Gln Thr Pro Thr Leu Gly Asn Arg Gln Thr Pro Arg 


        35                  40                  45 


 


Leu Gly Ile His Ala Arg Pro Arg Arg Arg Ala Thr Thr Ser Leu Leu 


    50                  55                  60 


 


Thr Leu Leu Leu Ala Phe Gly Lys Asn Ala Val Arg Cys Ala Leu Ile 


65                  70                  75                  80 


 


Gly Pro Gly Ser Leu Thr Ser Arg Thr Arg Pro Leu Thr Glu Pro Leu 


                85                  90                  95 


 


Gly Glu Lys Glu Arg Arg Glu Val Phe Phe Pro Pro Arg Pro Glu Arg 


            100                 105                 110 


 


Val Glu His Asn Val Glu Ser Ser Arg Trp Glu Pro Arg Arg Arg Gly 


        115                 120                 125 


 


Ala Cys Gly Ser Arg Gly Gly Asn Phe Pro Ser Pro Arg Gly Gly Ser 


    130                 135                 140 


 


Gly Val Ala Ser Leu Glu Arg Ala Glu Asn Ser Ser Thr Glu Pro Ala 


145                 150                 155                 160 


 


Lys Ala Ile Lys Pro Ile Asp Arg Lys Ser Val His Gln Ile Cys Ser 


                165                 170                 175 


 


Gly Pro Val Val Pro Ser Leu Arg Pro Asn Ala Val Lys Glu Leu Val 


            180                 185                 190 


 


Glu Asn Ser Leu Asp Ala Gly Ala Thr Asn Val Asp Leu Lys Leu Lys 


        195                 200                 205 


 


Asp Tyr Gly Val Asp Leu Ile Glu Val Ser Gly Asn Gly Cys Gly Val 


    210                 215                 220 


 


Glu Glu Glu Asn Phe Glu Gly Phe Thr Leu Lys His His Thr Cys Lys 


225                 230                 235                 240 


 


Ile Gln Glu Phe Ala Asp Leu Thr Gln Val Glu Thr Phe Gly Phe Arg 


                245                 250                 255 


 


Gly Glu Ala Leu Ser Ser Leu Cys Ala Leu Ser Asp Val Thr Ile Ser 


            260                 265                 270 


 


Thr Cys Arg Val Ser Ala Lys Val Gly Thr Arg Leu Val Phe Asp His 


        275                 280                 285 


 


Tyr Gly Lys Ile Ile Gln Lys Thr Pro Tyr Pro Arg Pro Arg Gly Met 


    290                 295                 300 


 


Thr Val Ser Val Lys Gln Leu Phe Ser Thr Leu Pro Val His His Lys 


305                 310                 315                 320 


 


Glu Phe Gln Arg Asn Ile Lys Lys Lys Arg Ala Cys Phe Pro Phe Ala 


                325                 330                 335 


 


Phe Cys Arg Asp Cys Gln Phe Pro Glu Ala Ser Pro Ala Met Leu Pro 


            340                 345                 350 


 


Val Gln Pro Val Glu Leu Thr Pro Arg Ser Thr Pro Pro His Pro Cys 


        355                 360                 365 


 


Ser Leu Glu Asp Asn Val Ile Thr Val Phe Ser Ser Val Lys Asn Gly 


    370                 375                 380 


 


Pro Gly Ser Ser Arg 


385 


 


 


<210> SEQ ID NO 24 


<211> LENGTH: 264 


<212> TYPE: PRT 


<213> ORGANISM: Homo sapiens 


 


<400> SEQUENCE: 24 


 


Met Cys Pro Trp Arg Pro Arg Leu Gly Arg Arg Cys Met Val Ser Pro 


 1               5                  10                  15 


 


Arg Glu Ala Asp Leu Gly Pro Gln Lys Asp Thr Arg Leu Asp Leu Pro 


            20                  25                  30 


 


Arg Ser Pro Ala Arg Ala Pro Arg Glu Gln Asn Ser Leu Gly Glu Val 


        35                  40                  45 


 


Asp Arg Arg Gly Pro Arg Glu Gln Thr Arg Ala Pro Ala Thr Ala Ala 


    50                  55                  60 


 


Pro Pro Arg Pro Leu Gly Ser Arg Gly Ala Glu Ala Ala Glu Pro Gln 


65                  70                  75                  80 


 


Glu Gly Leu Ser Ala Thr Val Ser Ala Cys Phe Gln Glu Gln Gln Glu 


                85                  90                  95 


 


Met Asn Thr Leu Gln Gly Pro Val Ser Phe Lys Asp Val Ala Val Asp 


            100                 105                 110 


 


Phe Thr Gln Glu Glu Trp Arg Gln Leu Asp Pro Asp Glu Lys Ile Ala 


        115                 120                 125 


 


Tyr Gly Asp Val Met Leu Glu Asn Tyr Ser His Leu Val Ser Val Gly 


    130                 135                 140 


 


Tyr Asp Tyr His Gln Ala Lys His His His Gly Val Glu Val Lys Glu 


145                 150                 155                 160 


 


Val Glu Gln Gly Glu Glu Pro Trp Ile Met Glu Gly Glu Phe Pro Cys 


                165                 170                 175 


 


Gln His Ser Pro Glu Pro Ala Lys Ala Ile Lys Pro Ile Asp Arg Lys 


            180                 185                 190 


 


Ser Val His Gln Ile Cys Ser Gly Pro Val Val Leu Ser Leu Ser Thr 


        195                 200                 205 


 


Ala Val Lys Glu Leu Val Glu Asn Ser Leu Asp Ala Gly Ala Thr Asn 


    210                 215                 220 


 


Ile Asp Leu Lys Leu Lys Asp Tyr Gly Val Asp Leu Ile Glu Val Ser 


225                 230                 235                 240 


 


Asp Asn Gly Cys Gly Val Glu Glu Glu Asn Phe Glu Gly Leu Ile Ser 


                245                 250                 255 


 


Phe Ser Ser Glu Thr Ser His Met 


            260 


 


 


<210> SEQ ID NO 25 


<211> LENGTH: 264 


<212> TYPE: PRT 


<213> ORGANISM: Homo sapiens 


 


<400> SEQUENCE: 25 


 


Met Cys Pro Trp Arg Pro Arg Leu Gly Arg Arg Cys Met Val Ser Pro 


 1               5                  10                  15 


 


Arg Glu Ala Asp Leu Gly Pro Gln Lys Asp Thr Arg Leu Asp Leu Pro 


            20                  25                  30 


 


Arg Ser Pro Ala Arg Ala Pro Arg Glu Gln Asn Ser Leu Gly Glu Val 


        35                  40                  45 


 


Asp Arg Arg Gly Pro Arg Glu Gln Thr Arg Ala Pro Ala Thr Ala Ala 


    50                  55                  60 


 


Pro Pro Arg Pro Leu Gly Ser Arg Gly Ala Glu Ala Ala Glu Pro Gln 


65                  70                  75                  80 


 


Glu Gly Leu Ser Ala Thr Val Ser Ala Cys Phe Gln Glu Gln Gln Glu 


                85                  90                  95 


 


Met Asn Thr Leu Gln Gly Pro Val Ser Phe Lys Asp Val Ala Val Asp 


            100                 105                 110 


 


Phe Thr Gln Glu Glu Trp Arg Gln Leu Asp Pro Asp Glu Lys Ile Ala 


        115                 120                 125 


 


Tyr Gly Asp Val Met Leu Glu Asn Tyr Ser His Leu Val Ser Val Gly 


    130                 135                 140 


 


Tyr Asp Tyr His Gln Ala Lys His His His Gly Val Glu Val Lys Glu 


145                 150                 155                 160 


 


Val Glu Gln Gly Glu Glu Pro Trp Ile Met Glu Gly Glu Phe Pro Cys 


                165                 170                 175 


 


Gln His Ser Pro Glu Pro Ala Lys Ala Ile Lys Pro Ile Asp Arg Lys 


            180                 185                 190 


 


Ser Val His Gln Ile Cys Ser Gly Pro Val Val Leu Ser Leu Ser Thr 


        195                 200                 205 


 


Ala Val Lys Glu Leu Val Glu Asn Ser Leu Asp Ala Gly Ala Thr Asn 


    210                 215                 220 


 


Ile Asp Leu Lys Leu Lys Asp Tyr Gly Val Asp Leu Ile Glu Val Ser 


225                 230                 235                 240 


 


Asp Asn Gly Cys Gly Val Glu Glu Glu Asn Phe Glu Gly Leu Ile Ser 


                245                 250                 255 


 


Phe Ser Ser Glu Thr Ser His Met 


            260

## Claims

1. A method for generating a yeast cell harboring a mutation in a gene of interest, comprising:
introducing a dominant negative allele of a mismatch repair gene selected from the group consisting of PMS2, PMSR2, PMSR3, MLH1, and MLH3 into a yeast cell comprising the gene of interest whereby the cell becomes hypermutable; and
testing the cell to determine whether the gene of interest harbors a mutation.

2. The method of claim 1 wherein the dominant negative allele is PMS2-134.

3. The method of claim 1 further comprising restoring genetic stability to the cell.

4. The method of claim 1 further comprising exposing the cell to a chemical mutagen.

5. The method of claim 1 wherein the step of testing comprises analyzing a nucleotide sequence of the gene of interest.

6. The method of claim 1 wherein the step of testing comprises analyzing mRNA transcribed from the gene of interest.

7. The method of claim 1 wherein the step of testing comprises analyzing a phenotype associated with the gene of interest.

8. The method of claim 1 wherein the step of testing comprises analyzing a protein encoded by the gene of interest.

9. A method for generating a yeast cell harboring a mutation in a gene of interest, comprising:
introducing (a) a dominant negative allele of a mismatch repair gene selected from the group consisting of PMS2, PMSR2, PMSR3, MLH1, and MLH and (b) a gene of interest into a yeast cell whereby the cell becomes hypermutable; and
testing the cell to determine whether the gene of interest harbors a mutation.

10. The method of claim 9 wherein the dominant negative allele is PMS2-134.

11. The method of claim 9 further comprising restoring genetic stability to the cell.

12. The method of claim 9 further comprising exposing the cell to a chemical mutagen.

13. The method of claim 9 wherein the step of testing comprises analyzing a nucleotide sequence of the gene of interest.

14. The method of claim 9 wherein the step of testing comprises analyzing mRNA transcribed from the gene of interest.

15. The method of claim 9 wherein the step of testing comprises analyzing a phenotype associated with the gene of interest.

16. The method of claim 9 wherein the step of testing comprises analyzing a protein encoded by the gene of interest.