Patent Publication Number: US-2003224463-A1

Title: Compositions and methods relating to DNA mismatch repair genes

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
     [0001] This application is a continuation from U.S. patent application Ser. No. 09/265,503 filed Mar. 10, 1999, which is a continuation from U.S. patent application Ser. No. 08/352,902, filed Dec. 4, 1994, which is a continuation-in-part of U.S. patent application Ser. No. 08/209,521, filed Mar. 8, 1994, which is a continuation-in-part from U.S. patent application Ser. No. 08/168,877, filed Dec. 17, 1993. All of the above patent applications are incorporated by reference. 
    
    
     [0002] This invention was made with government support under Agreement No. GM 32741 and Agreement No. HG00395/GM50006 awarded by the National Institute of Health in the General Sciences Division. The government has certain rights in the invention. 
    
    
     
       FIELD OF THE INVENTION  
       [0003] The present invention involves DNA mismatch repair genes. In particular, the invention relates to identification of mutations and polymorphisms in DNA mismatch repair genes, to identification and characterization of DNA mismatch-repair-defective tumors, and to detection of genetic susceptibility to cancer.  
       BACKGROUND  
       [0004] In recent years, with the development of powerful cloning and amplification techniques such as the polymerase chain reaction (PCR), in combination with a rapidly accumulating body of information concerning the structure and location of numerous human genes and markers, it has become practical and advisable to collect and analyze samples of DNA or RNA from individuals who are members of families which are identified as exhibiting a high frequency of certain genetically transmitted disorders. For example, screening procedures are routinely used to screen for genes involved in sickle cell anemia, cystic fibrosis, fragile X chromosome syndrome and multiple sclerosis. For some types of disorders, early diagnosis can greatly improve the person&#39;s long-term prognosis by, for example, adopting an aggressive diagnostic routine, and/or by making life style changes if appropriate to either prevent or prepare for an anticipated problem.  
       [0005] Once a particular human gene mutation is identified and linked to a disease, development of screening procedures to identify high-risk individuals can be relatively straight forward. For example, after the structure and abnormal phenotypic role of the mutant gene are understood, it is possible to design primers for use in PCR to obtain amplified quantities of the gene from individuals for testing. However, initial discovery of a mutant gene, i.e., its structure, location and linkage with a known inherited health problem, requires substantial experimental effort and creative research strategies.  
       [0006] One approach to discovering the role of a mutant gene in causing a disease begins with clinical studies on individuals who are in families which exhibit a high frequency of the disease. In these studies, the approximate location of the disease-causing locus is determined indirectly by searching for a chromosome marker which tends to segregate with the locus. A principal limitation of this approach is that, although the approximate genomic location of the gene can be determined, it does not generally allow actual isolation or sequencing of the gene. For example, Lindblom et al. 3  reported results of linkage analysis studies performed with SSLP (simple sequence length polymorphism) markers on individuals from a family known to exhibit a high incidence of hereditary non-polyposis colon cancer (HNPCC). Lindblom et al. found a “tight linkage” between a polymorphic marker on the short arm of human chromosome 3 (3p21-23) and a disease locus apparently responsible for increasing an individual&#39;s risk of developing colon cancer. Even though 3p21-23 is a fairly specific location relative to the entire genome, it represents a huge DNA region relative to the probable size of the mutant gene. The mutant gene could be separated from the markers identifying the locus by millions of bases. At best, such linkage studies have only limited utility for screening purposes because in order to predict one person&#39;s risk, genetic analysis must be performed with tightly linked genetic markers on a number of related individuals in the family. It is often impossible to obtain such information, particularly if affected family members are deceased. Also, informative markers may not exist in the family under analysis. Without knowing the gene&#39;s structure, it is not possible to sample, amplify, sequence and determine directly whether an individual carries the mutant gene.  
       [0007] Another approach to discovering a disease-causing mutant gene begins with design and trial of PCR primers, based on known information about the disease, for example, theories for disease state mechanisms, related protein structures and function, possible analogous genes in humans or other species, etc. The objective is to isolate and sequence candidate normal genes which are believed to sometimes occur in mutant forms rendering an individual disease prone. This approach is highly dependent on how much is known about the disease at the molecular level, and on the investigator&#39;s ability to construct strategies and methods for finding candidate genes. Association of a mutation in a candidate gene with a disease must ultimately be demonstrated by performing tests on members of a family which exhibits a high incidence of the disease. The most direct and definitive way to confirm such linkage in family studies is to use PCR primers which are designed to amplify portions of the candidate gene in samples collected from the family members. The amplified gene products are then sequenced and compared to the normal gene structure for the purpose of finding and characterizing mutations. A given mutation is ultimately implicated by showing that affected individuals have it while unaffected individuals do not, and that the mutation causes a change in protein function which is not simply a polymorphism.  
       [0008] Another way to show a high probability of linkage between a candidate gene mutation and disease is by determining the chromosome location of the gene, then comparing the gene&#39;s map location to known regions of disease-linked loci such as the one identified by Lindblom et al. Coincident map location of a candidate gene in the region of a previously identified disease-linked locus may strongly implicate an association between a mutation in the candidate gene and the disease.  
       [0009] There are other ways to show that mutations in a gene candidate may be linked to the disease. For example, artificially produced mutant forms of the gene can be introduced into animals. Incidence of the disease in animals carrying the mutant gene can then be compared to animals with the normal genotype. Significantly elevated incidence of disease in animals with the mutant genotype, relative to animals with the wild-type gene, may support the theory that mutations in the candidate gene are sometimes responsible for occurrence of the disease.  
       [0010] One type of disease which has recently received much attention because of the discovery of disease-linked gene mutations is Hereditary Nonpolyposis Colon Cancer (HNPCC). 1,2  Members of HNPCC families also display increased susceptibility to other cancers including endometrial, ovarian, gastric and breast. Approximately 10% of colorectal cancers are believed to be HNPCC. Tumors from HNPCC patients display an unusual genetic defect in which short, repeated DNA sequences, such as the dinucleotide repeat sequences found in human chromosomal DNA (“microsatellite DNA”), appear to be unstable. This genomic instability of short, repeated DNA sequences, sometimes called the “RER+” phenotype, is also observed in a significant proportion of a wide variety of sporadic tumors, suggesting that many sporadic tumors may have acquired mutations that are similar (or identical) to mutations that are inherited in HNPCC.  
       [0011] Genetic linkage studies have identified two HNPCC loci thought to account for as much as 90% of HNPCC. The loci map to human chromosome 2p 15-16 (2p21) and 3p21-23. Subsequent studies have identified human DNA mismatch repair gene hMSH2 as being the gene on chromosome 2p21, in which mutations account for a significant fraction of HNPCC cancers. 1, 2, 12  hMSH2 is one of several genes whose normal function is to identify and correct DNA mispairs including those that follow each round of chromosome replication.  
       [0012] The best defined mismatch repair pathway is the  E. coli  MutHLS pathway that promotes a long-patch (approximately 3 Kb) excision repair reaction which is dependent on the mutH, mutL, mutS and mutU (uvrD) gene products. The MutHLS pathway appears to be the most active mismatch repair pathway in  E. coli  and is known to both increase the fidelity of DNA replication and to act on recombination intermediates containing mispaired bases. The system has been reconstituted in vitro, and requires the mutH, mutL, mutS and uvrD (helicase II) proteins along with DNA polymerase III holoenzyme, DNA ligase, single-stranded DNA binding protein (SSB) and one of the single-stranded DNA exonucleases, Exo I, Exo VII or RecJ. hMSH2 is homologous to the bacterial mutS gene. A similar pathway in yeast includes the yeast MSH2 gene and two mutL-like genes referred to as PMS1 and MLH1.  
       [0013] With the knowledge that mutations in a human mutS type gene (hMSH2) sometimes cause cancer, and the discovery that HNPCC tumors exhibit microsatellite DNA instability, interest in other DNA mismatch repair genes and gene products, and their possible roles in HNPCC and/or other cancers, has intensified. It is estimated that as many as 1 in 200 individuals carry a mutation in either the hMSH2 gene or other related genes which encode for other proteins in the same DNA mismatch repair pathway.  
       [0014] An important objective of our work has been to identify human genes which are useful for screening and identifying individuals who are at elevated risk of developing cancer. Other objects are: to determine the sequences of exons and flanking intron structures in such genes; to use the structural information to design testing procedures for the purpose of finding and characterizing mutations which result in an absence of or defect in a gene product which confers cancer susceptibility; and to distinguish such mutations from “harmless” polymorphic variations. Another object is to use the structural information relating to exon and flanking intron sequences of a cancer-linked gene, to diagnose tumor types and prescribe appropriate therapy. Another object is to use the structural information relating to a cancer-linked gene to identify other related candidate human genes for study.  
       SUMMARY OF THE INVENTION  
       [0015] Based on our knowledge of DNA mismatch repair mechanisms in bacteria and yeast including conservation of mismatch repair genes, we reasoned that human DNA mismatch repair homologs should exist, and that mutations in such homologs affecting protein function, would be likely to cause genetic instability, possibly leading to an increased risk of developing certain forms of human cancer.  
       [0016] We have isolated and sequenced two human genes, hPMS1 and hMLH1 each of which encodes for a protein involved in DNA mismatch repair. hPMS1 and hMLH1 are homologous to mutL genes found in  E. coli . Our studies strongly support an association between mutations in DNA mismatch repair genes and susceptibility to HNPCC. Thus, DNA mismatch repair gene sequence information of the present invention, namely, cDNA and genomic structures relating to hMLH1 and hPMS1, make possible a number of useful methods relating to cancer risk determination and diagnosis. The invention also encompasses a large number of nucleotide and protein structures which are useful in such methods.  
       [0017] We mapped the location of hMLH1 to human chromosome 3p21-23. This is a region of the human genome that, based upon family studies, harbors a locus that predisposes individuals to HNPCC. Additionally, we have found a mutation in a conserved region of the hMLH1 cDNA in HNPCC-affected individuals from a Swedish family. The mutation is not found in unaffected individuals from the same family, nor is it a simple polymorphism. We have also found that a homologous mutation in yeast results in a defective DNA mismatch repair protein. We have also found a frameshift mutation in hMLH1 of affected individuals from an English family. Our discovery of a cancer-linked mutations in hMLH1, combined with the gene&#39;s map position which is coincident with a previously identified HNPCC-linked locus, plus the likely role of the hMLH1 gene in mutation avoidance makes the hMLH1 gene a prime candidate for underlying one form of common inherited human cancer, and a prime candidate to screen and identify individuals who have an elevated risk of developing cancer.  
       [0018] hMLH1 has 19 exons and 18 introns. We have determined the location of each of the 18 introns relative to hMLH1 cDNA. We have also determined the structure of all intron/exon boundary regions of hMLH1. Knowledge of the intron/exon boundary structures makes possible efficient screening regimes to locate mutations which negatively affect the structure and function of gene products. Further, we have designed complete sets of oligonucleotide primer pairs which can be used in PCR to amplify individual complete exons together with surrounding intron boundary structures.  
       [0019] We mapped the location of HPMS1 to human chromosome 7. Subsequent studies by others 39  have confirmed our prediction that mutations in this gene are linked to HNPCC.  
       [0020] The most immediate use of the present invention will be in screening tests on human individuals who are members of families which exhibit an unusually high frequency of early onset cancer, for example HNPCC. Accordingly, one aspect of the invention comprises a method of diagnosing cancer susceptibility in a subject by detecting a mutation in a mismatch repair gene or gene product in a tissue from the subject, wherein the mutation is indicative of the subject&#39;s susceptibility to cancer. In a preferred embodiment of the invention, the step of detecting comprises detecting a mutation in a human mutL homolog gene, for example, hMLH1 of hPMS1.  
       [0021] The method of diagnosing preferably comprises the steps of: 1) amplifying a segment of the mismatch repair gene or gene product from an isolated nucleic acid; 2) comparing the amplified segment with an analogous segment of a wild-type allele of the mismatch repair gene or gene product; and 3) detecting a difference between the amplified segment and the analogous segment, the difference being indicative of a mutation in the mismatch repair gene or gene product which confers cancer susceptibility.  
       [0022] Another aspect of the invention provides methods of determining whether the difference between the amplified segment and the analogous wild-type segment causes an affected phenotype, i.e., does the sequence alteration affect the individual&#39;s ability to repair DNA mispairs.  
       [0023] The method of diagnosing may include the steps of: 1) reverse transcribing all or a portion of an RNA copy of a DNA mismatch repair gene; and 2) amplifying a segment of the DNA produced by reverse transcription. An amplifying step in the present invention may comprise: selecting a pair of oligonucleotide primers capable of hybridizing to opposite strands of the mismatch repair gene, in an opposite orientation; and performing a polymerase chain reaction utilizing the oligonucleotide primers such that nucleic acid of the mismatch repair chain intervening between the primers is amplified to become the amplified segment.  
       [0024] In preferred embodiments of the methods summarized above, the DNA mismatch repair gene is hMLH1 or hPMS1. The segment of DNA corresponds to a unique portion of a nucleotide sequence selected from the group consisting of SEQ ID NOS: 6-24. “First stage” oligonucleotide primers selected from the group consisting of SEQ ID NOS: 44-82 are used in PCR to amplify the DNA segment. The invention also provides a method of using “second stage” nested primers (SEQ ID NOS: 83-122), for use with the first stage primers to allow more specific amplification and conservation of template DNA.  
       [0025] Another aspect of the present invention provides a method of identifying and classifying a DNA mismatch repair defective tumor comprising detecting in a tumor a mutation in a mismatch repair gene or gene product, preferably a mutL homolog (hMLH1 or hPMS1), the mutation being indicative of a defect in a mismatch repair system of the tumor.  
       [0026] The present invention also provides useful nucleotide and protein compositions. One such composition is an isolated nucleotide or protein structure including a segment sequentially corresponding to a unique portion of a human mutL homolog gene or gene product, preferably derived from either hMLH1 or hPMS1.  
       [0027] Other composition aspects of the invention comprise oligonucleotide primers capable of being used together in a polymerase chain reaction to amplify specifically a unique segment of a human mutL homolog gene, preferably hMLH1 or hPMS1.  
       [0028] Another aspect of the present invention provides a probe including a nucleotide sequence capable of binding specifically by Watson/Crick pairing to complementary bases in a portion of a human mutL homolog gene; and a label-moiety attached to the sequence, wherein the label-moiety has a property selected from the group consisting of fluorescent, radioactive and chemiluminescent.  
       [0029] We have also isolated and sequenced mouse MLH1 (mMLH1) and PMS1 (mPMS1) genes. We have used our knowledge of mouse mismatch repair genes to construct animal models for studying cancer. The models will be useful to identify additional oncogenes and to study environmental effects on mutagenesis.  
       [0030] Our knowledge of hMLH1 and hPMS1 gene sequences makes it possible to produce monoclonal and polyclonal antibodies for use in tests that detect the presence or absence of DNA mismatch repair protein in a tumor sample. Protein based testing is receiving significant attention in view of recent research showing that methylation of hMLH1 promoter DNA is the basis for DNA mismatch repair deficiency in some sporadic tumors. In this situation there is usually no detectable mutation in the hMLH1 cDNA. A screen for hMLH1 cDNA mutations would not show any abnormality. However, an immunoassay for hMLH1 protein shows absence of the protein in tumors with inactivation of the hMLH1 gene by mutation or by promotor methylation, and may be the screening test of choice for some applications. The protein structure information has been used to generate monoclonal antibodies that bind specifically to hMLH1 or hPMS1. The antibodies can be conjugated to labels such as fluorescent compounds, and then used as an immunohistochemical stain to detect DNA mismatch repair protein in a tumor sample.  
       [0031] In addition to diagnostic and therapeutic uses for the genes, our knowledge of hMLH1 and hPMS1 can be used to search for other genes of related function which are candidates for playing a role in certain forms of human cancer. 
     
    
    
     DESCRIPTION OF THE FIGURES  
     [0032]FIG. 1 is a flow chart showing an overview of the sequence of experimental steps we used to isolate, characterize and use human and mouse PMS1 and MLH1 genes.  
     [0033]FIG. 2 is an alignment of protein sequences for mutL homologs (SEQ ID NOS: 1-3) showing two highly-conserved regions (underlined) which we used to create degenerate PCR oligonucleotides for isolating additional mutL homologs.  
     [0034]FIG. 3 shows the entire cDNA nucleotide sequence (SEQ ID NO: 4) for the human MLH1 gene, and the corresponding predicted amino acid sequence (SEQ ID NO: 5) for the human MLH1 protein. The underlined DNA sequences are the regions of cDNA that correspond to the degenerate PCR primers that were originally used to amplify a portion of the MLH1 gene (nucleotides 118-135 and 343-359).  
     [0035]FIG. 4A shows the nucleotide sequences of the 19 exons which collectively correspond to the entire hMLH1 cDNA structure. The exons are flanked by intron boundary structures. Primer sites are underlined. The exons with their flanking intron structures correspond to SEQ ID NOS: 6-24. The exons, shown in non-underlined small case letters, corespond to SEQ ID NOS: 25-43.  
     [0036]FIG. 4B shows nucleotide sequences of primer pairs which have been used in PCR to amplify the individual exons. The “second stage” amplification primers (SEQ ID NOS: 83-122) are “nested” primers which are used to amplify target exons from the amplification product obtained with corresponding “first stage” amplification primers (SEQ ID NOS: 44-82). The structures in FIG. 4B correspond to the structures in Tables 2 and 3.  
     [0037]FIG. 5 is an alignment of the predicted amino acid sequences for human and yeast (SEQ ID NOS: 5 and 123, respectively) MLH1 proteins. Amino acid identities are indicated by boxes and gaps are indicated by dashes.  
     [0038]FIG. 6 is a phylogenetic tree of MutL-related proteins.  
     [0039]FIG. 7 is a two-panel photograph. The first panel (A) is a metaphase spread showing hybridization of the hMLH1 gene of chromosome 3. The second panel (B) is a composite of chromosome 3 from multiple metaphase spreads aligned with a human chromosome 3 ideogram. The region of hybridization is indicated in the ideogram by a vertical bar.  
     [0040]FIG. 8 is a comparison of sequence chromatograms from affected and unaffected individuals showing identification of a C to T transition mutation that produces a non-conservative amino acid substitution at position 44 of the hMLH1 protein.  
     [0041]FIG. 9 is an amino acid sequence alignment (SEQ ID NOS: 124-131) of the highly-conserved region of the MLH family of proteins surrounding the site of the predicted amino acid substitution. Bold type indicates the position of the predicted serine to phenylalanine amino acid substitution in affected individuals. Also highlighted are the serine or alanine residues conserved at this position in MutL-like proteins. Bullets indicate positions of highest amino acid conservation. For the MLH 1 protein, the dots indicate that the sequence has not been obtained. Sequences were aligned as described below in reference to the phylogenetic tree of FIG. 6.  
     [0042]FIG. 10 shows the entire nucleotide sequence for hPMS1 (SEQ ID NO: 132).  
     [0043]FIG. 11 is an alignment of the predicted amino acid sequences for human and yeast PMS1 proteins (SEQ ID NOS: 133 and 134, respectively). Amino acid identities are indicated by boxes and gaps are indicated by dashes.  
     [0044]FIG. 12 is a partial nucleotide sequence (SEQ ID NO: 149) from human MLH1.  
     [0045]FIG. 13 shows partial nucleotide sequences (SEQ ID NOS: 150 and 151) from human PMS1 cDNA.  
     [0046]FIG. 14 is a partial nucleotide sequence (SEQ ID NO: 152) from human MLH1.  
     [0047]FIG. 15 is a partial nucleotide sequence (SEQ ID NO: 153) from human PMS1. 
    
    
     DEFINITIONS  
     [0048] gene—“Gene” means a nucleotide sequence that contains a complete coding sequence. Generally, “genes” also include nucleotide sequences found upstream (e.g. promoter sequences, enhancers, etc.) or downstream (e.g. transcription termination signals, polyadenylation sites, etc.) of the coding sequence that affect the expression of the encoded polypeptide.  
     [0049] gene product—A “gene product” is either a DNA or RNA (mRNA) copy of a portion of a gene, or a corresponding amino acid sequence translated from mRNA.  
     [0050] wild-type—The term “wild-type”, when applied to nucleic acids and proteins of the present invention, means a version of a nucleic acid or protein that functions in a manner indistinguishable from a naturally-occurring, normal version of that nucleic acid or protein (i.e. a nucleic acid or protein with wild-type activity). For example, a “wild-type” allele of a mismatch repair gene is capable of functionally replacing a normal, endogenous copy of the same gene within a host cell without detectably altering mismatch repair in that cell. Different wild-type versions of the same nucleic acid or protein may or may not differ structurally from each other.  
     [0051] non-wild-type—The term “non-wild-type” when applied to nucleic acids and proteins of the present invention, means a version of a nucleic acid or protein that functions in a manner distinguishable from a naturally-occurring, normal version of that nucleic acid or protein. Non-wild-type alleles of a nucleic acid of the invention may differ structurally from wild-type alleles of the same nucleic acid in any of a variety of ways including, but not limited to, differences in the amino acid sequence of an encoded polypeptide and/or differences in expression levels of an encoded nucleotide transcript of polypeptide product.  
     [0052] For example, the nucleotide sequence of a non-wild-type allele of a nucleic acid of the invention may differ from that of a wild-type allele by, for example, addition, deletion, substitution, and/or rearrangement of nucleotides. Similarly, the amino acid sequence of a non-wild-type mismatch repair protein may differ from that of a wild-type mismatch repair protein by, for example, addition, substitution, and/or rearrangement of amino acids.  
     [0053] Particular non-wild-type nucleic acids or proteins that, when introduced into a normal host cell, interfere with the endogenous mismatch repair pathway, are termed “dominant negative” nucleic acids or proteins.  
     [0054] homologous—The term “homologous” refers to nucleic acids or polypeptides that are highly related at the level of nucleotide or amino acid sequence. Nucleic acids or polypeptides that are homologous to each other are termed “homologues”.  
     [0055] The term “homologous” necessarily refers to a comparison between two sequences. In accordance with the invention, two nucleotide sequences are considered to be homologous if the polypeptides they encode are at least about 50-60% identical, preferably about 70% identical, for at least one stretch of at least 20 amino acids. Preferably, homologous nucleotide sequences are also characterized by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. Both the identity and the approximate spacing of these amino acids relative to one another must be considered for nucleotide sequences to be considered to be homologous. For nucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4-5 uniquely specified amino acids.  
     [0056] upstream/downstream—The terms “upstream” and “downstream” are art-understood terms referring to the position of an element of nucleotide sequence. “Upstream” signifies an element that is more 5′ than the reference element. “Downstream” refers to an element that is more 3′ than a reference element.  
     [0057] intron/exon—The terms “exon” and “intron” are art-understood terms referring to various portions of genomic gene sequences. “Exons” are those portions of a genomic gene sequence that encode protein. “Introns” are sequences of nucleotides found between exons in genomic gene sequences.  
     [0058] affected—The term “affected”, as used herein, refers to those members of a kindred that either have developed a characteristic cancer (e.g. colon cancer in an HNPCC lineage) and/or are predicted, on the basis of, for example, genetic studies, to carry an inherited mutation that confers susceptibility to cancer.  
     [0059] unique—A “unique” segment, fragment or portion of a gene or protein means a portion of a gene or protein which is different sequentially from any other gene or protein segment in an individual&#39;s genome. As a practical matter, a unique segment or fragment of a gene will typically be a nucleotide of at least about 13 bases in length and will be sufficiently different from other gene segments so that oligonucleotide primers may be designed and used to selectively and specifically amplify the segment. A unique segment of a protein is typically an amino acid sequence which can be translated from a unique segment of a gene.  
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     DESCRIPTION OF THE INVENTION  
     [0126] We have discovered mammalian genes which are involved in DNA mismatch repair. One of the genes, hPMS1, encodes a protein which is homologous to the yeast DNA mismatch repair protein PMS1. We have mapped the locations of hPMS1 to human chromosome 7 and the mouse PMS1 gene to mouse chromosome 5, band G. Another gene, hMLH1 (MutL Homolog) encodes a protein which is homologous to the yeast DNA mismatch repair protein MLH1. We have mapped the locations of hMLH1 to human chromosome 3p21.3-23 and to mouse chromosome 9, band E.  
     [0127] Studies 1,2  have demonstrated involvement of a human DNA mismatch repair gene homolog, hMSH2, on chromosome 2p in HNPCC. Based upon linkage data, a second HNPCC locus has been assigned to chromosome 3p21-23. 3  Examination of tumor DNA from the chromosome 3-linked kindreds revealed dinucleotide repeat instability similar to that observed for other HNPCC families 6  and several types of sporadic tumors. 7-10  Because dinucleotide repeat instability is characteristic of a defect in DNA mismatch repair, 5, 11, 12  we reasoned that HNPCC linked to chromosome 3p21-23 could result from a mutation in a second DNA mismatch repair gene.  
     [0128] Repair of mismatched DNA in  Escherichia coli  requires a number of genes including mutS, mutL and mutH, defects in any one of which result in elevated spontaneous mutation rates. 13  Genetic analysis in the yeast  Saccharomyces cerevisiae  has identified three DNA mismatch repair genes: a mutS homolog, MSH2, 14  and two mutL homologs, PMS1 16  and MLH1. 4  Each of these three genes play an indispensable role in DNA replication fidelity, including the stabilization of dinucleotide repeats. 5    
     [0129] We believe that hMLH1 is the HNPCC gene previously linked to chromosome 3p based upon the similarity of the hMLH1 gene product to the yeast DNA mismatch repair protein, MLH1, 4  the coincident location of the hMLH1 gene and the HNPCC locus on chromosome 3, and hMLH1 missense mutations which we found in affected individuals from chromosome 3-linked HNPCC families.  
     [0130] Our knowledge of the human and mouse MLH1 and PMS1 gene structures has many important uses. The gene sequence information can be used to screen individuals for cancer risk. Knowledge of the gene structures makes it possible to easily design PCR primers which can be used to selectively amplify portions of hMLH1 and hPMS1 genes for subsequent comparison to the normal sequence and cancer risk analysis. This type of testing also makes it possible to search for and characterize hMLH1 and hPMS1 cancer-linked mutations for the purpose of eventually focusing the cancer screening effort on specific gene loci. Specific characterization of cancer-linked mutations in hMLH1 and hPMS1 makes possible the production of other valuable diagnostic tools such as allele specific probes which may be used in screening tests to determine the presence or absence of specific gene mutations.  
     [0131] Additionally, the gene sequence information for hMLH1 and/or hPMS1 can be used, for example, in a two hybrid system, to search for other genes of related function which are candidates for cancer involvement.  
     [0132] The hMLH1 and hPMS1 gene structures are useful for making proteins which are used to develop antibodies directed to specific portions or the complete hMLH1 and hPMS1 proteins. Such antibodies can then be used to isolate the corresponding protein and possibly related proteins for research and diagnostic purposes.  
     [0133] The mouse MLH1 and PMS1 gene sequences are useful for producing mice that have mutations in the respective gene. The mutant mice are useful for studying the gene&#39;s function, particularly its relationship to cancer.  
     Methods for Isolating and Characterizing Mammalian MLH1 and PMS1 Genes  
     [0134] We have isolated and characterized four maimalian genes, i.e., human MLH1 (hMLH1), human PMS1 (hPMS1), mouse MLH1 (mPMS1) and mouse PMS1 (mPMS1). Due to the structural similarity between these genes, the methods we have employed to isolate and characterize them are generally the same. FIG. 1 shows in broad terms, the experimental approach which we used to isolate and characterize the four genes. The following discussion refers to the step-by-step procedure shown in FIG. 1.  
     [0135] Step 1 Design of Degenerate Oligonucleotide Pools for PCR  
     [0136] Earlier reports indicated that portions of three MutL-like proteins, two from bacteria, MutL and HexB, and one from yeast, PMS1 are highly conserved. 16,18,19  After inspection of the amino acid sequences of HexB, MutL and PMS1 proteins, as shown in FIG. 2, we designed pools of degenerate oligonucleotide pairs corresponding to two highly-conserved regions, KELVEN and GFRGEA, of the MutL-like proteins. The sequences (SEQ ID NOS: 139 and 140, respectively) of the degenerate oligonucleotides which we used to isolate the four genes are:  
                          5′-CTTGAT TCTAGA GC(T/C)TCNCCNC(T/G)(A/G)AANCC-3′           and               5′-AGCTCG GAGCTC AA(A/G)GA(A/G)(T/C)TNGTNGANAA-3′.          
 
     [0137] The underlined sequences within the primers are XbaI and SacI restriction endonuclease sites respectively. They were introduced in order to facilitate the cloning of the PCR-amplified fragments. In the design of the oligonucleotides, we took into account the fact that a given amino acid can be coded for by more than one DNA triplet (codon). The degeneracy within these sequences are indicated by multiple nucleotides within parentheses or N, for the presence of any base at that position.  
     [0138] Step 2 Reverse Transcription and PCR on Poly A+ Selected mRNA isolated from human cells  
     [0139] We isolated messenger (poly A+ enriched) RNA from cultured human cells, synthesized double-stranded cDNA from the mRNA, and performed PCR with the degenerate oligonucleotides. 4  After trying a number of different PCR conditions, for example, adjusting the annealing temperature, we successfully amplified a DNA of the size predicted (˜210 bp) for a MutL-like protein.  
     [0140] Step 3 Cloning and Sequencing of PCR-Generated Fragments; Identification of Two Gene Fragments Representing Human PMS1 and MLH1  
     [0141] We isolated the PCR amplified material (˜210 bp) from an agarose gel and cloned this material into a plasmid (pUC19). We determined the DNA sequence of several different clones. The amino acid sequence inferred from the DNA sequence of two clones showed strong similarity to other known MutL-like proteins. 4,16,18,19  The predicted amino acid sequence for one of the clones was most similar to the yeast PMS1 protein. Therefore we named it hPMS1, for human PMS1. The second clone was found to encode a polypeptide that most closely resembles yeast MLH1 protein and was named, hMLH1, for human MLH1.  
     [0142] Step 4 Isolation of Complete Human and Mouse PMS1 and MLH1 cDNA Clones using the PCR Fragments as Probes  
     [0143] We used the 210 bp PCR-generated fragments of the hMLH1 and hPMS1 cDNAs, as probes to screen both human and mouse cDNA libraries (from Stratagene, or as described in reference 30). A number of cDNAs were isolated that corresponded to these two genes. Many of the cDNAs were truncated at the 5′ end. Where necessary, PCR techniques 31 were used to obtain the 5′-end of the gene in addition to further screening of cDNA libraries. Complete composite cDNA sequences were used to predict the amino acid sequence of the human and mouse, MLH1 and PMS1 proteins.  
     [0144] Step 5 Isolation of Human and Mouse, PMS1 and MLH1 Genomic Clones  
     [0145] Information on genomic and cDNA structure of the human MLH1 and PMS1 genes are necessary in order to thoroughly screen for mutations in cancer prone families. We have used human cDNA sequences as probes to isolate the genomic sequences of human PMS1 and MLH1. We have isolated four cosmids and two P1 clones for hPMS1, that together are likely to contain most, if not all, of the cDNA (exon) sequence. For hMLH1 we have isolated four overlapping λ-phage clones containing 5′-MLH1 genomic sequences and four PI clones (two full length clones and two which include the 5′ coding end plus portions of the promoter region) P1 clone. PCR analysis using pairs of oligonucleotides specific to the 5′ and 3′ ends of the hMLH1 cDNA, clearly indicates that the P1 clone contains the complete hMLH1 cDNA information. Similarly, genomic clones for mouse PMS1 and MLH1 genes have been isolated and partially characterized (described in Step 8).  
     [0146] Step 6 Chromosome Positional Mapping of the Human and Mouse, PMS1 and MLH1 Genes by Fluorescence In Situ Hybridization  
     [0147] We used genomic clones isolated from human and mouse PMS1 and MLH1 for chromosomal localization by fluorescence in situ hybridization (FISH). 20,21  We mapped the human MLH1 gene to chromosome 3p21.3-23, shown in FIG. 7 as discussed in more detail below. We mapped the mouse MLH1 gene to chromosome 9 band E, a region of synteny between mouse and human. 22  In addition to FISH techniques, we used PCR with a pair of hMLH1-specific oligonucleotides to analyze DNA from a rodent/human somatic cell hybrid mapping panel (Coriell Institute for Medial Research, Camden, N.J.). Our PCR results with the panel clearly indicate that hMLH1 maps to chromosome 3. The position of hMLH1 3p21.3-23 is coincident to a region known to harbor a second locus for HNPCC based upon linkage data.  
     [0148] We mapped the hPMS1 gene, as shown in FIG. 12, to the long (q) arm of chromosome 7 (either 7q11 or 7q22) and the mouse PMS1 to chromosome 5 band G, two regions of synteny between the human and the mouse. 22  We performed PCR using oligonucleotides specific to hPMS1 on DNA from a rodent/human cell panel. In agreement with the FISH data, the location of hPMS1 was confirmed to be on chromosome 7. These observations assure us that our human map position for hPMS1 to chromosome 7 is correct. The physical localization of hPMS1 is useful for the purpose of identifying families which may potentially have a cancer linked mutation in hPMS1.  
     [0149] Step 7 Using Genomic and cDNA Sequences to Identify Mutations in hPMS1 and hMLH1 Genes from HNPCC Families  
     [0150] We have analyzed samples collected from individuals in HNPCC families for the purpose of identifying mutations in hPMS1 or hMLH1 genes. Our approach is to design PCR primers based on our knowledge of the gene structures, to obtain exon/intron segments which we can compare to the known normal sequences. We refer to this approach as an “exon-screening”.  
     [0151] Using cDNA sequence information we have designed and are continuing to design hPMS1 and hMLH1 specific oligonucleotides to delineate exon/intron boundaries within genomic sequences. The hPMS1 and hMLH1 specific oligonucleotides were used to probe genomic clones for the presence of exons containing that sequence. Oligonucleotides that hybridized were used as primers for DNA sequencing from the genomic clones. Exon-intron junctions were identified by comparing genomic with cDNA sequences.  
     [0152] Amplification of specific exons from genomic DNA by PCR and sequencing of the products is one method to screen HNPCC families for mutations. 1,2  We have identified genomic clones containing hMLH1 cDNA information and have determined the structures of all intron/exon boundary regions which flanks the 19 exons of hMCH1.  
     [0153] We have used the exon-screening approach to examine the MLH1 gene of individuals from HNPCC families showing linkage to chromosome 3. 3  As will be discussed in more detail below, we identified a mutation in the MLH1 gene of one such family, consisting of a C to T substitution. We predict that the C to T mutation causes a serine to phenylalanine substitution in a highly-conserved region of the protein. We are continuing to identify HNPCC families from whom we can obtain samples in order to find additional mutations in hMLH1 and hPMS1 genes.  
     [0154] We are also using a second approach to identify mutations in hPMS1 and hMLH1. The approach is to design hPMS1 or hMLH1 specific oligonucleotide primers to produce first-strand cDNA by reverse transcription off RNA. PCR using gene-specific primers will allow us to amplify specific regions from these genes. DNA sequencing of the amplified fragments will allow us to detect mutations.  
     [0155] Step 8 Design Targeting Vectors to Disrupt Mouse PMS1 and MLH1 Genes in ES Cells; Study Mice Deficient in Mismatch Repair.  
     [0156] We constructed a gene targeting vector based on our knowledge of the genomic mouse PMS1 DNA structure. We used the vector to disrupt the PMS1 gene in mouse embryonic stem cells. 36  The cells were injected into mouse blastocysts which developed into mice that are chimeric (mixtures) for cells carrying the PMS1 mutation. The chimeric animals will be used to breed mice that are heterozygous and homozygous for the PMS1 mutation. These mice will be useful for studying the role of the PMS1 gene in the whole organism.  
     Human MLH1  
     [0157] The following discussion is a more detailed explanation of our experimental work relating to hMLH1. As mentioned above, to clone mammalian MLH genes, we used PCR techniques like those used to identify the yeast MSH1, MSH2 and MLH1 genes and the human MSH2 gene. 1, 2, 4, 14  As template in the PCR, we used double-stranded cDNA synthesized from poly (A+) enriched RNA prepared from cultured primary human fibroblasts. The degenerate oligonucleotides were targeted at the N-terminal amino acid sequences KELVEN and GFRGEA (see FIG. 3), two of the most conserved regions of the MutL family of proteins previously described for bacteria and yeast. 16,18,19  Two PCR products of the predicted size were identified, cloned and shown to encode a predicted amino acid sequence with homology to MutL-like proteins. These two fragments generated by PCR were used to isolate human cDNA and genomic DNA clones.  
     [0158] The oligonueleotide primers which we used to amplify human MutL-related sequences were 5′-CTTGATTCTAGAGC(T/C)TCNCCNC(T/G)(A/G)AANCC-3′ (SEQ ID NO: 139) and 5′-AGGTCGGAGCTCAA(A/G)GA(A/G)(T/C)TNGTNGANAA-3′ (SEQ ID NO: 140). PCR was carried out in 50 μL reactions containing cDNA template, 1.0 μM each primer, 5 IU of Taq polymerase (C) 50 mM KCl, 10 mM Tris buffer pH 7.5 and 1.5 mM MgCl. PCR was carried out for 35 cycles of 1 minute at 94 C.°, 1 minute at 43 C.° and 1.5 minutes at 62 C.°. Fragments of the expected size, approximately 212 bp, were cloned into pUC 19 and sequenced. The cloned MLH1 PCR products were labeled with a random primer labeling kit (RadPrime, Gibco BRL) and used to probe human cDNA and genomic cosmid libraries by standard procedures. DNA sequencing of double-stranded plasmid DNAs was performed as previously described.  
     [0159] The hMLH1 cDNA nucleotide sequence as shown in FIG. 3 encodes an open reading frame of 2268 bp. Also shown in FIG. 3 is the predicted protein sequence encoded for by the hMLH1 cDNA. The underlined DNA sequences are the regions of cDNA that correspond to the degenerate PCR primers that were originally used to amplify a portion of the MLH1 gene (nucleotides 118-135 and 343-359).  
     [0160]FIG. 4A shows 19 nucleotide sequences corresponding to portions of hMLH1. Each sequence includes one of the 19 exons, in its entirety, surrounded by flanking intron sequences. Target PCR primer cites are underlined. More details relating to the derivation and uses of the sequences shown in FIG. 4A, are set forth below.  
     [0161] As shown in FIG. 5, the hMLH1 protein is comprised of 756 amino acids and shares 41% identity with the protein product of the yeast DNA mismatch repair gene, MLH1. 4  The regions of the hMLH1 protein most similar to yeast MLH1 correspond to amino acids 11 through 317, showing 55% identity, and the last 13 amino acids which are identical between the two proteins. FIG. 5 shows an alignment of the predicted human MLH1 and  S. cerevisiae  MLH1 protein sequences. Amino acid identities are indicated by boxes, and gaps are indicated by dashes. The pair wise protein sequence alignment was performed with DNAStar MegAlign using the clustal method. 27  Pair wise alignment parameters were a ktuple of 1, gap penalty of 3, window of 5 and diagonals of 5. Furthermore, as shown in FIG. 13, the predicted amino acid sequences of the human and mouse MLH1 proteins show at least 74% identity.  
     [0162]FIG. 6 shows a phylogenetic tree of MutL-related proteins. The phylogenetic tree was constructed using the predicted amino acid sequences of 7 MutL-related proteins: human MLH1; mouse MLH1;  S. cerevisiae  MLH1;  S. cerevisiae  PMS1;  E. coli ; MutL;  S. typhimurium  MutL and  S. pneumoniae  HexB. Required sequences were obtained from GenBank release 7.3. The phylogenetic tree was generated with the PILEUP program of the Genetics Computer Group software using a gap penalty of 3 and a length penalty of 0.1. The recorded DNA sequences of hMLH1 and hPMS1 have been submitted to GenBank.  
     hMLH1 Intron Location and Intron/Exon Boundary Structures  
     [0163] In our previous U.S. patent application Ser. No. 08/209,521, we described the nucleotide sequence of a complimentary DNA (cDNA) clone of a human gene, hMLH1. The cDNA sequence of hMLH1 (SEQ ID NO: 4) is presented in this application in FIG. 3. We note that there may be some variability between individuals hMLH1 cDNA structures, resulting from polymorphisms within the human population, and the degeneracy of the genetic code.  
     [0164] In the present application, we report the results of our genomic sequencing studies. Specifically, we have cloned the human genomic region that includes the hMLH1 gene, with specific focus on individual exons and surrounding intron/exon boundary structures. Toward the ultimate goal of designing a comprehensive and efficient approach to identify and characterize mutations which confer susceptibility to cancer, we believe it is important to know the wild-type sequences of intron structures which flank exons in the hMLH1 gene. One advantage of knowing the sequence of introns near the exon boundaries, is that it makes it possible to design primer pairs for selectively amplifying entire individual exons. More importantly, it is also possible that a mutation in an intron region, which, for example, may cause a mRNA splicing error, could result in a defective gene product, i.e., susceptibility to cancer, without showing any abnormality in an exon region of the gene. We believe a comprehensive screening approach requires searching for mutations, not only in the exon or cDNA, but also in the intron structures which flank the exon boundaries.  
     [0165] We have cloned the human genomic region that includes hMLH1 using approaches which are known in the art, and other known approaches could have been used. We used PCR to screen a P1 human genomic library for the hMLH1 gene. We obtained four clones, two that contained the whole gene and two which lacked the C-terminus. We characterized one of the full length clones by cycle sequencing, which resulted in our definition of all intron/exon junction sequences for both sides of the 19 hMLH1 exons. We then designed multiple sets of PCR primers to amplify each individual exon (first stage primers) and verified the sequence of each exon and flanking intron sequence by amplifying several different genomic DNA samples and sequencing the resulting fragments using an ABI 373 sequencer. In addition, we have determined the sizes of each hMLH1 exon using PCR methods. Finally, we devised a set of nested PCR primers (second stage primers) for reamplification of individual exons. We have used the second stage primers in a multi-plex method for analyzing HNPCC families and tumors for hMLH1 mutations. Generally, in the nested PCR primer approach, we perform a first multi-plex amplification with four to eight sets of “first stage” primers, each directed to a different exon. We then reamplify individual exons from the product of the first amplification step, using a single set of second stage primers. Examples and further details relating to our use of the first and second stage primers are set forth below.  
     [0166] Through our genomic sequencing studies, we have identified all nineteen exons within the hMLH1 gene, and have mapped the intron/exon boundaries. One aspect of the invention, therefore, is the individual exons of the hMLH1 gene. Table 1 presents the nucleotide coordinates (i.e., the point of insertion of each intron within the coding region of the gene) of the hMLH1 exons (SEQ ID NOS: 25-43). The presented coordinates are based on the hMLH1 cDNA sequence, assigning position “1” to the “A” of the start “ATG” (which A is nucleotide 1 in SEQ ID NO: 4.  
                           TABLE 1                                   Intron Number   cDNA Sequence Coordinates                          intron 1   116 &amp; 117           intron 2   207 &amp; 208           intron 3   306 &amp; 307           intron 4   380 &amp; 381           intron 5   453 &amp; 454           intron 6   545 &amp; 546           intron 7   592 &amp; 593           intron 8   677 &amp; 678           intron 9   790 &amp; 791           intron 10   884 &amp; 885           intron 11   1038 &amp; 1039           intron 12   1409 &amp; 1410           intron 13   1558 &amp; 1559           intron 14   1667 &amp; 1668           intron 15   1731 &amp; 1732           intron 16   1896 &amp; 1897           intron 17   1989 &amp; 1990           intron 18   2103 &amp; 2104                      
 
     [0167] We have also determined the nucleotide sequence of intron regions which flank exons of the hMLH1 gene. SEQ ID NOS: 6-24 are individual exon sequences bounded by their respective upstream and downstream intron sequences. The same nucleotide structures are shown in FIG. 4A, where the exons are numbered from N-terminus to C-terminus with respect to the chromosomal locus. The 5-digit numbers indicate the primers used to amplify the exon. All sequences are numbered assuming the A of the ATG codon is nucleotide 1. The numbers in ( ) are the nucleotide coordinates of the coding sequence found in the indicated exon. Uppercase is intron. Lowercase is exon or non-translated sequences found in the mRNA/cDNA clone. Lowercase and underlined sequences correspond to primers. The stop codon at 2269-2271 is in italics and underlined.  
     [0168] Table 2 presents the sequences of primer pairs (“first stage” primers) which we have used to amplify individual exons together with flanking intron structures.  
                                   TABLE 2                           PRIMER       PRIMER               EXON NO.   LOCATION   PRIMER NO.   SEQ ID NO   PRIMER NUCLEOTIDE SEQUENCE                                                        1   upstream   18442   44   5′ aggcactgaggtgattggc           1   downstream   19109   45   5′ tcgtagcccttaagtgagc               2   upstream   19689   46   5′ taatatgtacattagagtagttg       2   downstream   19688   47   5′ cagagaaaggtcctgactc               3   upstream   19687   48   5′ agagatttggaaaatgagtaac       3   downstream   19786   49   5′ acaatgtcatcacaggagg               4   upstream   18492   50   5′ aacctttccctttggtgagg       4   downstream   18421   51   5′ gattactctgagacctaggc               5   upstream   18313   52   5′ gattttctcttttccccttggg       5   downstream   18179   53   5′ caaacaaagcttcaacaatttac               6   upstream   18318   54   5′ gggttttattttcaagtacttctatg       6   downstream   18317   55   5′ gctcagcaactgttcaatgtatgagc               7   upstream   19009   56   5′ ctagtgtgtgtttttggc       7   downstream   19135   57   5′ cataaccttatctccacc               8   upstream   18197   58   5′ ctcagccatgagacaataaatcc       8   downstream   18924   59   5′ ggttcccaaataatgtgatgg               9   upstream   18765   60   5′ caaaagcttcagaatctc       9   downstream   18198   61   5′ ctgtgggtgtttcctgtgagtgg               10   upstream   18305   62   5′ catgactttgtgtgaatgtacacc       10   downstream   18306   63   5′ gaggagagcctgatagaacatctg               11   upstream   18182   64   5′ gggctttttctccccctcec       11   downstream   19041   6S   5′ aaaatctgggctctcaeg               12   upstream   18579   66   5′ aattatacctcatactagc       12   downstream   18178   67   5′ gttttattacagaataaaggagg       12   downstream   19070   68   5′ aagccaaagttagaaggca               13   upstream   18420   69   5′ tgcaacccacaaaatttggc       13   downstream   18443   70   5′ ctttctceatttccaaaacc               14   upstream   19028   71   5′ tggtgtctctagttctgg       14   downstream   18897   72   5′ cattgttgtagtagctctgc               15   upstream   19025   73   5′ cccatttgtcccaaetgg       15   downstream   18575   74   5′ cggtcagttgaaatgtcag               16   upstream   18184   75   5′ catttggatgctccgttaaagc       16   downstream   18314   76   5′ cacccggctggaaattttatttg               17   upstream   18429   77   5′ ggaaaggcactggagaaatggg       17   downstream   18315   78   5′ ccctccagcaeacatgcatgtaccg               18   upstream   18444   79   5′ taagtagtctgtgatctccg       18   downstream   18581   80   5′ atgtatgaggtcctgtcc               19   upstream   18638   81   5′ gacaccagtgtatgttgg       19   downstream   18637   82   5′ tgagaaagaagaacacatccc                  
 
     [0169] Additionally, we have designed a set of “second stage” amplification primers, the structures of which are shown below in Table 3. We use the second stage primers in conjunction with the first stage primers in a nested amplification protocol, as described below.  
                                   TABLE 3                           PRIMER       PRIMER               EXON NO.   LOCATION   PRIMER NO.   SEQ ID NO   PRIMER NUCLEOTIDE SEQUENCE                                                        1   upstream   19295   83    5′ tgtaaaacgacggccagtcactgaggtgattggctgaa           1   downstream   19446   84   *5′ tagcccttaagtgagcccg               2   upstream   18685   85    5′ tgtaaaacgacggccagttacattagagtagttgcaga       2   downstream   19067   86   *5′ aggtcctgactcttccatg               3   upstream   18687   87    5′ tgtaaaacgaeggccagtttggaaaatgagtaacatgatt       3   downstream   19068   88   *5′ tgtcatcacaggaggatat               4   upstream   19294   89    5′ tgtaaaacgacggccagtctttccctttggtgaggtga       4   downstream   19077   90   *5′ tactctgagacctaggccca               5   upstream   19301   91    5′ tgtaaaacgacggccagttctcttttccccttgggattag       5   downstream   19046   92   *5′ acaaagcttcaacaatttactct               6   upstream   19711   93    5′ tgtaaaacgacggccagtgttttattttcaagtacttctatgaatt       6   downstream   19079   94   *5′ cagcaaetgttcaatgtatgagcact               7   upstream   19293   95  5′ tgtaaaaegacggccagtgtgtgtgtttttggcaac       7   downstream   19435   96   *5′ aaccttatctccaccagc               8   upstream   19329   97    5′ tgtaaaacgacggccagtagccatgagacaataaatccttg       8   downstream   19450   98   *5′ tcccaaataatgtgatggaatg               9   upstream   19608   99    5′ tgtaaaacgacggccagtaagcttcagaatctctttt       9   downstream   19449   100   *5′ tgggtgtttcctgtgagtggatt               10   upstream   19297   101    5′ tgtaaaacgacggccagtactttgtgtgaatgtacacctgtg       10   downstream   19081   102   *5′ gagagcctgatagaacatctgttg               11   upstream   19486   103    5′ tgtaaaaegacggccagtctttttctccccctcccacta       11   downstream   19455   104   *5′ tctgggctctcacgtct               12   upstream   20546   105   *5′ cttattctgagtctctcc       12   downstream   20002   106    5′ tgtaaaacgacggccagtgtttgctcagaggctgc       12   upstream   19829   107   *5′ gatggttcgtacagattcccg       12   downstream   19385   108    5′ tgtaaaacgacggccagtttattacagaataaaggaggtag               13   upstream   19300   109    5′ tgtaaaacgacggccagtaacccacaaaatttggctaag       13   downstream   19078   110   *5′ tctccatttccaaaaccttg               14   upstream   19456   111   *5′ tgtctctagttctggtgc       14   downstream   19472   112    5′ tgtaaaacgacggccagttgttgtagtagctctgcttg               15   upstream   19697   113   *5′ atttgtcccaactggttgta       15   downstream   19466   114    5′ tgtaaaacgacggccagttcagttgaaatgtcagaaagtg               16   upstream   19269   115    5′ tgtaaaacgacggccagt       16   downstream   19047   116   *5′ ccggctggaaattttatttggag               17   upstream   19298   117    5′ tgtaaaacgacggccagtaggcactggagaaatgggatttg       17   downstream   19080   118   *5′ tccagcacacatgcatgtaccgaaat               18   upstream   19436   119   *5′ gtagtctgtgatctccgttt       18   downstream   19471   120    5′ tgtaaaacgacggccagttatgaggtcctgtcctag               19   upstream   19447   121   *5′ accagtgtatgttgggatg       19   downstream   19330   122    5′ tgtaaaacgacggccagtgaaagaagaacacatcccaca                  
 
     [0170] In Table 3 an asteric (*) indicates that the 5′ nucleotide is biotinylated. Exons 1-7, 10, 13 and 16-19 can be specifically amplified in PCR reactions containing either 1.5 mM or 3 mM MgCl 2 . Exons 11 and 14 can only be specifically amplified in PCR reactions containing 1.5 mM MgCl 2  and exons 8, 9, 12 and 15 can only be specifically amplified in PCR reactions containing 3 mM MgCl 2 . With respect to exon 12, the second stage amplification primers have been designed so that exon 12 is reamplified in two halves. The 20546 and 20002 primer set amplifies the N-terminal half. The primer set 19829 and 19835 amplifies the C-terminal half. An alternate primer for 18178 is 19070.  
     [0171] The hMLH1 sequence information provided by our studies and disclosed in this application and preceding related applications, may be used to design a large number of different oligonucleotide primers for use in identifying hMLH1 mutations that correlate with cancer susceptibility and/or with tumor development in an individual, including primers that will amplify more than one exon (and/or flanking intron sequences) in a single product band.  
     [0172] One of ordinary skill in the art would be familiar with considerations important to the design of PCR primers for use to amplify the desired fragment or gene. 37  These considerations may be similar, though not necessarily identical to those involved in design of sequencing primers, as discussed above. Generally it is important that primers hybridize relatively specifically (i.e. have a T m  of greater than about 55-degrees° C., and preferably around 60-degrees° C.). For most cases, primers between about 17 and 25 nucleotides in length work well. Longer primers can be useful for amplifying longer fragments. In all cases, it is desirable to avoid using primers that are complementary to more than one sequence in the human genome, so that each pair of PCR primers amplifies only a single, correct fragment. Nevertheless, it is only absolutely necessary that the correct band be distinguishable from other product bands in the PCR reaction.  
     [0173] The exact PCR conditions (e.g. salt concentration, number of cycles, type of DNA polymerase, etc.) can be varied as known in the art to improve, for example, yield or specificity of the reaction. In particular, we have found it valuable to use nested primers in PCR reactions in order to reduce the amount of required DNA substrate and to improve amplification specificity.  
     [0174] Two examples follow. The first example illustrates use of a first stage primer pair (SEQ ID NOS: 69 and 70) to amplify intron/exon segment (SEQ ID NO: 18). The second example illustrates use of second stage primers to amplify a target intron/exon segment from the product of a first PCR amplification step employing first stage primers.  
     EXAMPLE 1  
     Amplification of hMLH1 Genomic Clones from a P1 Phage Library  
     [0175] 25 ng genomic DNA (or 1 ng of a P1 phage can be used) was used in PCR reactions including:  
     [0176] 0.05 mM dNTPs  
     [0177] 50 mM KCl  
     [0178] 3 mM Mg  
     [0179] 10 mM Tris-HCl pH 8.5  
     [0180] 0 . 01 % gelatin  
     [0181] 5 μM primers  
     [0182] Reactions were performed on a Perkin-Elmer Cetus model 9600 thermal cycler. Reactions were incubated at 95-degrees° C. for 5 minutes, followed by 35 cycles (30 cycles from a PI phage) of:  
     [0183] 94-degrees° C. for 30 seconds  
     [0184] 55-degrees° C. for 30 seconds  
     [0185] 72-degrees° C. for 1 minute.  
     [0186] A final 7 minute extension reaction was then performed at 72′-degrees C. Desirable P1 clones were those from which an approximately bp product band was produced.  
     EXAMPLE 2  
     Amplification of hMLH1 Sequences from Genomic DNA Using Nested PCR Primers  
     [0187] We performed two-step PCR amplification of hMLH1 sequences from genomic DNA as follows. Typically, the first amplification was performed in a 25 microliter reaction including:  
     [0188] 25 ng of chromosomal DNA  
     [0189] Perkin-Elmer PCR buffer II (any suitable buffer could be used)  
     [0190] 3 mM MgCl 2    
     [0191] 50 μM each dNTP  
     [0192] Taq DNA polymerase  
     [0193] 5 μM primers (SEQ ID NOS: 69, 70)  
     [0194] and incubated at 95-degrees° C. for 5 minutes, followed by 20 cycles of:  
     [0195] 94-degrees° C. for 30 seconds  
     [0196] 55-degrees° C. for 30 seconds.  
     [0197] The product band was typically small enough (less than an approximately 500 bp) that separate extension steps were not performed as part of each cycle. Rather, a single extension step was performed, at 72-degrees° C. for 7 minutes, after the 20 cycles were completed. Reaction products were stored at 4-degrees° C.  
     [0198] The second amplification reaction, usually 25 or 50 microliters in volume, included:  
     [0199] 1 or 2 microliters (depending on the volume of the reaction) of the first amplification reaction product  
     [0200] Perkin-Elmer PCR buffer II (any suitable buffer could be used)  
     [0201] 3 mM or MgCl 2    
     [0202] 50 μM each dNTP  
     [0203] Taq DNA polymerase  
     [0204] 5 μM nested primers (SEQ ID NOS: 109, 110),  
     [0205] and was incubated at 95-degrees° C. for 5 minutes, followed by 20-25 cycles of:  
     [0206] 94-degrees° C. for 30 seconds  
     [0207] 55-degrees° C. for 30 seconds  
     [0208] a single extension step was performed, at 72-degrees° C. for 7 minutes, after the cycles were completed. Reaction products were stored at 4-degrees° C.  
     [0209] Any set of primers capable of amplifying a target hMLH1 sequence can be used in the first amplification reaction. We have used each of the primer sets presented in Table 2 to amplify an individual hMLH1 exon in the first amplification reaction. We have also used combinations of those primer sets, thereby amplifying multiple individual hMLH1 exons in the first amplification reaction.  
     [0210] The nested primers used in the first amplification step were designed relative to the primers used in the first amplification reaction. That is, where a single set of primers is used in the first amplification reaction, the primers used in the second amplification reaction should be identical to the primers used in the first reaction except that the primers used in the second reaction should not include the 5′-most nucleotides of the first amplification reaction primers, and should extend sufficiently more at the 3′ end that the T m  of the second amplification primers is approximately the same as the T m  of the first amplification reaction primers. Our second reaction primers typically lacked the 3 5′-most nucleotides of the first amplification reaction primers, and extended approximately 3-6 nucleotides farther on the 3′ end. SEQ ID NOS: 109, 110 are examples of nested primer pairs that could be used in a second amplification reaction when SEQ ID NOS: 69 and 70 were used in the first amplification reaction.  
     [0211] We have also found that it can be valuable to include a standard sequence at the 5′ end of one of the second amplification reaction primers to prime sequencing reactions. Additionally, we have found it useful to biotinylate that last nucleotide of one or both of the second amplification reaction primers so that the product band can easily be purified using magnetic beads 40  and then sequencing reactions can be performed directly on the bead-associated products. 41-45    
     [0212] For additional discussion of multiplex amplification and sequencing methods, see References by Zu et al. and Espelund et al. 46,47    
     hMLH1 Link to Cancer  
     [0213] As a first step to determine whether hMLH1 was a candidate for the 1HNPCC locus on human chromosome 3p21-23, 3  we mapped hMLH1 by fluorescence in situ hybridization (FISH). 20,21  We used two separate genomic fragments (data not shown) of the hMLH1 gene in FISH analysis. Examination of several metaphase chromosome spreads localized hMLH1 to chromosome 3p21.3-23.  
     [0214] Panel A of FIG. 7 shows hybridization of hMLH1 probes in a metaphase spread. Biotinylated hMLH1 genomic probes were hybridized to banded human metaphase chromosomes as previously described. 20,21  Detection was performed with fluorescein isothiocyanate (FITC)-conjugated avidin (green signal); chromosomes, shown in blue, were counterstained with 4′6-diamino-2-phenylindole (DAPI). Images were obtained with a cooled CCD camera, enhanced, pseudocoloured and merged with the following programs: CCD Image Capture; NIH Image 1.4; Adobe Photoshop and Genejoin Maxpix respectively. Panel B of FIG. 7 shows a composite of chromosome 3 from multiple metaphase spreads aligned with the human chromosome 3 ideogram. Region of hybridization (distal portion of 3p21.3-23) is indicated in the ideogram by a vertical bar.  
     [0215] As independent confirmation of the location of hMLH1 on chromosome 3, we used both PCR with a pair of hMLH1-specific oligonucleotides and Southern blotting with a hMLH1-specific probe to analyze DNA from the NIGMS2 rodent/human cell panel (Coriell Inst. for Med. Res., Camden, N.J., USA). Results of both techniques indicated chromosome 3 linkage. We also mapped the mouse MLH1 gene by FISH to chromosome 9 band E. This is a position of synteny to human chromosome 3p. 22  Therefore, the hMLH1 gene localizes to 3p21.3-23, within the genomic region implicated in chromosome 3-linked HNPCC families. 3    
     [0216] Next, we analyzed blood samples from affected and unaffected individuals from two chromosome-3 candidate HNPCC families  3  for mutations. One family, Family 1, showed significant linkage (lod score=3.01 at recombination fraction of 0) between HNPCC and a marker on 3p. For the second family, Family 2, the reported lod score (1.02) was below the commonly accepted level of significance, and thus only suggested linkage to the same marker on 3p. Subsequent linkage analysis of Family 2 with the microsatellite marker D3S1298 on 3p21.3 gave a more significant lod score of 1.88 at a recombination fraction of 0. Initially, we screened for mutations in two PCR-amplified exons of the hMLH1 gene by direct DNA sequencing (FIG. 4). We examined these two exons from three affected individuals of Family 1, and did not detect any differences from the expected sequence. In Family 2, we observed that four individuals affected with colon cancer are heterozygous for a C to T substitution in an exon encoding amino acids 41-69, which corresponds to a highly-conserved region of the protein (FIG. 9). For one affected individual, we screened PCR-amplified cDNA for additional sequence differences. The combined sequence information obtained from the two exons and cDNA of this one affected individual represents 95% (i.e. all but the first 116 bp) of the open reading frame. We observed no nucleotide changes other than the C to T substitution. In addition, four individuals from Family 2, predicted to be carriers based upon linkage data, and as yet unaffected with colon cancer, were found to be heterozygous for the same C to T substitution. Two of these predicted carriers are below and two are above the mean age of onset (50 years) in this particular family. Two unaffected individuals examined from this same family, both predicted by linkage data to be non carriers, showed the expected normal sequence at this position. Linkage analysis that includes the C to T substitution in Family 2 gives a lod score of 2.23 at a recombination fraction 0. Using low stringency cancer diagnostic criteria, we calculated a lod score of 2.53. These data indicate the C to T substitution shows significant linkage to the HNPCC in Family 2.  
     [0217]FIG. 8 shows sequence chromatograms indicating a C to T transition mutation that produces a non-conservative amino acid substitution at position 44 of the hMLH1 protein. Sequence analysis of one unaffected (top panels, plus and minus strands) and one affected individual (lower panels, plus and minus strands) is presented. The position of the heterozygous nucleotide is indicated by an arrow. Analysis of the sequence chromatographs indicates that there is sufficient T signal in the C peak and enough A signal in the G peak for the affected individuals to be heterozygous at this site.  
     [0218] To determine whether this C to T substitution was a polymorphism, we sequenced this same exon amplified from the genomic DNA from 48 unrelated individuals and observed only the normal sequence. We have examined an additional 26 unrelated individuals using allele specific oligonucleotide (ASO) hybridization analysis. 33  The ASO sequences (SEQ ID NOS: 141 and 142, respectively) which we used are:  
     [0219] 5′-ACTTGTGGATTTTGC-3′ and 5′-ACTTGTGAATTTTGC-3′.  
     [0220] Based upon direct DNA sequencing and ASO analysis, none of these 74 unrelated individuals carry the C to T substitution. Therefore, the C to T substitution observed in Family 2 individuals is not likely to be a polymorphism. As mentioned above, we did not detect this same C to T substitution in affected individuals from a second chromosome 3-linked family, Family 1.3 We are continuing to study individuals of Family 1 for mutations in hMLH1.  
     [0221] Table 4 below summarizes our experimental analysis of blood samples from affected and unaffected individuals from Family 2 and unrelated individuals.  
                       TABLE 4                               Number of Individuals with               C to T Mutation/       FAMILY 2   Status   Number of Individuals Tested                      Affected   4/4           Predicted Carriers   4/4           Predicted Non-carriers   0/2           Unrelated Individuals    0/74                  
 
     [0222] Based on several criteria, we suggest that the observed C to T substitution in the coding region of hMLH1 represents the mutation that is the basis for HNPCC in Family 2. 3  First, DNA sequence and ASO analysis did not detect the C to T substitution in 74 unrelated individuals. Thus, the C to T substitution is not simply a polymorphism. Second, the observed C to T substitution is expected to produce a serine to phenylalanine change at position 44 (See FIG. 9). This amino acid substitution is a non-conservative change in a conserved region of the protein (FIGS. 3 and 9). Secondary structure predictions using Chou-Fasman parameters suggest a helix-turn-beta sheet structure with position 44 located in the turn. The observed Ser to Phe substitution, at position 44 lowers the prediction for this turn considerably, suggesting that the predicted amino acid substitution alters the conformation of the hMLH1 protein. The suggestion that the Ser to Phe substitution is a mutation which confers cancer susceptibility is further supported by our experiments which show that an analogous substitution (alanine to phenylalanine) in a yeast MLH1 gene results in a nonfunctional mismatch repair protein. In bacteria and yeast, a mutation affecting DNA mismatch repair causes comparable increases in the rate of spontaneous mutation including additions and deletions within dinucleotide repeats. 4,5,11,13,14,15,16  In humans, mutation of hMSH2 is the basis of chromosome-2 HNPCC, 1,2  tumors which show microsatellite instability and an apparent defect in mismatch repair. 12  Chromosome 3-linked HNPCC is also associated with instability of dinucleotide repeats. 3  Combined with these observations, the high degree of conservation between the human MLH1 protein and the yeast DNA mismatch repair protein MLH1 suggests that hMLH1 is likely to function in DNA mismatch repair. During isolation of the hMLH1 gene, we identified the hPMS1 gene. This observation suggests that mammalian DNA mismatch repair, like that in yeast, 4  may require at least two MutL-like proteins.  
     [0223] It should be noted that it appears that different HNPCC families show different mutations in the MLH1 gene. As explained above, affected individuals in Family 1 showed “tight linkage” between HNPCC and a locus in the region of 3p21-23. However, affected individuals in Family 1 do not have the C to T mutation found in Family 2. It appears that the affected individuals in Family 1 have a different mutation in their MLH1 gene. Further, we have used the structure information and methods described in this application to find and characterize another hMLH1 mutation which apparently confers cancer susceptibility in heterozygous carriers of the mutant gene in a large English HNPCC family. The hMLH1 mutation in the English family is a +1 T frameshift which is predicted to lead to the synthesis of a truncated hMLH1 proteiri. Unlike, for example, sickle cell anemia, in which essentially all known affected individuals have the same mutation multiple hMLH1 mutations have been discovered and linked to cancer. Therefore, knowledge of the entire cDNA sequence for hMLH1 (and probably hPMS1), as well as genomic sequences particularly those that surround exons, will be useful and important for characterizing mutations in families identified as exhibiting a high frequency of cancer.  
     [0224] Subsequent to our discovery of a cancer conferring mutation in hMLH1, studies by others have resulted in the characterization of at least 5 additional mutations in hMLH1, each of which appears to have conferred cancer susceptibility to individuals in at least one HNPCC family. For example, Papadopoulos et al. indentified such as a mutation, characterized by an in-frame deletion of 165 base pairs between codons 578 to 632. In another family, Papadopoulos et al. observed an hMLH1 mutation, characterized by a frame shift and substitution of new amino acids, namely, a 4 base pair deletion between codons 727 and 728. Papadopoulos et al. also reports an hMLH1 cancer linked mutation, characterized by an extension of the COOH terminus, namely, a 4 base pair insertion between codons 755 and 756. 38    
     [0225] In summary, we have shown that DNA mismatch repair gene hMLH1 which is likely to be the hereditary nonpolyposis colon cancer gene previously localized by linkage analysis to chromosome 3p21-23. 3  Availability of the hMLH1 gene sequence will facilitate the screening of HNPCC families for cancer-linked mutations. In addition, although loss of heterozygosity (LOH) of linked markers is not a feature of either the 2p or 3p forms of HNPCC, 3,6  LOH involving the 3p21.3-23 region has been observed in several human cancers. 24,26  This suggests the possibility that hMLH1 mutation may play some role in these tumors.  
     Human PMS1  
     [0226] Human PMS1 was isolated using the procedures discussed with reference to FIG. 1. FIG. 10 shows the entire hPMS1 cDNA nucleotide sequence. FIG. 11 shows an alignment of the predicted human and yeast PMS1 protein sequences. We determined by FISH analysis that human PMS1 is located on chromosome 7. Subsequent to our discovery of hPMS1, others have identified mutations in the gene which appear to confer HNPCC susceptibility. 39    
     Mouse MLH1  
     [0227] Using the procedure outlined above with reference to FIG. 1, we have determined a partial nucleotide sequence of mouse MLH1 cDNA, as shown in FIG. 12 (SEQ ID NO: 135). FIG. 13 shows the corresponding predicted amino acid sequence for mMLH1 protein (SEQ ID NO: 136) in comparison to the predicted hMLH1 protein sequence (SEQ ID NO: 5). Comparison of the mouse and human MLH1 proteins as well as the comparison of hMLH1 with yeast MLH1 proteins, as shown in FIG. 9, indicate a high degree of conservation.  
     Mouse PMS1  
     [0228] Using the procedures discussed above with reference to FIG. 1, we isolated and sequenced the mouse PMS1 gene, as shown in FIG. 14 (SEQ ID NO: 137). This cDNA sequence encodes a predicted protein of 864 amino acids (SEQ ID NO: 138), as shown in FIG. 15, where it is compared to the predicted amino acid sequence for hPMS1 (SEQ ID NO: 133). The degree of identity between the predicted mouse and human PMS1 proteins is high, as would be expected between two mammals. Similarly, as noted above, there is a strong similarity between the human PMS1 protein and the yeast DNA mismatch repair protein PMS1, as shown in FIG. 11. The fact that yeast PMS1 and MLH1 function in yeast to repair DNA mismatches, strongly suggests that human and mice PMS1 and MLH 1 are also mismatch repair proteins.  
     Uses for Mouse MLH1 and PMS1  
     [0229] We believe our isolation and characterization of mMLH1 and mPMS1 genes will have many research applications. For example, as already discussed above, we have used our knowledge of the mPMS1 gene to produce antibodies which react specifically with hPMS1. We have already explained that antibodies directed to the human proteins, MLH1 or PMS1 may be used for both research purposes as well as diagnostic purposes.  
     [0230] We also believe that our knowledge of mPMS1 and mMLH1 will be useful for constructing mouse models in order to study the consequences of DNA mismatch repair defects. We expect that mPMS1 or mMLH1 defective mice will be highly prone to cancer because chromosome 2p and 3p-associated HNPCC are each due to a defect in a mismatch repair gene. 1,2  As noted above, we have already produced chimeric mice which carry an mPMS1 defective gene. We are currently constructing mice heterozygous for mPMS1 or mMLH1 mutation. These heterozygous mice should provide useful animal models for studying human cancer, in particular HNPCC. The mice will be useful for analysis of both intrinsic and extrinsic factors that determine cancer risk and progression. Also, cancers associated with mismatch repair deficiency may respond differently to conventional therapy in comparison to other cancers. Such animal models will be useful for determining if differences exist, and allow the development of regimes for the effective treatment of these types of tumors. Such animal models may also be used to study the relationship between hereditary versus dietary factors in carcinogenesis.  
     Distinguishing Mutations From Polymorphisms  
     [0231] For studies of cancer susceptibility and for tumor identification and characterization, it is important to distinguish “mutations” from “polymorphisms”. A “mutation” produces a “non-wild-type allele” of a gene. A non-wild-type allele of a gene produces a transcript and/or a protein product that does not function normally within a cell. “Mutations” can be any alteration in nucleotide sequence including insertions, deletions, substitutions, and rearrangements.  
     [0232] “Polymorphisms”, on the other hand, are sequence differences that are found within the population of normally-functioning (i.e., “wild-type”) genes. Some polymorphisms result from the degeneracy of the nucleic acid code. That is, given that most amino acids are encoded by more than one triplet codon, many different nucleotide sequences can encode the same polypeptide. Other polymorphisms are simply sequence differences that do not have a significant effect on the function of the gene or encoded polypeptide. For example, polypeptides can often tolerate small insertions or deletions, or “conservative” substitutions in their amino acid sequence without significantly altering function of the polypeptide.  
     [0233] “Conservative” substitutions are those in which a particular amino acid is substituted by another amino acid of similar chemical characteristics. For example, the amino acids are often characterized as “non-polar (hydrophobic)” including alanine, leucine, isoleucine, valine, proline, phenylaline, tryptophan, and methionine; “polar neutral”, including glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; “positively charged (basic)”, including arginine, lysine, and histidine; and “negatively charged (acidic)”, including aspartic acid and glutamic acid. A substitution of one amino acid for another amino acid in the same group is generally considered to be “conservative”, particularly if the side groups of the two relevant amino acids are of a similar size.  
     [0234] The first step in identifying a mutation or polymorphism in a mismatch repair gene sequence involves identification, using available techniques including those described herein, of a mismatch repair gene, (or gene fragment) sequence that differs from a known, normal (e.g. wild-type) sequence of the same mismatch repair gene (or gene fragment). For example, a hMLH1 gene (or gene fragment) sequence could be identified that differs in at least one nucleotide position from a known normal (e.g. wild-type) hMLH1 sequence such as any of SEQ ID NOS: 6-24.  
     [0235] Mutations can be distinguished from polymorphisms using any of a variety of methods, perhaps the most direct of which is data collection and correlation with tumor development. That is, for example, a subject might be identified whose hMLH1 gene sequence differs from a sequence reported in SEQ ID. NOS: 6-24, but who does not have cancer and has no family history of cancer. Particularly if other, preferably senior, members of that subject&#39;s family have hMLH1 gene sequences that differ from SEQ ID NOS: 6-24 in the same way(s), it is likely that subject&#39;s hMLH1 gene sequence could be categorized as a “polymorphism”. If other, unrelated individuals are identified with the same hMLH1 gene sequence and no family history of cancer, the categorization may be confirmed.  
     [0236] Mutations that are responsible for conferring genetic susceptibility to cancer can be identified because, among other things, such mutations are likely to be present in all tissues of an affected individual and in the germ line of at least one of that individual&#39;s parents, and are not likely to be found in unrelated families with no history of cancer.  
     [0237] When distinguishing mutations from polymorphisms, it can sometimes be valuable to evaluate a particular sequence difference in the presence of at least one known mismatch repair gene mutation. In some instances, a particular sequence change will not have a detectable effect (i.e., will appear to be a polymorphism) when assayed alone, but will, for example, increase the penetrance of a known mutation, such that individuals carrying both the apparent polymorphism difference and a known mutation have higher probability of developing cancer than do individuals carrying only the mutation. Sequence differences that have such an effect are properly considered to be mutations, albeit weak ones.  
     [0238] As discussed above and previously (U.S. patent application Ser. Nos. 08/168,877 and 08/209,521), mutations in mismatch repair genes or gene products produced non-wild-type versions of those genes or gene products. Some mutations can therefore be distinguished from polymorphisms by their functional characteristics in in vivo or in vitro mismatch repair assays. Any available mismatch repair assay can be used to analyze these characteristics. 49-63  It is generally desirable to utilize more than one mismatch repair assay before classifying a sequence change as a polymorphism, since some mutations will have effects that will not be observed in all assays.  
     [0239] For example, a mismatch repair gene containing a mutation would not be expected to be able to replace an endogenous copy of the same gene in a host cell without detectably affecting mismatch repair in that cell; whereas a mismatch repair gene containing a sequence polymorphism would be expected to be able to replace an endogenous copy of the same gene in a host cell without detectably affecting mismatch repair in that cell. We note that for such “replacement” studies, it is generally desirable to introduce the gene to be tested into a host cell of the same (or at least closely related) species as the cell from which the test gene was derived, to avoid complications due to, for example, the inability of a gene product from one species to interact with other mismatch repair gene products from another species. Similarly, a mutant mismatch repair protein would not be expected to function normally in an in vitro mismatch repair system (preferably from a related organism); whereas a polymorphic mismatch repair protein would be expected to function normally.  
     [0240] The methods described herein and previously allow identification of different kinds of mismatch repair gene mutations. The following examples illustrate protocols for distinguishing mutations from polymorphisms in DNA mismatch repair genes.  
     EXAMPLE 3  
     [0241] We have developed a system for testing in yeast,  S. cerevisiae  the functional significance of mutations found in either the hMLH1 or hPMS1 genes. The system is described in this application using as an example, the serine (SER) to phenylalanine (PHE) causing mutation in hMLH1 that we found in a family with HNPCC, as described above. We have derived a yeast strain that it is essentially deleted for its MLH1 gene and hence is a strong mutator (i.e., 1000 fold above the normal rate in a simple genetic marker assay involving reversion from growth dependence on a given amino acid to independence (reversion of the hom3-10 allele, Prolla, Christie and Liskay, Mol Cell Biol, 14:407-415, 1994). When we placed the normal yeast MLH1 gene (complete with all known control regions) on a yeast plasma that is stably maintained as a single copy into the MLH1-deleted strain, the mutator phenotype is fully corrected using the reversion to amino acid independence assay. However, if we introduce a deleted copy of the yeast MLH1 there is no correction. We next tested the mutation that in the HNPCC family caused a SER to PHE alteration. We found that the resultant mutant yeast protein cannot correct the mutator phenotype, strongly suggesting that the alteration from the wild-type gene sequence probably confers cancer susceptibility, and is therefore classified as a mutation, not a polymorphism. We subsequently tested proteins engineered to contain other amino acids at the “serene” position and found that most changes result in a fully mutant, or at least partially mutant phenotype.  
     [0242] As other “point” mutations in MLH1 and PMS1 genes are found in cancer families, they can be engineered into the appropriate yeast homolog gene and their consequence on protein function studied. In addition, we have identified a number of highly conserved amino acids in both the MLH1 and PMS1 genes. We also have evidence that hMLH1 interacts with yeast PMS1. This finding raises the possibility that mutations observed in the hMLH1 gene can be more directly tested in the yeast system. We plan to systematically make mutations that will alter the amino acid at these conserved positions and determine what amino acid substitutions are tolerated and which are not. By collecting mutation information relating to hMLH1 and hPMS1, both by determining and documenting actual found mutations in HNPCC families, and by artificially synthesizing mutants for testing in experimental systems, it may be eventually possible to practice a cancer susceptibility testing protocol which, once the individuals hMLH1 or hPMS1 structure is determined, only requires comparison of that structure to known mutation versus polymorphism data.  
     EXAMPLE 4  
     [0243] Another method which we have employed to study physical interactions between hMLH1 and hPMS1, can also be used to study whether a particular alteration in a gene product results in a change in the degree of protein-protein interaction. Information concerning changes in protein-protein interaction may demonstrate or confirm whether a particular genomic variation is a mutation or a polymorphism. Following our labs findings on the interaction between yeast MLH1 and PMS1 proteins in vitro and in vivo, (U.S. patent application Ser. No. 08/168,877), the interaction between the human counterparts of these two DNA mismatch repair proteins was tested. The human MLH1 and human PMS1 proteins were tested for in vitro interaction using maltose binding protein (MBP) affinity chromatography. hMLH1 protein was prepared as an MBP fusion protein, immobilized on an amylose resin column via the MBP, and tested for binding to hPMS1, synthesized in vitro. The hPMS1 protein bound to the MBP-hMLH1 matrix, whereas control proteins showed no affinity for the matrix. When the hMLH1 protein, translated in vitro, was passed over an MBP-hPMS1 fusion protein matrix, the hMLH1 protein bound to the MBP-hPMS1 matrix, whereas control proteins did not.  
     [0244] Potential in vivo interactions between hMLH1 and HPMS1 were tested using the yeast “two hybrid” system. 28  Our initial results indicate that hMLH1 and hPMS1 interact in vivo in yeast. The same system can also be used to detect changes in protein-protein interaction which result from changes in gene or gene product structure and which have yet to be classified as either a polymorphism or a mutation which confers cancer susceptibility.  
     Detection of HNPCC Families and Their Mutation(s)  
     [0245] It has been estimated that approximately 1,000,000 individuals in the United States carry (are heterozygous for) an HNPCC mutant gene. 29  Furthermore, estimates suggest that 50-60% of HNPCC families segregate mutations in the MSH2 gene that resides on chromosome 2p. 1,2  Another significant fraction appear to be associated with the HNPCC gene that maps to chromosome 3p21-22, presumably due to hMLH1 mutations such as the C to T transition discussed above. Identification of families that segregate mutant alleles of either the hMSH2 or hMLH1 gene, and the determination of which individuals in these families actually have the mutation will be of great utility in the early intervention into the disease. Such early intervention will likely include early detection through screening and aggressive follow-up treatment of affected individuals. In addition, determination of the genetic basis for both familial and sporadic tumors could direct the method of therapy in the primary tumor, or in recurrences.  
     [0246] Initially, HNPCC candidate families will be diagnosed partly through the study of family histories, most likely at the local level, e.g., by hospital oncologists. One criterion for HNPCC is the observation of microsatellite instability in individual&#39;s tumors. 3,6  The presenting patient would be tested for mutations in hMSH2, hMLH1, hPMS1 and other genes involved in DNA mismatch repair as they are identified. This is most easily done by sampling blood from the individual. Also highly useful would be freshly frozen tumor tissue. It is important to note for the screening procedure, that affected individuals are heterozygous for the offending mutation in their normal tissues.  
     [0247] The available tissues, e.g., blood and tumor, are worked up for PCR-based mutation analysis using one or both of the following procedures:  
     [0248] 1) Linkage Analysis with a Microsatellite Marker Tightly Linked to the hMLH1 Gene.  
     [0249] One approach to identify cancer prone families with a hMLH1 mutation is to perform linkage analysis with a highly polymorphic marker located within or tightly linked to hMLH1. Microsatellites are highly polymorphic and therefore are very useful as markers in linkage analysis. Because we possess the hMLH1 gene on a single large genomic fragment in a P1 phage clone (˜100 kbp), it is very likely that one or more microsatellites, e.g., tracts of dinucleotide repeats, exist within, or very close to, the hMLH1 gene. At least one such microsatellite has been reported. 38  Once such markers have been identified, PCR primers will be designed to amplify the stretches of DNA containing the microsatellites. DNA of affected and unaffected individuals from a family with a high frequency of cancer will be screened to determine the segregation of the MLH1 markers and the presence of cancer. The resulting data can be used to calculate a lod score and hence determine the likelihood of linkage between hMLH1 and the occurrence of cancer. Once linkage is established in a given family, the same polymorphic marker can be used to test other members of the kindred for the likelihood of their carrying the hMLH1 mutation.  
     [0250] 2) Sequencing of Reverse Transcribed cDNA.  
     [0251] a) RNA from affected individuals, unaffected and unrelated individuals is reverse transcribed (RT&#39;d), followed by PCR to amplify the cDNA in 4-5 overlapping portions. 34,37  It should be noted that for the purposes of PCR, many different oligonucleotide primer pair sequences may potentially be used to amplify relevant portions of an individual&#39;s hMLH1 or hPMS1 gene for genetic screening purposes. With the knowledge of the cDNA structures for the genes, it is a straight-forward exercise to construct primer pairs which are likely to be effective for specifically amplifying selected portions of the gene. While primer sequences are typically between 20 to 30 bases long, it may be possible to use shorter primers, potentially as small as approximately 13 bases, to amplify specifically selected gene segments. The principal limitation on how small a primer sequence may be is that it must be long enough to hybridize specifically to the targeted gene segment. Specificity of PCR is generally improved by lengthening primers and/or employing nested pairs of primers.  
     [0252] The PCR products, in total representing the entire cDNA, are then sequenced and compared to known wild-type sequences. In most cases a mutation will be observed in the affected individual. Ideally, the nature of mutation will indicate that it is likely to inactivate the gene product. Otherwise, the possibility that the alteration is not simply a polymorphism must be determined.  
     [0253] b) Certain mutations, e.g., those affecting splicing or resulting in translation stop codons, can destabilize the messenger RNA produced from the mutant gene and hence comprise the normal RT-based mutation detection method. One recently reported technique can circumvent this problem by testing whether the mutant cDNA can direct the synthesis of normal length protein in a coupled in vitro transcription/translation system. 32    
     [0254] 3) Direct Sequencing of Genomic DNA.  
     [0255] A second route to detect mutations relies on examining the exons and the intron/exon boundaries by PCR cycle sequencing directly off a DNA template. 1,2  This method requires the use of oligonucleotide pairs, such as those described in Tables 2 and 3 above, that amplify individual exons for direct PCR cycle sequencing. The method depends upon genomic DNA sequence information at each intron/exon boundary (50 bp, or greater, for each boundary). The advantage of the technique is two fold. First, because DNA is more stable than RNA, the condition of the material used for PCR is not as important as it is for RNA-based protocols. Second, most any mutation within the actual transcribed region of the gene, including those in an intron affecting splicing, will be detectable.  
     [0256] For each candidate gene, mutation detection may require knowledge of both the entire cDNA structure, and all intron/exon boundaries of the genomic structure. With such information, the type of causal mutation in a particular family can be determined. In turn, a more specific and efficient mutation detection scheme can be adapted for the particular family. Screening for the disease (HNPCC) is complex because it has a genetically heterogeneous basis in the sense that more than one gene is involved, and for each gene, multiple types of mutations are involved. 2  Any given family is highly likely to segregate one particular mutation. However, as the nature of the mutation in multiple families is determined, the spectrum of the most prevalent mutations in the population will be determined. In general, determination of the most frequent mutations will direct and streamline mutation detection.  
     [0257] Because HNPCC is so prevalent in the human population, carrier detection at birth could become part of standardized neonatal testing. Families at risk can be identified and all members not previously tested can be tested. Eventually, all affected kindreds could be determined.  
     Mode of Mutation Screening and Testing  
     [0258] DNA-Based Testing  
     [0259] Initial testing, including identifying likely HNPCC families by standard diagnosis and family history study, will likely be done in local and smaller DNA diagnosis laboratories. However, large scale testing of multiple family members, and certainly population wide testing, will ultimately require large efficient centralized commercial facilities.  
     [0260] Tests will be developed based on the determination of the most common mutations for the major genes underlying HNPCC, including at least the hMSH2 gene on chromosome 2p and the MLH1 gene on chromosome 3p. A variety of tests are likely to be developed. For example, one possibility is a set of tests employing oligonucleotide hybridizations that distinguish the normal vs. mutant alleles. 33  As already noted, our knowledge of the nucleotide structures for hMLH1, hPMS1 and hMSH2 genes makes possible the design of numerous oligonucleotide primer pairs which may be used to amplify specific portions of an individual&#39;s mismatch repair gene for genetic screening and cancer risk analysis. Our knowledge of the genes&#39; structures also makes possible the design of labeled probes which can be quickly used to determine the presence or absence of all or a portion of one of the DNA mismatch repair genes. For example, allele-specific oligomer probes (ASO) may be designed to distinguish between alleles. ASOs are short DNA segments that are identical in sequence except for a single base difference that reflects the difference between normal and mutant alleles. Under the appropriate DNA hybridization conditions, these probes can recognize a single base difference between two otherwise identical DNA sequences. Probes can be labeled radioactively or with a variety of non-radioactive reporter molecules, for example, fluorescent or chemiluminescent moieties. Labeled probes are then used to analyze the PCR sample for the presence of the disease-causing allele. The presence or absence of several different disease-causing genes can readily be determined in a single sample. The length of the probe must be long enough to avoid non-specific binding to nucleotide sequences other than the target. All tests will depend ultimately on accurate and complete structural information relating to hMLH1, hMSH2, hPMS1 and other DNA mismatch repair genes implicated in HNPCC.  
     [0261] Protein Detection-Based Screening  
     [0262] Tests based on the functionality of the protein product, per se, may also be used. The protein-examining tests will most likely utilize antibody reagents specific to either the hMLH1, hPMS1 or hMSH2 proteins or other related “cancer” gene products as they are identified.  
     [0263] For example, a frozen tumor specimen can be sectioned and prepared for antibody staining using indirect fluorescence techniques. Certain gene mutations are expected to alter or destabilize the protein structure sufficiently such as to give an altered or reduced signal after antibody staining. It is likely that such tests will be performed in cases where gene involvement in a family&#39;s cancer has yet to be established. Monoclonal antibodies are developed against the human MLH1 and PMS1 proteins. MLH1 and PMS1 human proteins are overexpressed in bacteria. Proteins are purified and injected into mice. Protein specific monoclonal antibodies are derived which can be used for diagnostic and research purposes. For example, two different monoclonal antibodies directed to hMLH1 are now available from PharMingen under catalog #13271A, clone: G168-15 and #13291A, clone: G168-728. Both of PharMingen&#39;s monoclonal antibodies were generated based on purified, recombinant hMLH1 protein, that was provided by applicants to PharMingen in the form of a fusion to bacterial protein.  
     [0264] Recent research suggests that methylation of hMLH1 promoter DNA is involved in mismatch repair gene inactivation in some sporadic tumors. 64, 65 In this situation, there is usually no detectable mutation in the hMLH1 cDNA. A screening test for hMLH1 cDNA mutations would not show any abnormality. However, an immunoassay for hMLH1 protein will show presence or absence of the protein. It is believed that 90-percent or more of sporadic colon tumors and endometrial tumors that are DNA mismatch repair defective, do not have mutations in any known gene, but instead are MLH1 protein deficient due to methylation of the promotor region of the gene. It appears that the most reliable method for detecting absence of hMLH1 in sporadic tumors is to use labeled antibodies in an immunohistochemical staining procedure of tumor sections. It is likely that immunohistochemical staining of tumor tissue sections with monoclonal antibodies directed to hMLH1 protein will become a standard diagnostic method at least for colon and endometrial tumors.  
     Identification and Characterization of DNA Mismatch Repair Tumors  
     [0265] In addition to their usefulness in diagnosing cancer susceptibility in a subject, nucleotide sequences that are homologous to a bacterial mismatch repair gene can be valuable for, among other things, use in the identification and characterization of mismatch-repair-defective tumors. Such identification and characterization is valuable because mismatch-repair-defective tumors may respond better to particular therapy regimens. For example, mismatch-repair-defective tumors might be sensitive to DNA damaging agents, especially when administered in combination with other therapeutic agents.  
     [0266] Defects in mismatch repair genes need not be present throughout an individual&#39;s tissues to contribute to tumor formation in that individual. Spontaneous mutation of a mismatch repair gene in a particular cell or tissue can contribute to tumor formation in that tissue. In fact, at least in some cases, a single mutation in a mismatch repair gene is not sufficient for tumor development. In such instances, an individual with a single mutation in a mismatch repair gene is susceptible to cancer, but will not develop a tumor until a secondary mutation occurs. Additionally, in some instances, the same mismatch repair gene mutation that is strictly tumor-associated in an individual will be responsible for conferring cancer susceptibility in a family with a hereditary predisposition to cancer development.  
     [0267] In yet another aspect of the invention, the sequence information we have provided can be used with methods known in the art to analyze tumors (or tumor cell lines) and to identify tumor-associated mutations in mismatch repair genes. Preferably, it is possible to demonstrate that these tumor-associated mutations are not present in non-tumor tissues from the same individual. The information described in this application is particularly useful for the identification of mismatch repair gene mutations within tumors (or tumor cell lines) that display genomic instability of short repeated DNA elements.  
     [0268] The sequence information and testing protocols of the present invention can also be used to determine whether two tumors are related, i.e., whether a second tumor is the result of metastasis from an earlier found first tumor which exhibits a particular DNA mismatch repair gene mutation.  
     Isolating Additional Genes of Related Function  
     [0269] Proteins that interact physically with either hMLH1 and/or hPMS1, are likely to be involved in DNA mismatch repair. By analogy to hMLH1 and hMSH2, mutations in the genes which encode for such proteins would be strong candidates for potential cancer linkage. A powerful molecular genetic approach using yeast, referred to as a “two-hybrid system”, allows the relatively rapid detection and isolation of genes encoding proteins that interact with a gene product of interest, e.g., hMLH1. 28    
     [0270] The two-hybrid system involves two plasmid vectors each intended to encode a fusion protein. Each of the two vectors contains a portion, or domain, of a transcription activator. The yeast cell used in the detection scheme contains a “reporter” gene. The activator alone cannot activate transcription. However, if the two domains are brought into close proximity then transcription may occur. The cDNA for the protein of interest, e.g., hMLH1 is inserted within a reading frame in one of the vectors. This is termed the “bait”. A library of human cDNAs, inserted into a second plasmid vector so as to make fusions with the other domain of the transcriptional activator, is introduced into the yeast cells harboring the “bait” vector. If a particular yeast cell receives a library member that contains a human cDNA encoding a protein that interacts with hMLH1 protein, this interaction will bring the two domains of the transcriptional activator into close proximity, activate transcription of the reporter gene and the yeast cell will turn blue. Next, the insert is sequenced to determine whether it is related to any sequence in the data base. The same procedure can be used to identify yeast proteins in DNA mismatch repair or a related process. Performing the yeast and human “hunts” in parallel has certain advantages. The function of novel yeast homologs can be quickly determined in yeast by gene disruption and subsequent examination of the genetic consequences of being defective in the new found gene. These yeast studies will help guide the analysis of novel human “hMLH1-or hPMS1-interacting” proteins in much the same way that the yeast studies on PMS1 and MLH1 have influenced our studies of the human MLH1 and PMS1 genes.  
     Production of Antibodies  
     [0271] By using our knowledge of the DNA sequences for hMLH1 and hPMS1, we can synthesize all or portions of the predicted protein structures for the purpose of producing antibodies. One important use for antibodies directed to hMLH1 and hPMS1 proteins will be for capturing other proteins which may be involved in DNA mismatch repair. For example, by employing coimmuno-precipitation techniques, antibodies directed to either hMLH1 or hPMS1 may be precipitated along with other associated proteins which are functionally and/or physically related. Another important use for antibodies will be for the purpose of isolating hMLH1 and hPMS1 proteins from tumor tissue. The hMLH1 and hPMS1 proteins from tumors can then be characterized for the purpose of determining appropriate treatment strategies.  
     [0272] We are in the process of developing monoclonal antibodies directed to the hMLH1 and hPMS1 proteins.  
     EXAMPLE 5  
     [0273] We have also used the following procedure to produce polyclonal antibodies directed to the human and mouse forms of PMS1 protein.  
     [0274] We inserted a 3′ fragment of the mouse PMS1 cDNA in the bacterial expression plasmid vector, pET (Novagen, Madison, Wis.). The expected expressed portion of the mouse PMS1 protein corresponds to a region of approximately 200 amino acids at the end of the PMS1 protein. This portion of the mPMS1 is conserved with yeast PMS1 but is not conserved with either the human or the mouse MLH1 proteins. One reason that we selected this portion of the PMS1 protein for producing antibodies is that we did not want the resulting antibodies to cross-react with MLH1. The mouse PMS1 protein fragment was highly expressed in  E. coli ., purified from a polyacrylamide gel and the eluted protein was then prepared for animal injections. Approximately 2 mg of the PMS1 protein fragment was sent to the Pocono Rabbit Farm (PA) for injections into rabbits. Sera from rabbits multiple times was tittered against the PMS1 antigen using standard ELISA techniques. Rabbit antibodies specific to mouse PMS1 protein were affinity-purified using columns containing immobilized mouse PMS1 protein. The affinity-purified polyclonal antibody preparation was tested further using Western blotting and dot blotting. We found that the polyclonal antibodies recognized, not only the mouse PMS1 protein, but also the human PMS1 protein which is very similar. Based upon the Western blots, there is no indication that other proteins were recognized strongly by our antibody, including either the human or mouse MLH1 proteins.  
     DNA Mismatch Repair Defective Mice  
     EXAMPLE 6  
     [0275] In order to create a experimental model system for studying DNA mismatch repair defects and resultant cancer in a whole animal system we have derived DNA mismatch repair defective mice using embryonic stem (ES) cell technology. Using genomic DNA containing a portion of the mPMS1 gene we constructed a vector that upon homologous recombination causes disruption of the chromosomal mPMS1 gene. Mouse ES cells from the 129 mouse strain were confirmed to contain a disrupted mPMS1 allele. The ES cells were injected into C57/BL6 host blastocysts to produce animals that were chimeric or a mixture of 129 and C57/BL6 cells. The incorporation of the ES cells was determined by the presence of patches of agouti coat coloring (indicative of ES cell contribution). All male chimeras were bred with C57/BL6 female mice.  
     [0276] Subsequently, twelve offspring (F 2 ) were born in which the agouti coat color was detected indicating the germline transmission of genetic material from the ES cells. Analysis of DNA extracted from the tail tips of the twelve offspring indicated that six of the animals were heterozygous (contained one wild-type and one mutant allele) for the mPMS1 mutation. Of the six heterozygous animals, three were female, (animals F 2 -8, F 2 -11 and F 2 -12) and three were males (F 2 , F 2 -10 and F 2 -13). Four breeding pens were set up to obtain mice that were homozygous for mPMS1 mutation, and additional heterozygous mice. Breeding pen #1 which contained animals F 2 -11 and F 2 -10, yielded a total of thirteen mice in three litters, four of which have been genotyped. Breeding pen #2 (animals F 2 -8 and F 2 -13) gave twenty-two animals and three litters, three of which have been genotyped. Of the seven animals genotyped, three homozygous female animals have been identified. One animal died at six weeks of age from unknown causes. The remaining homozygous females are alive and healthy at twelve weeks of age. The results indicate that mPMS1 homozygous defective mice are viable.  
     [0277] Breeding pens #3 and #4 were used to backcross the mPMS1 mutation into the C57/BL6 background. Breeding pen #3 (animal F 2 -12 crossed to a C57/BL6 mouse) produced twenty-one animals in two litters, nine of which have been genotyped. Breeding pen #4 (animal F 2 -6 crossed with a C57/BL6 mouse) gave eight mice. In addition, the original male chimera (breeding pen #5) has produced thirty-one additional offspring.  
     [0278] To genotype the animals, a series of PCR primers have been developed that are used to identify mutant and wild-type mPMS1 genes. They are: (SEQ ID NOS: 143-148, respectively)  
                                          Primer 1:   5′ TTCGGTGACAGATFFTGTAAATG-3′                           Primer 2:   5′ TTTACGGAGCCCTGGC-3′                       Primer 3:   5′ TCACCATAAAAATAGTTTCCCG-3′                       Primer 4:   5′ TCCTGGATCATATTTTCTGAGC-3′                       Primer 5:   5′ TTTCAGGTATGTCCTGTTACCC-3′                       Primer 6:   5′ TGAGGCAGCTTTTAAGAAACTC-3′                       Primers 1 @ 2   (5′ targeted)                       Primers 1 @ 3   (5′ untargeted)                       Primers 4 @ 5   (3′ targeted)                       Primers 4 @ 6   (3′ untargeted)          
 
     [0279] The mice we have developed provide an animal model system for studying the consequences of defects in DNA mismatch repair and resultant HNPCC. The long term survival of mice homozygous and heterozygous for the mPMS1 mutation and the types and timing of tumors in these mice will be determined. The mice will be screened daily for any indication of cancer onset as indicated by a hunched appearance in combination with deterioration in coat condition. These mice carrying mPMS1 mutation will be used to test the effects of other factors, environmental and genetic, on tumor formation. For example, the effect of diet on colon and other type of tumors can be compared for normal mice versus those carrying mPMS1 mutation either in the heterozygous or homozygous genotype. In addition, the mPMS1 mutation can be put into different genetic backgrounds to learn about interactions between genes of the mismatch repair pathway and other genes involved in human cancer, for example, p53. Mice carrying mPMS1 mutations will also be useful for testing the efficacy of somatic gene therapy on the cancers that arise in mice, for example, the expected colon cancers. Further, isogenic fibroblast cell lines from the homozygous and heterozygous mPMS1 mice can be established for use in various cellular studies, including the determination of spontaneous mutation rates.  
     [0280] We are currently constructing a vector for disrupting the mouse mMLH1 gene to derive mice carrying mutation in mMLH1. We will compare mice carrying defects in mPMS1 to mice carrying defects in mMLH1. In addition, we will construct mice that carry mutations in both genes to see whether there is a synergistic effect of having mutations in two HNPCC genes. Other studies on the mMLH1 mutant mice will be as described above for the mPMS1 mutant mice.  
     [0281] While the present invention has been particularly shown and described with reference to the foregoing preferred embodiments, those skilled in the art will understand that many variations may be made therein without departing from the spirit and scope of the invention as defined in the following claims. The description of the invention should be understood to include all novel and non-obvious combinations of elements described herein, and claims may be presented in this or a later application to any novel and non-obvious combination of these elements. Where the claims recite “a” or “a first” element or the equivalent thereof, such claims should be understood to include incorporation of one or more such elements, neither requiring nor excluding two or more such elements.  
    
     
       
         1 
         
           
             153  
           
           
             1  
             361  
             PRT  
             Salmonella typhimurium  
           
            1 

Met Pro Ile Gln Val Leu Pro Pro Gln Leu Ala Asn Gln Ile Ala Ala 
1               5                   10                  15 

Gly Glu Val Val Glu Arg Pro Ala Ser Val Val Lys Glu Leu Val Glu 
            20                  25                  30 

Asn Ser Leu Asp Ala Gly Ala Thr Arg Val Asp Ile Asp Ile Glu Arg 
        35                  40                  45 

Gly Gly Ala Lys Leu Ile Arg Ile Arg Asp Asn Gly Cys Gly Ile Lys 
    50                  55                  60 

Lys Glu Glu Leu Ala Leu Ala Leu Ala Arg His Ala Thr Ser Lys Ile 
65                  70                  75                  80 

Ala Ser Leu Asp Asp Leu Glu Ala Ile Ile Ser Leu Gly Phe Arg Gly 
                85                  90                  95 

Glu Ala Leu Ala Ser Ile Ser Ser Val Ser Arg Leu Thr Leu Thr Ser 
            100                 105                 110 

Arg Thr Ala Glu Gln Ala Glu Ala Trp Gln Ala Tyr Ala Glu Gly Arg 
        115                 120                 125 

Asp Met Asp Val Thr Val Lys Pro Ala Ala His Pro Val Gly Thr Thr 
    130                 135                 140 

Leu Glu Val Leu Asp Leu Phe Tyr Asn Thr Pro Ala Arg Arg Lys Phe 
145                 150                 155                 160 

Met Arg Thr Glu Lys Thr Glu Phe Asn His Ile Asp Glu Ile Ile Arg 
                165                 170                 175 

Arg Ile Ala Leu Ala Arg Phe Asp Val Thr Leu Asn Leu Ser His Asn 
            180                 185                 190 

Gly Lys Leu Val Arg Gln Tyr Arg Ala Val Ala Lys Asp Gly Gln Lys 
        195                 200                 205 

Glu Arg Arg Leu Gly Ala Ile Cys Gly Thr Pro Phe Leu Glu Gln Ala 
    210                 215                 220 

Leu Ala Ile Glu Trp Gln His Gly Asp Lys Thr Lys Arg Gly Trp Val 
225                 230                 235                 240 

Ala Asp Pro Asn His Thr Thr Thr Ala Leu Thr Glu Ile Gln Tyr Cys 
                245                 250                 255 

Tyr Val Asn Gly Arg Met Met Arg Asp Arg Leu Ile Asn His Ala Ile 
            260                 265                 270 

Arg Gln Ala Cys Glu Asp Lys Leu Gly Ala Asp Gln Gln Pro Ala Phe 
        275                 280                 285 

Val Leu Tyr Leu Glu Ile Asp Pro His Gln Val Asp Val Asn Val His 
    290                 295                 300 

Pro Ala Lys His Glu Val Arg Phe His Gln Ser Arg Leu Val His Asp 
305                 310                 315                 320 

Phe Ile Tyr Gln Gly Val Leu Ser Val Leu Gln Gln Gln Thr Glu Thr 
                325                 330                 335 

Ala Leu Pro Leu Glu Glu Ile Ala Pro Ala Pro Arg His Val Gln Glu 
            340                 345                 350 

Asn Arg Ile Ala Ala Gly Arg Asn His 
        355                 360 

 
           
             2  
             538  
             PRT  
             Streptococcus pneumoniae  
           
            2 

Met Ser His Ile Ile Glu Leu Pro Glu Met Leu Ala Asn Gln Ile Ala 
1               5                   10                  15 

Ala Gly Glu Val Ile Glu Arg Pro Ala Ser Val Cys Lys Glu Leu Val 
            20                  25                  30 

Glu Asn Ala Ile Asp Ala Gly Ser Ser Gln Ile Ile Ile Glu Ile Glu 
        35                  40                  45 

Glu Ala Gly Leu Lys Lys Val Gln Ile Thr Asp Asn Gly His Gly Ile 
    50                  55                  60 

Ala His Asp Glu Val Glu Leu Ala Leu Arg Arg His Ala Thr Ser Lys 
65                  70                  75                  80 

Ile Lys Asn Gln Ala Asp Leu Phe Arg Ile Arg Thr Leu Gly Phe Arg 
                85                  90                  95 

Gly Glu Ala Leu Pro Ser Ile Ala Ser Val Ser Val Leu Thr Leu Leu 
            100                 105                 110 

Thr Ala Val Asp Gly Ala Ser His Gly Thr Lys Leu Val Ala Arg Gly 
        115                 120                 125 

Gly Glu Val Glu Glu Val Ile Pro Ala Thr Ser Pro Val Gly Thr Lys 
    130                 135                 140 

Val Cys Val Glu Asp Leu Phe Phe Asn Thr Pro Ala Arg Leu Lys Tyr 
145                 150                 155                 160 

Met Lys Ser Gln Gln Ala Glu Leu Ser His Ile Ile Asp Ile Val Asn 
                165                 170                 175 

Arg Leu Gly Leu Ala His Pro Glu Ile Ser Phe Ser Leu Ile Ser Asp 
            180                 185                 190 

Gly Lys Glu Met Thr Arg Thr Ala Gly Thr Gly Gln Leu Arg Gln Ala 
        195                 200                 205 

Ile Ala Gly Ile Tyr Gly Leu Val Ser Ala Lys Lys Met Ile Glu Ile 
    210                 215                 220 

Glu Asn Ser Asp Leu Asp Phe Glu Ile Ser Gly Phe Val Ser Leu Pro 
225                 230                 235                 240 

Glu Leu Thr Arg Ala Asn Arg Asn Tyr Ile Ser Leu Phe Ile Asn Gly 
                245                 250                 255 

Arg Tyr Ile Lys Asn Phe Leu Leu Asn Arg Ala Ile Leu Asp Gly Phe 
            260                 265                 270 

Gly Ser Lys Leu Met Val Gly Arg Phe Pro Leu Ala Val Ile His Ile 
        275                 280                 285 

His Ile Asp Pro Tyr Leu Ala Asp Val Asn Val His Pro Thr Lys Gln 
    290                 295                 300 

Glu Val Arg Ile Ser Lys Glu Lys Glu Leu Met Thr Leu Val Ser Glu 
305                 310                 315                 320 

Ala Ile Ala Asn Ser Leu Lys Glu Gln Thr Leu Ile Pro Asp Ala Leu 
                325                 330                 335 

Glu Asn Leu Ala Lys Ser Thr Val Arg Asn Arg Glu Lys Val Glu Gln 
            340                 345                 350 

Thr Ile Leu Pro Leu Ser Phe Pro Glu Leu Glu Phe Phe Gly Gln Met 
        355                 360                 365 

His Gly Thr Tyr Leu Phe Ala Gln Gly Arg Asp Gly Leu Tyr Ile Ile 
    370                 375                 380 

Asp Gln His Ala Ala Gln Glu Arg Val Lys Tyr Glu Glu Tyr Arg Glu 
385                 390                 395                 400 

Ser Ile Gly Asn Val Asp Gln Ser Gln Gln Gln Leu Leu Val Pro Tyr 
                405                 410                 415 

Ile Phe Glu Phe Pro Ala Asp Asp Ala Leu Arg Leu Lys Glu Arg Met 
            420                 425                 430 

Pro Leu Leu Glu Glu Val Gly Val Phe Leu Ala Glu Tyr Gly Glu Asn 
        435                 440                 445 

Gln Phe Ile Leu Arg Glu His Pro Ile Trp Met Ala Glu Glu Glu Ile 
    450                 455                 460 

Glu Ser Gly Ile Tyr Glu Met Cys Asp Met Leu Leu Leu Thr Lys Glu 
465                 470                 475                 480 

Val Ser Ile Lys Lys Tyr Arg Ala Glu Leu Ala Ile Met Met Ser Cys 
                485                 490                 495 

Lys Arg Ser Ile Lys Ala Asn His Arg Ile Asp Asp His Ser Ala Arg 
            500                 505                 510 

Gln Leu Leu Tyr Gln Leu Ser Gln Cys Asp Asn Pro Tyr Asn Cys Pro 
        515                 520                 525 

His Gly Arg Pro Val Leu Val His Phe Thr 
    530                 535 

 
           
             3  
             607  
             PRT  
             Saccharomyces cerevisiae  
           
            3 

Met Phe His His Ile Glu Asn Leu Leu Ile Glu Thr Glu Lys Arg Cys 
1               5                   10                  15 

Lys Gln Lys Glu Gln Arg Tyr Ile Pro Val Lys Tyr Leu Phe Ser Met 
            20                  25                  30 

Thr Gln Ile His Gln Ile Asn Asp Ile Asp Val His Arg Ile Thr Ser 
        35                  40                  45 

Gly Gln Val Ile Thr Asp Leu Thr Thr Ala Val Lys Glu Leu Val Asp 
    50                  55                  60 

Asn Ser Ile Asp Ala Asn Ala Asn Gln Ile Glu Ile Ile Phe Lys Asp 
65                  70                  75                  80 

Tyr Gly Leu Glu Ser Ile Glu Cys Ser Asp Asn Gly Asp Gly Ile Asp 
                85                  90                  95 

Pro Ser Asn Tyr Glu Phe Leu Ala Leu Lys His Tyr Thr Ser Lys Ile 
            100                 105                 110 

Ala Lys Phe Gln Asp Val Ala Lys Val Gln Thr Leu Gly Phe Arg Gly 
        115                 120                 125 

Glu Ala Leu Ser Ser Leu Cys Gly Ile Ala Lys Leu Ser Val Ile Thr 
    130                 135                 140 

Thr Thr Ser Pro Pro Lys Ala Asp Lys Glu Leu Tyr Asp Met Val Gly 
145                 150                 155                 160 

His Ile Thr Ser Lys Thr Thr Thr Ser Arg Asn Lys Gly Thr Thr Val 
                165                 170                 175 

Leu Val Ser Gln Leu Phe His Asn Leu Pro Val Arg Gln Lys Glu Phe 
            180                 185                 190 

Ser Lys Thr Phe Lys Arg Gln Phe Thr Lys Cys Leu Thr Val Ile Gln 
        195                 200                 205 

Gly Tyr Ala Ile Ile Asn Ala Ala Ile Lys Phe Ser Val Trp Asn Ile 
    210                 215                 220 

Thr Pro Lys Gly Lys Lys Asn Leu Ile Leu Ser Thr Met Arg Asn Ser 
225                 230                 235                 240 

Ser Met Arg Lys Asn Ile Ser Ser Val Phe Gly Ala Gly Gly Met Arg 
                245                 250                 255 

Gly Glu Leu Glu Val Asp Leu Val Leu Asp Leu Asn Pro Phe Lys Asn 
            260                 265                 270 

Arg Met Leu Gly Lys Tyr Thr Asp Asp Pro Asp Phe Leu Asp Leu Asp 
        275                 280                 285 

Tyr Lys Ile Arg Val Lys Gly Tyr Ile Ser Gln Asn Ser Phe Gly Cys 
    290                 295                 300 

Gly Arg Asn Ser Lys Asp Arg Gln Phe Ile Tyr Val Asn Lys Arg Pro 
305                 310                 315                 320 

Val Glu Tyr Ser Thr Leu Leu Lys Cys Cys Asn Glu Val Tyr Lys Thr 
                325                 330                 335 

Phe Asn Asn Val Gln Phe Pro Ala Val Phe Leu Asn Leu Glu Leu Pro 
            340                 345                 350 

Met Ser Leu Ile Asp Val Asn Val Thr Pro Asp Lys Arg Val Ile Leu 
        355                 360                 365 

Leu His Asn Glu Arg Ala Val Ile Asp Ile Phe Lys Thr Thr Leu Ser 
    370                 375                 380 

Asp Tyr Tyr Asn Arg Gln Glu Leu Ala Leu Pro Lys Arg Met Cys Ser 
385                 390                 395                 400 

Gln Ser Glu Gln Gln Ala Gln Lys Arg Leu Leu Thr Glu Val Phe Asp 
                405                 410                 415 

Asp Asp Phe Lys Lys Met Glu Val Val Gly Gln Phe Asn Leu Gly Phe 
            420                 425                 430 

Ile Ile Val Thr Arg Lys Val Asp Asn Lys Ser Asp Leu Phe Ile Val 
        435                 440                 445 

Asp Gln His Ala Ser Asp Glu Lys Tyr Asn Phe Glu Thr Leu Gln Ala 
    450                 455                 460 

Val Thr Val Phe Lys Ser Gln Lys Leu Ile Ile Pro Gln Pro Val Glu 
465                 470                 475                 480 

Leu Ser Val Ile Asp Glu Leu Val Val Leu Asp Asn Leu Pro Val Phe 
                485                 490                 495 

Glu Lys Asn Gly Phe Lys Leu Lys Ile Asp Glu Glu Glu Glu Phe Gly 
            500                 505                 510 

Ser Arg Val Lys Leu Leu Ser Leu Pro Thr Ser Lys Gln Thr Leu Phe 
        515                 520                 525 

Asp Leu Gly Asp Phe Asn Glu Leu Ile His Leu Ile Lys Glu Asp Gly 
    530                 535                 540 

Gly Leu Arg Arg Asp Asn Ile Arg Cys Ser Lys Ile Arg Ser Met Phe 
545                 550                 555                 560 

Ala Met Arg Ala Cys Arg Ser Ser Ile Met Ile Gly Lys Pro Leu Asn 
                565                 570                 575 

Lys Lys Thr Met Thr Arg Val Val His Asn Leu Ser Glu Leu Asp Lys 
            580                 585                 590 

Pro Trp Asn Cys Pro His Gly Arg Pro Thr Met Arg His Leu Met 
        595                 600                 605 

 
           
             4  
             2484  
             DNA  
             Homo sapiens  
           
            4 

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 

 
           
             5  
             756  
             PRT  
             Homo sapiens  
           
            5 

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 

 
           
             6  
             397  
             DNA  
             Homo sapiens  
           
            6 

tggctggatg ctaagctaca gctgaaggaa gaacgtgagc acgaggcact gaggtgattg     60 

gctgaaggca cttccgttga gcatctagac gtttccttgg ctcttctggc gccaaaatgt    120 

cgttcgtggc aggggttatt cggcggctgg acgagacagt ggtgaaccgc atcgcggcgg    180 

gggaagttat ccagcggcca gctaatgcta tcaaagagat gattgagaac tggtacggag    240 

ggagtcgagc cgggctcact taagggctac gacttaacgg gccgcgtcac tcaatggcgc    300 

ggacacgcct ctttccccgg gcagaggcat gtacagcgca tgcccacaac ggcggaggcc    360 

gccgggttcc ctacgtgcca taagccttct ccttttc                             397 

 
           
             7  
             393  
             DNA  
             Homo sapiens  
           
            7 

aaacacgtta atgaggcact attgtttgta tttggagttt gttatcattg cttggctcat     60 

attaaaatat gtacattaga gtagttgcag actgataaat tattttctgt ttgatttgcc    120 

agtttagatg caaaatccac aagtattcaa gtgattgtta aagagggagg cctgaagttg    180 

attcagatcc aagacaatgg caccgggatc agggtaagta aaacctcaaa gtagcaggat    240 

gtttgtgcgc ttcatggaag agtcaggacc tttctctgtt ctggaaacta ggcttttgca    300 

gatgggattt tttcactgaa aaattcaaca ccaacaataa atatttattg agtacctatt    360 

atttgcgggg cactgttcag gggatgtgtc agt                                 393 

 
           
             8  
             352  
             DNA  
             Homo sapiens  
           
            8 

tttcctggat taatcaagaa atggaattca aagagatttg gaaaatgagt aacatgatta     60 

tttactcatc tttttggtat ctaacagaaa gaagatctgg atattgtatg tgaaaggttc    120 

actactagta aactgcagtc ctttgaggat ttagccagta tttctaccta tggctttcga    180 

ggtgaggtaa gctaaagatt caagaaatgt gtaaaatatc ctcctgtgat gacattgtct    240 

gtcatttgtt agtatgtatt tctcaacata gataaataag gtttggtacc ttttacttgt    300 

taaatgtatg caaatctgag caaacttaat gaactttaac tttcaaagac tg            352 

 
           
             9  
             287  
             DNA  
             Homo sapiens  
           
            9 

tggaagcagc agcagataac ctttcccttt ggtgaggtga cagtgggtga cccagcagtg     60 

agtttttctt tcagtctatt ttcttttctt ccttaggctt tggccagcat aagccatgtg    120 

gctcatgtta ctattacaac gaaaacagct gatggaaagt gtgcatacag gtatagtgct    180 

gacttctttt actcatatat attcattctg aaatgtattt tgggcctagg tctcagagta    240 

atcctgtctc aacaccagtg ttatctttgg cagagatctt gagtacg                  287 

 
           
             10  
             336  
             DNA  
             Homo sapiens  
           
            10 

ttgatatgat tttctctttt ccccttggga ttagtatcta tctctctact ggatattaat     60 

ttgttatatt ttctcattag agcaagttac tcagatggaa aactgaaagc ccctcctaaa    120 

ccatgtgctg gcaatcaagg gacccagatc acggtaagaa tggtacatgg gagagtaaat    180 

tgttgaagct ttgtttgtat aaatattgga ataaaaaata aaattgcttc taagttttca    240 

gggtaataat aaaatgaatt tgcactagtt aatggaggtc ccaagatatc ctctaagcaa    300 

gataaatgac tattggcttt ttggcatggc agcctg                              336 

 
           
             11  
             275  
             DNA  
             Homo sapiens  
           
            11 

gcttttgcca ggaccatctt gggttttatt ttcaagtact tctatgaatt tacaagaaaa     60 

atcaatcttc tgttcaggtg gaggaccttt tttacaacat agccacgagg agaaaagctt    120 

taaaaaatcc aagtgaagaa tatgggaaaa ttttggaagt tgttggcagg tacagtccaa    180 

aatctgggag tgggtctctg agatttgtca tcaaagtaat gtgttctagt gctcatacat    240 

tgaacagttg ctgagctaga tggtgaaaag taaaa                               275 

 
           
             12  
             389  
             DNA  
             Homo sapiens  
           
            12 

cagcaaccta taaaagtaga gaggagtctg tgttttgacg cagcaccttt agcattttta     60 

tttggatgaa gtttctgctg gtttattttt ctgtgggtaa aatattaata ggctgtatgg    120 

agatattttt ctttatatgt acctttgttt agattactca actccactaa tttatttaac    180 

taaaaggggg ctctgacatc tagtgtgtgt ttttggcaac tcttttctta ctcttttgtt    240 

tttcttttcc aggtattcag tacacaatgc aggcattagt ttctcagtta aaaaagtaag    300 

ttcttggttt atgggggatg gttttgtttt atgaaaagaa aaaaggggat ttttaatagt    360 

ttgctggtgg agataaggtt atgatgttt                                      389 

 
           
             13  
             381  
             DNA  
             Homo sapiens  
           
            13 

atgtttcagt ctcagccatg agacaataaa tccttgtgtc ttctgctgtt tgtttatcag     60 

caaggagaga cagtagctga tgttaggaca ctacccaatg cctcaaccgt ggacaatatt    120 

cgctccatct ttggaaatgc tgttagtcgg tatgtcgata acctatataa aaaaatcttt    180 

tacatttatt atcttggttt atcattccat cacattattt gggaaccttt caagatatta    240 

tgtgtgttaa gagtttgctt tagtcaaata cacaggcttg ttttatgctt cagatttgtt    300 

aatggagttc ttatttcacg taatcaacac tttctaggtg tatgtaatct cctagattct    360 

gtggcgtgaa tcatgtgttc t                                              381 

 
           
             14  
             526  
             DNA  
             Homo sapiens  
           
            14 

actgagtagg gtaggtgggt gagtgggtgg gtgggtgggt gggtggatgg atggatggga     60 

ggatgggtgg gtgaatgggt gaacagacaa atggatggat gaatggacag gcacaggagg    120 

acctcaaatg gaccaagtct tcggggccct catttcacaa agttagttta tgggaaggaa    180 

ccttgtgttt ttaaattctg attcttttgt aatgtttgag ttttgagtat tttcaaaagc    240 

ttcagaatct cttttctaat agagaactga tagaaattgg atgtgaggat aaaaccctag    300 

ccttcaaaat gaatggttac atatccaatg caaactactc agtgaagaag tgcatcttct    360 

tactcttcat caaccgtaag ttaaaaagaa ccacatggga aatccactca caggaaacac    420 

ccacagggaa ttttatggga ccatggaaaa atttctgagt ccataggttt gattaaacat    480 

ggagaaacct catggcaaag tttggtttta ttgggaagca tgtata                   526 

 
           
             15  
             434  
             DNA  
             Homo sapiens  
           
            15 

atagtgggct ggaaagtggc cacaggtaaa ggtgcacctt tcttcctggg gatgtgatgt     60 

gcatatcact acagaaatgt ctttcctgag gtgatgtcat gactttgtgt gaatgtacac    120 

ctgtgacctc acccctcagg acagttttga actggttgct ttctttttat tgtttagatc    180 

gtctggtaga atcaacttcc ttgagaaaag ccatagaaac agtgtatgca gcctatttgc    240 

ccaaaaacac acacccattc ctgtacctca ggtaatgtag caccaaactc ctcaaccaag    300 

actcacaagg aacagatgtt ctatcaggct ctcctctttg aaagagatga gcatgctaat    360 

agtacaatca gagtgaatcc catacaccac tggcaaaagg atgttctgtc ccttcttaca    420 

ggtacaaggc acag                                                      434 

 
           
             16  
             458  
             DNA  
             Homo sapiens  
           
            16 

cttacgcaaa gctacacagc tcttaagtag cagtgccaat atttgaacac actcagactc     60 

gagcctgagg ttttgaccac tgtgtcatct ggcctcaaat cttctggcca ccacatacac    120 

catatgtggg ctttttctcc ccctcccact atctaaggta attgttctct cttattttcc    180 

tgacagttta gaaatcagtc cccagaatgt ggatgttaat gtgcacccca caaagcatga    240 

agttcacttc ctgcacgagg agagcatcct ggagcgggtg cagcagcaca tcgagagcaa    300 

gctcctgggc tccaattcct ccaggatgta cttcacccag gtcagggcgc ttctcatcca    360 

gctacttctc tggggccttt gaaatgtgcc cggccagacg tgagagccca gatttttgct    420 

gttatttagg aacttttttt gaagtattac ctggatag                            458 

 
           
             17  
             618  
             DNA  
             Homo sapiens  
           
            17 

gataattata cctcatacta gcttctttct tagtactgct ccatttgggg acctgtatat     60 

ctatacttct tattctgagt ctctccacta tatatatata tatatatata tttttttttt    120 

tttttttttt taatacagac tttgctacca ggacttgctg gcccctctgg ggagatggtt    180 

aaatccacaa caagtctgac ctcgtcttct acttctggaa gtagtgataa ggtctatgcc    240 

caccagatgg ttcgtacaga ttcccgggaa cagaagcttg atgcatttct gcagcctctg    300 

agcaaacccc tgtccagtca gccccaggcc attgtcacag aggataagac agatatttct    360 

agtggcaggg ctaggcagca agatgaggag atgcttgaac tcccagcccc tgctgaagtg    420 

gctgccaaaa atcagagctt ggagggggat acaacaaagg ggacttcaga aatgtcagag    480 

aagagaggac ctacttccag caaccccagg tatggccttt tgggaaaagt acagcctacc    540 

tcctttattc tgtaataaaa ctgccttcta actttggctt ttcatgaatc acttgcatct    600 

tctctctgcc gacttccc                                                  618 

 
           
             18  
             478  
             DNA  
             Homo sapiens  
           
            18 

ctgtgctcca gcacaggtca tccagctctg tagaccagcg cagagaagtt gcttgctccc     60 

aaatgcaacc cacaaaattt ggctaagttt aaaaacaaga ataataatga tctgcacttc    120 

cttttcttca ttgcagaaag agacatcggg aagattctga tgtggaaatg gtggaagatg    180 

attcccgaaa ggaaatgact gcagcttgta ccccccggag aaggatcatt aacctcacta    240 

gtgttttgag tctccaggaa gaaattaatg agcagggaca tgagggtacg taaacgctgt    300 

ggcctgcctg ggatgcatag ggcctcaact gccaaggttt tggaaatgga gaaagcagtc    360 

atgttgtcag agtggcacta cagttttgat gggcaagctc ctcttccttt actaacccac    420 

aatagcatca gcttaaagac aatttttgat tgggagaaaa gggagaaaat aatctctg      478 

 
           
             19  
             377  
             DNA  
             Homo sapiens  
           
            19 

cagttttcac caggaggctc aaatcaggcc tttgcttact tggtgtctct agttctggtg     60 

cctggtgctt tggtcaatga agtggggttg gtaggattct attacttacc tgttttttgg    120 

ttttattttt tgttttgcag ttctccggga gatgttgcat aaccactcct tcgtgggctg    180 

tgtgaatcct cagtgggcct tggcacagca tcaaaccaag ttataccttc tcaacaccac    240 

caagcttagg taaatcagct gagtgtgtga acaagcagag ctactacaac aatggtccag    300 

ggagcacagg cacaaaagct aaggagagca gcatgaaggt agttgggaag ggcacaggct    360 

ttggagtcag cacatgt                                                   377 

 
           
             20  
             325  
             DNA  
             Homo sapiens  
           
            20 

cccctggttg aagcgttgga atcccactct ttggaagatt gtgttagact gttaaccaga     60 

ttccacagcc aggcagaact atgtctgtct catccatgtg tcagggatta cgtctcccat    120 

ttgtcccaac tggttgtatc tcaagcatga attcagcttt tccttaaagt cacttcattt    180 

ttattttcag tgaagaactg ttctaccaga tactcattta tgattttgcc aattttggtg    240 

ttctcaggtt atcggtaagt ttagatcctt ttcacttctg acatttcaac tgaccgcccc    300 

gcaaacagta gctctccact aaata                                          325 

 
           
             21  
             341  
             DNA  
             Homo sapiens  
           
            21 

catttatggt ttctcacctg ccattctgat agtggattct tgggaattca ggcttcattt     60 

ggatgctccg ttaaagcttg ctccttcatg ttcttgcttc ttcctaggag ccagcaccgc    120 

tctttgacct tgccatgctt gccttagata gtccagagag tggctggaca gaggaagatg    180 

gtcccaaaga aggacttgct gaatacattg ttgagtttct gaagaagaag gctgagatgc    240 

ttgcagacta tttctctttg gaaattgatg aggtgtgaca gccattctta tacttctgtt    300 

gtattctcca aataaaattt ccagccgggt gcattggctc a                        341 

 
           
             22  
             260  
             DNA  
             Homo sapiens  
           
            22 

cagataggag gcacaaggcc tgggaaaggc actggagaaa tgggatttgt ttaaactatg     60 

acagcattat ttcttgttcc cttgtccttt ttcctgcaag caggaaggga acctgattgg    120 

attacccctt ctgattgaca actatgtgcc ccctttggag ggactgccta tcttcattct    180 

tcgactagcc actgaggtca gtgatcaagc agatactaag catttcggta catgcatgtg    240 

tgctggaggg aaagggcaaa                                                260 

 
           
             23  
             340  
             DNA  
             Homo sapiens  
           
            23 

ctatatcttc ccagcaatat tcacagtccg tttacagttt taacgcctaa agtatcacat     60 

ttcgtttttt agctttaagt agtctgtgat ctccgtttag aatgagaatg tttaaattcg    120 

tacctatttt gaggtattga atttctttgg accaggtgaa ttgggacgaa gaaaaggaat    180 

gttttgaaag cctcagtaaa gaatgcgcta tgttctattc catccggaag cagtacatat    240 

ctgaggagtc gaccctctca ggccagcagg tacagtggtg atgcacactg gcaccccagg    300 

actaggacag gacctcatac atcttaggag atgaaacttg                          340 

 
           
             24  
             563  
             DNA  
             Homo sapiens  
           
            24 

aatcctcttg tgttcaggcc tgtggatccc tgagaggcta gcccacaaga tccacttcaa     60 

aagccctaga taacaccaag tctttccaga cccagtgcac atcccatcag ccaggacacc    120 

agtgtatgtt gggatgcaaa cagggaggct tatgacatct aatgtgtttt ccagagtgaa    180 

gtgcctggct ccattccaaa ctcctggaag tggactgtgg aacacattgt ctataaagcc    240 

ttgcgctcac acattctgcc tcctaaacat ttcacagaag atggaaatat cctgcagctt    300 

gctaacctgc ctgatctata caaagtcttt gagaggtgtt aaatatggtt atttatgcac    360 

tgtgggatgt gttcttcttt ctctgtattc cgatacaaag tgttgtatca aagtgtgata    420 

tacaaagtgt accaacataa gtgttggtag cacttaagac ttatacttgc cttctgatag    480 

tattccttta tacacagtgg attgattata aataaataga tgtgtcttaa cataatttct    540 

tatttaattt tattatgtat ata                                            563 

 
           
             25  
             137  
             DNA  
             Homo sapiens  
           
            25 

cttggctctt ctggcgccaa aatgtcgttc gtggcagggg ttattcggcg gctggacgag     60 

acagtggtga accgcatcgc ggcgggggaa gttatccagc ggccagctaa tgctatcaaa    120 

gagatgattg agaactg                                                   137 

 
           
             26  
             91  
             DNA  
             Homo sapiens  
           
            26 

tttagatgca aaatccacaa gtattcaagt gattgttaaa gagggaggcc tgaagttgat     60 

tcagatccaa gacaatggca ccgggatcag g                                    91 

 
           
             27  
             99  
             DNA  
             Homo sapiens  
           
            27 

aaagaagatc tggatattgt atgtgaaagg ttcactacta gtaaactgca gtcctttgag     60 

gatttagcca gtatttctac ctatggcttt cgaggtgag                            99 

 
           
             28  
             74  
             DNA  
             Homo sapiens  
           
            28 

gctttggcca gcataagcca tgtggctcat gttactatta caacgaaaac agctgatgga     60 

aagtgtgcat acag                                                       74 

 
           
             29  
             73  
             DNA  
             Homo sapiens  
           
            29 

agcaagttac tcagatggaa aactgaaagc ccctcctaaa ccatgtgctg gcaatcaagg     60 

gacccagatc acg                                                        73 

 
           
             30  
             92  
             DNA  
             Homo sapiens  
           
            30 

gtggaggacc ttttttacaa catagccacg aggagaaaag ctttaaaaaa tccaagtgaa     60 

gaatatggga aaattttgga agttgttggc ag                                   92 

 
           
             31  
             43  
             DNA  
             Homo sapiens  
           
            31 

gtattcagta cacaatgcag gcattagttt ctcagttaaa aaa                       43 

 
           
             32  
             89  
             DNA  
             Homo sapiens  
           
            32 

caaggagaga cagtagctga tgttaggaca ctacccaatg cctcaaccgt ggacaatatt     60 

cgctccatct ttggaaatgc tgttagtcg                                       89 

 
           
             33  
             113  
             DNA  
             Homo sapiens  
           
            33 

agaactgata gaaattggat gtgaggataa aaccctagcc ttcaaaatga atggttacat     60 

atccaatgca aactactcag tgaagaagtg catcttctta ctcttcatca acc           113 

 
           
             34  
             94  
             DNA  
             Homo sapiens  
           
            34 

atcgtctggt agaatcaact tccttgagaa aagccataga aacagtgtat gcagcctatt     60 

tgcccaaaaa cacacaccca ttcctgtacc tcag                                 94 

 
           
             35  
             154  
             DNA  
             Homo sapiens  
           
            35 

tttagaaatc agtccccaga atgtggatgt taatgtgcac cccacaaagc atgaagttca     60 

cttcctgcac gaggagagca tcctggagcg ggtgcagcag cacatcgaga gcaagctcct    120 

gggctccaat tcctccagga tgtacttcac ccag                                154 

 
           
             36  
             371  
             DNA  
             Homo sapiens  
           
            36 

actttgctac caggacttgc tggcccctct ggggagatgg ttaaatccac aacaagtctg     60 

acctcgtctt ctacttctgg aagtagtgat aaggtctatg cccaccagat ggttcgtaca    120 

gattcccggg aacagaagct tgatgcattt ctgcagcctc tgagcaaacc cctgtccagt    180 

cagccccagg ccattgtcac agaggataag acagatattt ctagtggcag ggctaggcag    240 

caagatgagg agatgcttga actcccagcc cctgctgaag tggctgccaa aaatcagagc    300 

ttggaggggg atacaacaaa ggggacttca gaaatgtcag agaagagagg acctacttcc    360 

agcaacccca g                                                         371  
           
             37  
             149  
             DNA  
             Homo sapiens  
           
            37 

aaagagacat cgggaagatt ctgatgtgga aatggtggaa gatgattccc gaaaggaaat     60 

gactgcagct tgtacccccc ggagaaggat cattaacctc actagtgttt tgagtctcca    120 

ggaagaaatt aatgagcagg gacatgagg                                      149 

 
           
             38  
             109  
             DNA  
             Homo sapiens  
           
            38 

ttctccggga gatgttgcat aaccactcct tcgtgggctg tgtgaatcct cagtgggcct     60 

tggcacagca tcaaaccaag ttataccttc tcaacaccac caagcttag                109 

 
           
             39  
             64  
             DNA  
             Homo sapiens  
           
            39 

tgaagaactg ttctaccaga tactcattta tgattttgcc aattttggtg ttctcaggtt     60 

atcg                                                                  64 

 
           
             40  
             165  
             DNA  
             Homo sapiens  
           
            40 

gagccagcac cgctctttga ccttgccatg cttgccttag atagtccaga gagtggctgg     60 

acagaggaag atggtcccaa agaaggactt gctgaataca ttgttgagtt tctgaagaag    120 

aaggctgaga tgcttgcaga ctatttctct ttggaaattg atgag                    165 

 
           
             41  
             93  
             DNA  
             Homo sapiens  
           
            41 

gaagggaacc tgattggatt accccttctg attgacaact atgtgccccc tttggaggga     60 

ctgcctatct tcattcttcg actagccact gag                                  93 

 
           
             42  
             114  
             DNA  
             Homo sapiens  
           
            42 

gtgaattggg acgaagaaaa ggaatgtttt gaaagcctca gtaaagaatg cgctatgttc     60 

tattccatcc ggaagcagta catatctgag gagtcgaccc tctcaggcca gcag          114 

 
           
             43  
             360  
             DNA  
             Homo sapiens  
           
            43 

agtgaagtgc ctggctccat tccaaactcc tggaagtgga ctgtggaaca cattgtctat     60 

aaagccttgc gctcacacat tctgcctcct aaacatttca cagaagatgg aaatatcctg    120 

cagcttgcta acctgcctga tctatacaaa gtctttgaga ggtgttaaat atggttattt    180 

atgcactgtg ggatgtgttc ttctttctct gtattccgat acaaagtgtt gtatcaaagt    240 

gtgatataca aagtgtacca acataagtgt tggtagcact taagacttat acttgccttc    300 

tgatagtatt cctttataca cagtggattg attataaata aatagatgtg tcttaacata    360 

 
           
             44  
             19  
             DNA  
             Homo sapiens  
           
            44 

aggcactgag gtgattggc                                                  19 

 
           
             45  
             19  
             DNA  
             Homo sapiens  
           
            45 

tcgtagccct taagtgagc                                                  19 

 
           
             46  
             22  
             DNA  
             Homo sapiens  
           
            46 

aatatgtaca ttagagtagt tg                                              22 

 
           
             47  
             19  
             DNA  
             Homo sapiens  
           
            47 

cagagaaagg tcctgactc                                                  19 

 
           
             48  
             22  
             DNA  
             Homo sapiens  
           
            48 

agagatttgg aaaatgagta ac                                              22 

 
           
             49  
             19  
             DNA  
             Homo sapiens  
           
            49 

acaatgtcat cacaggagg                                                  19 

 
           
             50  
             20  
             DNA  
             Homo sapiens  
           
            50 

aacctttccc tttggtgagg                                                 20 

 
           
             51  
             20  
             DNA  
             Homo sapiens  
           
            51 

gattactctg agacctaggc                                                 20 

 
           
             52  
             22  
             DNA  
             Homo sapiens  
           
            52 

gattttctct tttccccttg gg                                              22 

 
           
             53  
             23  
             DNA  
             Homo sapiens  
           
            53 

caaacaaagc ttcaacaatt tac                                             23 

 
           
             54  
             26  
             DNA  
             Homo sapiens  
           
            54 

gggttttatt ttcaagtact tctatg                                          26 

 
           
             55  
             26  
             DNA  
             Homo sapiens  
           
            55 

gctcagcaac tgttcaatgt atgagc                                          26 

 
           
             56  
             18  
             DNA  
             Homo sapiens  
           
            56 

ctagtgtgtg tttttggc                                                   18 

 
           
             57  
             18  
             DNA  
             Homo sapiens  
           
            57 

cataacctta tctccacc                                                   18 

 
           
             58  
             23  
             DNA  
             Homo sapiens  
           
            58 

ctcagccatg agacaataaa tcc                                             23 

 
           
             59  
             21  
             DNA  
             Homo sapiens  
           
            59 

ggttcccaaa taatgtgatg g                                               21 

 
           
             60  
             18  
             DNA  
             Homo sapiens  
           
            60 

caaaagcttc agaatctc                                                   18 

 
           
             61  
             23  
             DNA  
             Homo sapiens  
           
            61 

ctgtgggtgt ttcctgtgag tgg                                             23 

 
           
             62  
             24  
             DNA  
             Homo sapiens  
           
            62 

catgactttg tgtgaatgta cacc                                            24 

 
           
             63  
             24  
             DNA  
             Homo sapiens  
           
            63 

gaggagagcc tgatagaaca tctg                                            24 

 
           
             64  
             20  
             DNA  
             Homo sapiens  
           
            64 

gggctttttc tccccctccc                                                 20 

 
           
             65  
             18  
             DNA  
             Homo sapiens  
           
            65 

aaaatctggg ctctcacg                                                   18 

 
           
             66  
             19  
             DNA  
             Homo sapiens  
           
            66 

aattatacct catactagc                                                  19 

 
           
             67  
             23  
             DNA  
             Homo sapiens  
           
            67 

gttttattac agaataaagg agg                                             23 

 
           
             68  
             19  
             DNA  
             Homo sapiens  
           
            68 

aagccaaagt tagaaggca                                                  19 

 
           
             69  
             20  
             DNA  
             Homo sapiens  
           
            69 

tgcaacccac aaaatttggc                                                 20 

 
           
             70  
             20  
             DNA  
             Homo sapiens  
           
            70 

ctttctccat ttccaaaacc                                                 20 

 
           
             71  
             18  
             DNA  
             Homo sapiens  
           
            71 

tggtgtctct agttctgg                                                   18 

 
           
             72  
             20  
             DNA  
             Homo sapiens  
           
            72 

cattgttgta gtagctctgc                                                 20 

 
           
             73  
             18  
             DNA  
             Homo sapiens  
           
            73 

cccatttgtc ccaactgg                                                   18 

 
           
             74  
             19  
             DNA  
             Homo sapiens  
           
            74 

cggtcagttg aaatgtcag                                                  19 

 
           
             75  
             22  
             DNA  
             Homo sapiens  
           
            75 

catttggatg ctccgttaaa gc                                              22 

 
           
             76  
             23  
             DNA  
             Homo sapiens  
           
            76 

cacccggctg gaaattttat ttg                                             23 

 
           
             77  
             22  
             DNA  
             Homo sapiens  
           
            77 

ggaaaggcac tggagaaatg gg                                              22 

 
           
             78  
             25  
             DNA  
             Homo sapiens  
           
            78 

ccctccagca cacatgcatg taccg                                           25 

 
           
             79  
             20  
             DNA  
             Homo sapiens  
           
            79 

taagtagtct gtgatctccg                                                 20 

 
           
             80  
             18  
             DNA  
             Homo sapiens  
           
            80 

atgtatgagg tcctgtcc                                                   18 

 
           
             81  
             18  
             DNA  
             Homo sapiens  
           
            81 

gacaccagtg tatgttgg                                                   18 

 
           
             82  
             20  
             DNA  
             Homo sapiens  
           
            82 

gagaaagaag aacacatccc                                                 20 

 
           
             83  
             38  
             DNA  
             Homo sapiens  
           
            83 

tgtaaaacga cggccagtca ctgaggtgat tggctgaa                             38 

 
           
             84  
             19  
             DNA  
             Homo sapiens  
             
               misc_feature  
               (1)..(1)  
               The residue is biotinylated  
             
           
            84 

tagcccttaa gtgagcccg                                                  19 

 
           
             85  
             38  
             DNA  
             Homo sapiens  
           
            85 

tgtaaaacga cggccagtta cattagagta gttgcaga                             38 

 
           
             86  
             19  
             DNA  
             Homo sapiens  
             
               misc_feature  
               (1)..(1)  
               The residue is biotinylated  
             
           
            86 

aggtcctgac tcttccatg                                                  19 

 
           
             87  
             40  
             DNA  
             Homo sapiens  
           
            87 

tgtaaaacga cggccagttt ggaaaatgag taacatgatt                           40 

 
           
             88  
             19  
             DNA  
             Homo sapiens  
             
               misc_feature  
               (1)..(1)  
               The residue is biotinylated  
             
           
            88 

tgtcatcaca ggaggatat                                                  19 

 
           
             89  
             38  
             DNA  
             Homo sapiens  
           
            89 

tgtaaaacga cggccagtct ttccctttgg tgaggtga                             38 

 
           
             90  
             20  
             DNA  
             Homo sapiens  
             
               misc_feature  
               (1)..(1)  
               The residue is biotinylated  
             
           
            90 

tactctgaga cctaggccca                                                 20 

 
           
             91  
             40  
             DNA  
             Homo sapiens  
           
            91 

tgtaaaacga cggccagttc tcttttcccc ttgggattag                           40 

 
           
             92  
             23  
             DNA  
             Homo sapiens  
             
               misc_feature  
               (1)..(1)  
               The residue is biotinylated  
             
           
            92 

acaaagcttc aacaatttac tct                                             23 

 
           
             93  
             46  
             DNA  
             Homo sapiens  
           
            93 

tgtaaaacga cggccagtgt tttattttca agtacttcta tgaatt                    46 

 
           
             94  
             26  
             DNA  
             Homo sapiens  
             
               misc_feature  
               (1)..(1)  
               The residue is biotinylated  
             
           
            94 

cagcaactgt tcaatgtatg agcact                                          26 

 
           
             95  
             36  
             DNA  
             Homo sapiens  
           
            95 

tgtaaaacga cggccagtgt gtgtgttttt ggcaac                               36 

 
           
             96  
             18  
             DNA  
             Homo sapiens  
             
               misc_feature  
               (1)..(1)  
               The residue is biotinylated  
             
           
            96 

aaccttatct ccaccagc                                                   18 

 
           
             97  
             41  
             DNA  
             Homo sapiens  
           
            97 

tgtaaaacga cggccagtag ccatgagaca ataaatcctt g                         41 

 
           
             98  
             22  
             DNA  
             Homo sapiens  
             
               misc_feature  
               (1)..(1)  
               The residue is biotinylated  
             
           
            98 

tcccaaataa tgtgatggaa tg                                              22 

 
           
             99  
             37  
             DNA  
             Homo sapiens  
           
            99 

tgtaaaacga cggccagtaa gcttcagaat ctctttt                              37 

 
           
             100  
             23  
             DNA  
             Homo sapiens  
             
               misc_feature  
               (1)..(1)  
               The residue is biotinylated  
             
           
            100 

tgggtgtttc ctgtgagtgg att                                             23 

 
           
             101  
             42  
             DNA  
             Homo sapiens  
           
            101 

tgtaaaacga cggccagtac tttgtgtgaa tgtacacctg tg                        42 

 
           
             102  
             24  
             DNA  
             Homo sapiens  
             
               misc_feature  
               (1)..(1)  
               The residue is biotinylated  
             
           
            102 

gagagcctga tagaacatct gttg                                            24 

 
           
             103  
             39  
             DNA  
             Homo sapiens  
           
            103 

tgtaaaacga cggccagtct ttttctcccc ctcccacta                            39 

 
           
             104  
             17  
             DNA  
             Homo sapiens  
             
               misc_feature  
               (1)..(1)  
               The residue is biotinylated  
             
           
            104 

tctgggctct cacgtct                                                    17 

 
           
             105  
             18  
             DNA  
             Homo sapiens  
             
               misc_feature  
               (1)..(1)  
               The residue is biotinylated  
             
           
            105 

cttattctga gtctctcc                                                   18 

 
           
             106  
             35  
             DNA  
             Homo sapiens  
           
            106 

tgtaaaacga cggccagtgt ttgctcagag gctgc                                35 

 
           
             107  
             21  
             DNA  
             Homo sapiens  
             
               misc_feature  
               (1)..(1)  
               The residue is biotinylated  
             
           
            107 

gatggttcgt acagattccc g                                               21 

 
           
             108  
             41  
             DNA  
             Homo sapiens  
           
            108 

tgtaaaacga cggccagttt attacagaat aaaggaggta g                         41 

 
           
             109  
             39  
             DNA  
             Homo sapiens  
           
            109 

tgtaaaacga cggccagtaa cccacaaaat ttggctaag                            39 

 
           
             110  
             20  
             DNA  
             Homo sapiens  
             
               misc_feature  
               (1)..(1)  
               The residue is biotinylated  
             
           
            110 

tctccatttc caaaaccttg                                                 20 

 
           
             111  
             18  
             DNA  
             Homo sapiens  
             
               misc_feature  
               (1)..(1)  
               The residue is biotinylated  
             
           
            111 

tgtctctagt tctggtgc                                                   18 

 
           
             112  
             38  
             DNA  
             Homo sapiens  
           
            112 

tgtaaaacga cggccagttg ttgtagtagc tctgcttg                             38 

 
           
             113  
             20  
             DNA  
             Homo sapiens  
             
               misc_feature  
               (1)..(1)  
               The residue is biotinylated  
             
           
            113 

atttgtccca actggttgta                                                 20 

 
           
             114  
             39  
             DNA  
             Homo sapiens  
           
            114 

tgtaaaacga cggccagttc agttgaaatg tcagaagtg                            39 

 
           
             115  
             18  
             DNA  
             Homo sapiens  
           
            115 

tgtaaaacga cggccagt                                                   18 

 
           
             116  
             23  
             DNA  
             Homo sapiens  
             
               misc_feature  
               (1)..(1)  
               The residue is biotinylated  
             
           
            116 

ccggctggaa attttatttg gag                                             23 

 
           
             117  
             41  
             DNA  
             Homo sapiens  
           
            117 

tgtaaaacga cggccagtag gcactggaga aatgggattt g                         41 

 
           
             118  
             26  
             DNA  
             Homo sapiens  
             
               misc_feature  
               (1)..(1)  
               The residue is biotinylated  
             
           
            118 

tccagcacac atgcatgtac cgaaat                                          26 

 
           
             119  
             20  
             DNA  
             Homo sapiens  
             
               misc_feature  
               (1)..(1)  
               The residue is biotinylated  
             
           
            119 

gtagtctgtg atctccgttt                                                 20 

 
           
             120  
             36  
             DNA  
             Homo sapiens  
           
            120 

tgtaaaacga cggccagtta tgaggtcctg tcctag                               36 

 
           
             121  
             19  
             DNA  
             Homo sapiens  
             
               misc_feature  
               (1)..(1)  
               The residue is biotinylated  
             
           
            121 

accagtgtat gttgggatg                                                  19 

 
           
             122  
             39  
             DNA  
             Homo sapiens  
           
            122 

tgtaaaacga cggccagtga aagaagaaca catcccaca                            39 

 
           
             123  
             770  
             PRT  
             Saccharomyces cerevisiae  
           
            123 

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 Gln Leu Gly Asp Tyr 
465                 470                 475                 480 

Lys Val Pro Ser Ile Ala Asp Asp Glu Lys Asn Ala Leu Pro Ile Ser 
                485                 490                 495 

Lys Asp Gly Tyr Ile Arg Val Pro Lys Glu Arg Val Asn Val Asn Leu 
            500                 505                 510 

Thr Ser Ile Lys Lys Leu Arg Glu Lys Val Asp Asp Ser Ile His Arg 
        515                 520                 525 

Glu Leu Thr Asp Ile Phe Ala Asn Leu Asn Tyr Val Gly Val Val Asp 
    530                 535                 540 

Glu Glu Arg Arg Leu Ala Ala Ile Gln His Asp Leu Lys Leu Phe Leu 
545                 550                 555                 560 

Ile Asp Tyr Gly Ser Val Cys Tyr Glu Leu Phe Tyr Gln Ile Gly Leu 
                565                 570                 575 

Thr Asp Phe Ala Asn Phe Gly Lys Ile Asn Leu Gln Ser Thr Asn Val 
            580                 585                 590 

Ser Asp Asp Ile Val Leu Tyr Asn Leu Leu Ser Glu Phe Asp Glu Leu 
        595                 600                 605 

Asn Asp Asp Ala Ser Lys Glu Lys Ile Ile Ser Lys Ile Trp Asp Met 
    610                 615                 620 

Ser Ser Met Leu Asn Glu Tyr Tyr Ser Ile Glu Leu Val Asn Asp Gly 
625                 630                 635                 640 

Leu Asp Asn Asp Leu Lys Ser Val Lys Leu Lys Ser Leu Pro Leu Leu 
                645                 650                 655 

Leu Lys Gly Tyr Ile Pro Ser Leu Val Lys Leu Pro Phe Phe Ile Tyr 
            660                 665                 670 

Arg Leu Gly Lys Glu Val Asp Trp Glu Asp Glu Gln Glu Cys Leu Asp 
        675                 680                 685 

Gly Ile Leu Arg Glu Ile Ala Leu Leu Tyr Ile Pro Asp Met Val Pro 
    690                 695                 700 

Lys Val Asp Thr Leu Asp Ala Ser Leu Ser Glu Asp Glu Lys Ala Gln 
705                 710                 715                 720 

Phe Ile Asn Arg Lys Glu His Ile Ser Ser Leu Leu Glu His Val Leu 
                725                 730                 735 

Phe Pro Cys Ile Lys Arg Arg Phe Leu Ala Pro Arg His Ile Leu Lys 
            740                 745                 750 

Asp Val Val Glu Ile Ala Asn Leu Pro Asp Leu Tyr Lys Val Phe Glu 
        755                 760                 765 

Arg Cys 
    770 

 
           
             124  
             64  
             PRT  
             Homo sapiens  
           
            124 

Val Asn Arg Ile Ala Ala Gly Glu Val Ile Gln Arg Pro Ala Asn Ala 
1               5                   10                  15 

Ile Lys Glu Met Ile Glu Asn Cys Leu Asp Ala Lys Phe Thr Ser Ile 
            20                  25                  30 

Gln Val Ile Val Lys Glu Gly Gly Leu Lys Leu Ile Gln Ile Gln Asp 
        35                  40                  45 

Asn Gly Thr Gly Ile Arg Lys Glu Asp Leu Asp Ile Val Cys Glu Arg 
    50                  55                  60 

 
           
             125  
             64  
             PRT  
             Homo sapiens  
           
            125 

Val Asn Arg Ile Ala Ala Gly Glu Val Ile Gln Arg Pro Ala Asn Ala 
1               5                   10                  15 

Ile Lys Glu Met Ile Glu Asn Cys Leu Asp Ala Lys Ser Thr Ser Ile 
            20                  25                  30 

Gln Val Ile Val Lys Glu Gly Gly Leu Lys Leu Ile Gln Ile Gln Asp 
        35                  40                  45 

Asn Gly Thr Gly Ile Arg Lys Glu Asp Leu Asp Ile Val Cys Glu Arg 
    50                  55                  60 

 
           
             126  
             52  
             PRT  
             Mus musculus  
           
            126 

Pro Ala Asn Ala Ile Lys Glu Met Ile Glu Asn Cys Leu Asp Ala Lys 
1               5                   10                  15 

Ser Thr Asn Ile Gln Val Val Val Lys Glu Gly Gly Leu Lys Leu Ile 
            20                  25                  30 

Gln Ile Gln Asp Asn Gly Thr Gly Ile Arg Lys Glu Asp Leu Asp Ile 
        35                  40                  45 

Val Cys Glu Arg 
    50 

 
           
             127  
             64  
             PRT  
             Saccharomyces cerevisiae  
           
            127 

Val Asn Lys Ile Ala Ala Gly Glu Ile Ile Ile Ser Pro Val Asn Ala 
1               5                   10                  15 

Leu Lys Glu Met Met Glu Asn Ser Ile Asp Ala Asn Ala Thr Met Ile 
            20                  25                  30 

Asp Ile Leu Val Lys Glu Gly Gly Ile Lys Val Leu Gln Ile Thr Asp 
        35                  40                  45 

Asn Gly Ser Gly Ile Asn Lys Ala Asp Leu Pro Ile Leu Cys Glu Arg 
    50                  55                  60 

 
           
             128  
             64  
             PRT  
             Saccharomyces cerevisiae  
           
            128 

Val His Arg Ile Thr Ser Gly Gln Val Ile Thr Asp Leu Thr Thr Ala 
1               5                   10                  15 

Val Lys Glu Leu Val Asp Asn Ser Ile Asp Ala Asn Ala Asn Gln Ile 
            20                  25                  30 

Glu Ile Ile Phe Lys Asp Tyr Gly Leu Glu Ser Ile Glu Cys Ser Asp 
        35                  40                  45 

Asn Gly Asp Gly Ile Asp Pro Ser Asn Tyr Glu Phe Leu Ala Leu Lys 
    50                  55                  60 

 
           
             129  
             64  
             PRT  
             Escherichia coli  
           
            129 

Ala Asn Gln Ile Ala Ala Gly Glu Val Val Glu Arg Pro Ala Ser Val 
1               5                   10                  15 

Val Lys Glu Leu Val Glu Asn Ser Leu Asp Ala Gly Ala Thr Arg Ile 
            20                  25                  30 

Asp Ile Asp Ile Glu Arg Gly Gly Ala Lys Leu Ile Arg Ile Arg Asp 
        35                  40                  45 

Asn Gly Cys Gly Ile Lys Lys Asp Glu Leu Ala Leu Ala Leu Ala Arg 
    50                  55                  60 

 
           
             130  
             64  
             PRT  
             Salmonella typhimurium  
           
            130 

Ala Asn Gln Ile Ala Ala Gly Glu Val Val Glu Arg Pro Ala Ser Val 
1               5                   10                  15 

Val Lys Glu Leu Val Glu Asn Ser Leu Asp Ala Gly Ala Thr Arg Val 
            20                  25                  30 

Asp Ile Asp Ile Glu Arg Gly Gly Ala Lys Leu Ile Arg Ile Arg Asp 
        35                  40                  45 

Asn Gly Cys Gly Ile Lys Lys Glu Glu Leu Ala Leu Ala Leu Ala Arg 
    50                  55                  60 

 
           
             131  
             64  
             PRT  
             Streptococcus pneumoniae  
           
            131 

Ala Asn Gln Ile Ala Ala Gly Glu Val Ile Glu Arg Pro Ala Ser Val 
1               5                   10                  15 

Cys Lys Glu Leu Val Glu Asn Ala Ile Asp Ala Gly Ser Ser Gln Ile 
            20                  25                  30 

Ile Ile Glu Ile Glu Glu Ala Gly Leu Lys Lys Val Gln Ile Thr Asp 
        35                  40                  45 

Asn Gly His Gly Ile Ala His Asp Glu Val Glu Leu Ala Leu Arg Arg 
    50                  55                  60 

 
           
             132  
             2687  
             DNA  
             Homo sapiens  
           
            132 

ccatggagcg agctgagagc tcgagtacag aacctgctaa ggccatcaaa cctattgatc     60 

ggaagtcagt ccatcagatt tgctctgggc aggtggtact gagtctaagc actgcggtaa    120 

aggagttagt agaaaacagt ctggatgctg gtgccactaa tattgatcta aagcttaagg    180 

actatggagt ggatcttatt gaagtttcag acaatggatg tggggtagaa gaagaaaact    240 

tcgaaggctt aactctgaaa catcacacat ctaagattca agagtttgcc gacctaactc    300 

aggttgaaac ttttggcttt cggggggaag ctctgagctc actttgtgca ctgagcgatg    360 

tcaccatttc tacctgccac gcatcggcga aggttggaac tcgactgatg tttgatcaca    420 

atgggaaaat tatccagaaa accccctacc cccgccccag agggaccaca gtcagcgtgc    480 

agcagttatt ttccacacta cctgtgcgcc ataaggaatt tcaaaggaat attaagaagg    540 

agtatgccaa aatggtccag gtcttacatg catactgtat catttcagca ggcatccgtg    600 

taagttgcac caatcagctt ggacaaggaa aacgacagcc tgtggtatgc acaggtggaa    660 

gccccagcat aaaggaaaat atcggctctg tgtttgggca gaagcagttg caaagcctca    720 

ttccttttgt tcagctgccc cctagtgact ccgtgtgtga agagtacggt ttgagctgtt    780 

cggatgctct gcataatctt ttttacatct caggtttcat ttcacaatgc acgcatggag    840 

ttggaaggag ttcaacagac agacagtttt tctttatcaa ccggcggcct tgtgacccag    900 

caaaggtctg cagactcgtg aatgaggtct accacatgta taatcgacac cagtatccat    960 

ttgttgttct taacatttct gttgattcag aatgcgttga tatcaatgtt actccagata   1020 

aaaggcaaat tttgctacaa gaggaaaagc ttttgttggc agttttaaag acctctttga   1080 

taggaatgtt tgatagtgat gtcaacaagc taaatgtcag tcagcagcca ctgctggatg   1140 

ttgaaggtaa cttaataaaa atgcatgcag cggatttgga aaagcccatg gtagaaaagc   1200 

aggatcaatc cccttcatta aggactggag aagaaaaaaa agacgtgtcc atttccagac   1260 

tgcgagaggc cttttctctt cgtcacacaa cagagaacaa gcctcacagc ccaaagactc   1320 

cagaaccaag aaggagccct ctaggacaga aaaggggtat gctgtcttct agcacttcag   1380 

gtgccatctc tgacaaaggc gtcctgagat ctcagaaaga ggcagtgagt tccagtcacg   1440 

gacccagtga ccctacggac agagcggagg tggagaagga ctcggggcac ggcagcactt   1500 

ccgtggattc tgaggggttc agcatcccag acacgggcag tcactgcagc agcgagtatg   1560 

cggccagctc cccaggggac aggggctcgc aggaacatgt ggactctcag gagaaagcgc   1620 

ctgaaactga cgactctttt tcagatgtgg actgccattc aaaccaggaa gataccggat   1680 

gtaaatttcg agttttgcct cagccaacta atctcgcaac cccaaacaca aagcgtttta   1740 

aaaaagaaga aattctttcc agttctgaca tttgtcaaaa gttagtaaat actcaggaca   1800 

tgtcagcctc tcaggttgat tgagctgtga aaattaataa gaaagttgtg cccctggact   1860 

tttctatgag ttctttagct aaacgaataa agcagttaca tcatgaagca cagcaaagtg   1920 

aaggggaaca gaattacagg aagtttaggg caaagatttg tcctggagaa aatcaagcag   1980 

ccgaagatga actaagaaaa gagataagta aaacgatgtt tgcagaaatg gaaatcattg   2040 

gtcagtttaa cctgggattt ataataacca aactgaatga ggatatcttc atagtggacc   2100 

agcatgccac ggacgagaag tataacttcg agatgctgca gcagcacacc gtgctccagg   2160 

ggcagaggct catagcacct cagactctca acttaactgc tgttaatgaa gctgttctga   2220 

tagaaaatct ggaaatattt agaaagaatg gctttgattt tgttatcgat gaaaatgctc   2280 

cagtcactga aagggctaaa ctgatttcct tgccaactag taaaaactgg accttcggac   2340 

cccaggacgt cgatgaactg atcttcatgc tgagcgacag ccctggggtc atgtgccgcc   2400 

cttcccgagt caagcagatg tttgcctcca gagcctgccg gaagtcggtg atgattggga   2460 

ctgctctcaa cacaagcgaa tgaagaaact gatcacccac atgggggaga tgggccaccc   2520 

ctggaactgt ccccatggaa ggccaccatg agacacatcg ccaacctggg tgtcatttct   2580 

cagaactgac cgtagtcact gtatggaata attggtttta tcgcagattt ttatgttttg   2640 

aaagacagag tcttcactaa ccttttttgt tttaaaatga aacctgc                 2687 

 
           
             133  
             862  
             PRT  
             Homo sapiens  
           
            133 

Met Glu Arg Ala Glu Ser Ser Ser Thr Glu Pro Ala Lys Ala Ile Lys 
1               5                   10                  15 

Pro Ile Asp Arg Lys Ser Val His Gln Ile Cys Ser Gly Gln Val Val 
            20                  25                  30 

Leu Ser Leu Ser Thr Ala Val Lys Glu Leu Val Glu Asn Ser Leu Asp 
        35                  40                  45 

Ala Gly Ala Thr Asn Ile Asp Leu Lys Leu Lys Asp Tyr Gly Val Asp 
    50                  55                  60 

Leu Ile Glu Val Ser Asp Asn Gly Cys Gly Val Glu Glu Glu Asn Phe 
65                  70                  75                  80 

Glu Gly Leu Thr Leu Lys His His Thr Ser Lys Ile Gln Glu Phe Ala 
                85                  90                  95 

Asp Leu Thr Gln Val Glu Thr Phe Gly Phe Arg Gly Glu Ala Leu Ser 
            100                 105                 110 

Ser Leu Cys Ala Leu Ser Asp Val Thr Ile Ser Thr Cys His Ala Ser 
        115                 120                 125 

Ala Lys Val Gly Thr Arg Leu Met Phe Asp His Asn Gly Lys Ile Ile 
    130                 135                 140 

Gln Lys Thr Pro Tyr Pro Arg Pro Arg Gly Thr Thr Val Ser Val Gln 
145                 150                 155                 160 

Gln Leu Phe Ser Thr Leu Pro Val Arg His Lys Glu Phe Gln Arg Asn 
                165                 170                 175 

Ile Lys Lys Glu Tyr Ala Lys Met Val Gln Val Leu His Ala Tyr Cys 
            180                 185                 190 

Ile Ile Ser Ala Gly Ile Arg Val Ser Cys Thr Asn Gln Leu Gly Gln 
        195                 200                 205 

Gly Lys Arg Gln Pro Val Val Cys Ile Gly Gly Ser Pro Ser Ile Lys 
    210                 215                 220 

Glu Asn Ile Gly Ser Val Phe Gly Gln Lys Gln Leu Gln Ser Leu Ile 
225                 230                 235                 240 

Pro Phe Val Gln Leu Pro Pro Ser Asp Ser Val Cys Glu Glu Tyr Gly 
                245                 250                 255 

Leu Ser Cys Ser Asp Ala Leu His Asn Leu Phe Tyr Ile Ser Gly Phe 
            260                 265                 270 

Ile Ser Gln Cys Thr His Gly Val Gly Arg Ser Ser Thr Asp Arg Gln 
        275                 280                 285 

Phe Phe Phe Ile Asn Arg Arg Pro Cys Asp Pro Ala Lys Val Cys Arg 
    290                 295                 300 

Leu Val Asn Glu Val Tyr His Met Tyr Asn Arg His Gln Tyr Pro Phe 
305                 310                 315                 320 

Val Val Leu Asn Ile Ser Val Asp Ser Glu Cys Val Asp Ile Asn Val 
                325                 330                 335 

Thr Pro Asp Lys Arg Gln Ile Leu Leu Gln Glu Glu Lys Leu Leu Leu 
            340                 345                 350 

Ala Val Leu Lys Thr Ser Leu Ile Gly Met Phe Asp Ser Asp Val Asn 
        355                 360                 365 

Lys Leu Asn Val Ser Gln Gln Pro Leu Leu Asp Val Glu Gly Asn Leu 
    370                 375                 380 

Ile Lys Met His Ala Ala Asp Leu Glu Lys Pro Met Val Glu His Gln 
385                 390                 395                 400 

Asp Gln Ser Pro Ser Leu Arg Ile Gly Glu Glu Lys Lys Asp Val Ser 
                405                 410                 415 

Ile Ser Arg Leu Arg Glu Ala Phe Ser Leu Arg His Thr Thr Glu Asn 
            420                 425                 430 

Lys Pro His Ser Pro Lys Thr Pro Glu Pro Arg Arg Ser Pro Leu Gly 
        435                 440                 445 

Gln Lys Arg Gly Met Leu Ser Ser Ser Thr Ser Gly Ala Ile Ser Asp 
    450                 455                 460 

Lys Gly Val Leu Arg Ser Gln Lys Glu Ala Val Ser Ser Ser His Gly 
465                 470                 475                 480 

Pro Ser Asp Pro Thr Asp Arg Ala Glu Val Glu Lys Asp Ser Gly His 
                485                 490                 495 

Gly Ser Thr Ser Val Asp Ser Glu Gly Phe Ser Ile Pro Asp Thr Gly 
            500                 505                 510 

Ser His Cys Ser Ser Glu Tyr Ala Ala Ser Ser Pro Gly Asp Arg Gly 
        515                 520                 525 

Ser Gln Glu His Val Asp Ser Gln Glu Lys Ala Pro Glu Thr Asp Asp 
    530                 535                 540 

Ser Phe Ser Asp Val Asp Cys His Ser Asn Gln Glu Asp Thr Gly Cys 
545                 550                 555                 560 

Lys Phe Arg Val Leu Pro Gln Pro Ile Asn Leu Ala Thr Pro Asn Thr 
                565                 570                 575 

Lys Arg Phe Lys Lys Glu Glu Ile Leu Ser Ser Ser Asp Ile Cys Gln 
            580                 585                 590 

Lys Leu Val Asn Thr Gln Asp Met Ser Ala Ser Gln Val Asp Val Ala 
        595                 600                 605 

Val Lys Ile Asn Lys Lys Val Val Pro Leu Asp Phe Ser Met Ser Ser 
    610                 615                 620 

Leu Ala Lys Arg Ile Lys Gln Leu His His Glu Ala Gln Gln Ser Glu 
625                 630                 635                 640 

Gly Glu Gln Asn Tyr Arg Lys Phe Arg Ala Lys Ile Cys Pro Gly Glu 
                645                 650                 655 

Asn Gln Ala Ala Glu Asp Glu Leu Arg Lys Glu Ile Ser Lys Thr Met 
            660                 665                 670 

Phe Ala Glu Met Glu Ile Ile Gly Gln Phe Asn Leu Gly Phe Ile Ile 
        675                 680                 685 

Thr Lys Leu Asn Glu Asp Ile Phe Ile Val Asp Gln His Ala Thr Asp 
    690                 695                 700 

Glu Lys Tyr Asn Phe Glu Met Leu Gln Gln His Thr Val Leu Gln Gly 
705                 710                 715                 720 

Gln Arg Leu Ile Ala Pro Gln Thr Leu Asn Leu Thr Ala Val Asn Glu 
                725                 730                 735 

Ala Val Leu Ile Glu Asn Leu Glu Ile Phe Arg Lys Asn Gly Phe Asp 
            740                 745                 750 

Phe Val Ile Asp Glu Asn Ala Pro Val Thr Glu Arg Ala Lys Leu Ile 
        755                 760                 765 

Ser Leu Pro Thr Ser Lys Asn Trp Thr Phe Gly Pro Gln Asp Val Asp 
    770                 775                 780 

Glu Leu Ile Phe Met Leu Ser Asp Ser Pro Gly Val Met Cys Arg Pro 
785                 790                 795                 800 

Ser Arg Val Lys Gln Met Phe Ala Ser Arg Ala Cys Arg Lys Ser Val 
                805                 810                 815 

Met Ile Gly Thr Ala Leu Asn Thr Ser Glu Met Lys Lys Leu Ile Thr 
            820                 825                 830 

His Met Gly Glu Met Gly His Pro Trp Asn Cys Pro His Gly Arg Pro 
        835                 840                 845 

Thr Met Arg His Ile Ala Asn Leu Gly Val Ile Ser Gln Asn 
    850                 855                 860 

 
           
             134  
             903  
             PRT  
             Saccharomyces cerevisiae  
           
            134 

Met Phe His His Ile Glu Asn Leu Leu Ile Glu Thr Glu Lys Arg Cys 
1               5                   10                  15 

Lys Gln Lys Glu Gln Arg Tyr Ile Pro Val Lys Tyr Leu Phe Ser Met 
            20                  25                  30 

Thr Gln Ile His Gln Ile Asn Asp Ile Asp Val His Arg Ile Thr Ser 
        35                  40                  45 

Gly Gln Val Ile Thr Asp Leu Thr Thr Ala Val Lys Glu Leu Val Asp 
    50                  55                  60 

Asn Ser Ile Asp Ala Asn Ala Asn Gln Ile Glu Ile Ile Phe Lys Asp 
65                  70                  75                  80 

Tyr Gly Leu Glu Ser Ile Glu Cys Ser Asp Asn Gly Asp Gly Ile Asp 
                85                  90                  95 

Pro Ser Asn Tyr Glu Phe Leu Ala Leu Lys His Tyr Thr Ser Lys Ile 
            100                 105                 110 

Ala Lys Phe Gln Asp Val Ala Lys Val Gln Thr Leu Gly Phe Arg Gly 
        115                 120                 125 

Glu Ala Leu Ser Ser Leu Cys Gly Ile Ala Lys Leu Ser Val Ile Thr 
    130                 135                 140 

Thr Thr Ser Pro Pro Lys Ala Asp Lys Leu Glu Tyr Asp Met Val Gly 
145                 150                 155                 160 

His Ile Thr Ser Lys Thr Thr Ser Arg Asn Lys Gly Thr Thr Val Leu 
                165                 170                 175 

Val Ser Gln Leu Phe His Asn Leu Pro Val Arg Gln Lys Glu Phe Ser 
            180                 185                 190 

Lys Thr Phe Lys Arg Gln Phe Thr Lys Cys Leu Thr Val Ile Gln Gly 
        195                 200                 205 

Tyr Ala Ile Ile Asn Ala Ala Ile Lys Phe Ser Val Trp Asn Ile Thr 
    210                 215                 220 

Pro Lys Gly Lys Lys Asn Leu Ile Leu Ser Thr Met Arg Asn Ser Ser 
225                 230                 235                 240 

Met Arg Lys Asn Ile Ser Ser Val Phe Gly Ala Gly Gly Met Phe Gly 
                245                 250                 255 

Leu Glu Glu Val Asp Leu Val Leu Asp Leu Asn Pro Phe Lys Asn Arg 
            260                 265                 270 

Met Leu Gly Lys Tyr Thr Asp Asp Pro Asp Phe Leu Asp Leu Asp Tyr 
        275                 280                 285 

Lys Ile Arg Val Lys Gly Tyr Ile Ser Gln Asn Ser Phe Gly Cys Gly 
    290                 295                 300 

Arg Asn Ser Lys Asp Arg Gln Phe Ile Tyr Val Asn Lys Arg Pro Val 
305                 310                 315                 320 

Glu Tyr Ser Thr Leu Leu Lys Cys Cys Asn Glu Val Tyr Lys Thr Phe 
                325                 330                 335 

Asn Asn Val Gln Phe Pro Ala Val Phe Leu Asn Leu Glu Leu Pro Met 
            340                 345                 350 

Ser Leu Ile Asp Val Asn Val Thr Pro Asp Lys Arg Val Ile Leu Leu 
        355                 360                 365 

His Asn Glu Arg Ala Val Ile Asp Ile Phe Lys Thr Thr Leu Ser Asp 
    370                 375                 380 

Tyr Tyr Asn Arg Gln Glu Leu Ala Leu Pro Lys Arg Met Cys Ser Gln 
385                 390                 395                 400 

Ser Glu Gln Gln Ala Gln Lys Arg Leu Lys Thr Glu Val Phe Asp Asp 
                405                 410                 415 

Arg Ser Thr Thr His Glu Ser Asp Asn Glu Asn Tyr His Thr Ala Arg 
            420                 425                 430 

Ser Glu Ser Asn Gln Ser Asn His Ala His Phe Asn Ser Thr Thr Gly 
        435                 440                 445 

Val Ile Asp Lys Ser Asn Gly Thr Glu Leu Thr Ser Val Met Asp Gly 
    450                 455                 460 

Asn Tyr Thr Asn Val Thr Asp Val Ile Gly Ser Glu Cys Glu Val Ser 
465                 470                 475                 480 

Val Asp Ser Ser Val Val Leu Asp Glu Gly Asn Ser Ser Thr Pro Thr 
                485                 490                 495 

Lys Lys Leu Pro Ser Ile Lys Thr Asp Ser Gln Asn Leu Ser Asp Leu 
            500                 505                 510 

Asn Leu Asn Asn Phe Ser Asn Pro Glu Phe Gln Asn Ile Thr Ser Pro 
        515                 520                 525 

Asp Lys Ala Arg Ser Leu Glu Lys Val Val Glu Glu Pro Val Tyr Phe 
    530                 535                 540 

Asp Ile Asp Gly Glu Lys Phe Gln Glu Lys Ala Val Leu Ser Gln Ala 
545                 550                 555                 560 

Asp Gly Leu Val Phe Val Asp Asn Glu Cys His Glu His Thr Asn Asp 
                565                 570                 575 

Cys Cys His Gln Glu Arg Arg Gly Ser Thr Asp Ile Glu Gln Asp Asp 
            580                 585                 590 

Glu Ala Asp Ser Ile Tyr Ala Glu Ile Glu Pro Val Glu Ile Asn Val 
        595                 600                 605 

Arg Thr Pro Leu Lys Asn Ser Arg Lys Ser Ile Ser Lys Asp Asn Tyr 
    610                 615                 620 

Arg Ser Leu Ser Asp Gly Leu Thr His Arg Lys Phe Glu Asp Glu Ile 
625                 630                 635                 640 

Leu Glu Tyr Asn Leu Ser Thr Lys Asn Phe Lys Glu Ile Ser Lys Asn 
                645                 650                 655 

Gly Lys Gln Met Ser Ser Ile Ile Ser Lys Arg Lys Ser Glu Ala Gln 
            660                 665                 670 

Glu Asn Ile Ile Lys Asn Lys Asp Glu Leu Glu Asp Phe Glu Gln Gly 
        675                 680                 685 

Glu Lys Tyr Leu Thr Leu Thr Val Ser Lys Asn Asp Phe Lys Lys Met 
    690                 695                 700 

Glu Val Val Gly Gln Phe Asn Leu Gly Phe Ile Ile Val Thr Arg Lys 
705                 710                 715                 720 

Val Asp Asn Lys Ser Lys Leu Phe Ile Val Asp Gln His Ala Ser Asp 
                725                 730                 735 

Glu Lys Tyr Asn Phe Glu Thr Leu Gln Ala Val Thr Val Phe Lys Ser 
            740                 745                 750 

Gln Lys Leu Ile Ile Pro Gln Pro Val Glu Leu Ser Val Ile Asp Glu 
        755                 760                 765 

Leu Val Val Leu Asp Asn Leu Pro Val Phe Glu Lys Asn Gly Phe Lys 
    770                 775                 780 

Leu Lys Ile Asp Glu Glu Glu Glu Phe Gly Ser Arg Val Lys Leu Leu 
785                 790                 795                 800 

Ser Leu Pro Thr Ser Lys Gln Thr Leu Phe Asp Leu Gly Asp Phe Asn 
                805                 810                 815 

Glu Leu Ile His Leu Ile Lys Glu Asp Gly Gly Leu Arg Arg Asp Asn 
            820                 825                 830 

Ile Arg Cys Ser Lys Ile Arg Ser Met Phe Ala Met Arg Ala Cys Arg 
        835                 840                 845 

Ser Ser Ile Met Ile Gly Lys Pro Leu Asn Lys Lys Thr Met Thr Arg 
    850                 855                 860 

Val Val His Asn Leu Ser Glu Leu Asp Lys Pro Trp Asn Cys Pro His 
865                 870                 875                 880 

Gly Arg Pro Thr Met Arg His Leu Met Glu Ile Arg Asp Trp Ser Ser 
                885                 890                 895 

Phe Ser Lys Asp Tyr Glu Ile 
            900 

 
           
             135  
             2577  
             DNA  
             Mus musculus  
           
            135 

ttccggccaa tgctatcaaa gagatgatag aaaactgttt agatgcaaaa tctacaaata     60 

ttcaagtggt tgttaaggaa ggtggcctga agctaattca gatccaagac aatggcactg    120 

gaatcaggaa ggaagatctg gatattgtgt gtgagaggtt cactacgagt aaactgcaga    180 

cttttgagga tttagccagt atttctacct atggctttcg tggtgagcat ttggcaagca    240 

taagtcatgt ggcccatgtc actattacaa ccaaaacagc tgatgggaaa tgtgcgtaca    300 

gagcaagtta ctcagatgga aagctgcaag cccctcctaa accctgtgca ggcaaccagg    360 

gcaccctgat cacggtggaa gacctttttt acaacataat cacaaggagg aaagctttaa    420 

aaaatccaag tgaagagtac ggaaaaattt tggaagttgt tggcaggtat tcaatacaca    480 

attcaggcat tagtatctca gttaaaaaac aaggtgagac agtatctgat gtcagaacac    540 

tgcccaatgc cacaaccgtg gacaacattc gctccatctt tggaaatgcg gttagtcgag    600 

aactgataga agttgggtgt gaggataaaa ccctagcttt caaaatgaat ggctatatat    660 

cgaatgcaaa gtattcagtg aagaagtgca ttttcctact cttcatcaac caccgtctgg    720 

tagaatcagc tgccttgaga aaagccattg aaactgtata tgcagcatac ttgccaaaaa    780 

cacacaccca ttcctgtacc tcagtttgaa atcagccctc agaacgtgac gtcaatgtac    840 

accccaccaa gacagaagtt cattttctgc acgaggagag cattctgcag cgtgtgcagc    900 

agcacattga gagcaagctg ctgggctcca attcctccag gatgtatttc acccagacct    960 

tgcttccagg acttgctggg cctctgggga ggcagctaga cccacgacag gggtggcttc   1020 

ctcatccact agtggaagtg gcgacaaggt ctacgcttac cagatgtcgc gtacggactc   1080 

ccgggatcag aagcttgacg cctttctgca gcctgtaacc agccttgtgc ccagccagcc   1140 

ccaggaccct cgccctgtcc gaggggccag gacagagggc tctcctgaaa gggccacgcg   1200 

ggaggatgag gagatgcttg ctctcccagc ccccgctgaa gcagctgctg agagtgagaa   1260 

cttggagagg gaatcactaa tggagacttc agacgcagcc cagaaagcgg cacccacttc   1320 

cagtccagga agctccagaa agagtcatcg ggaggactct gatgtggaaa tggtggaaaa   1380 

tgcttccggg aaggaaatga cagctgcttg ctaccccagg aggaggatca ttaacctcac   1440 

cagcgtcttg agtctccagg aagagattag tgagcggtgc catgagactc tccgggagat   1500 

actccgtaac cattcctttg tgggctgtgt gaatcctcag tgggccttgg cacagcacca   1560 

gaccaagcta tacctcctca acactaccaa gctcagtgaa gagctgttct accagatact   1620 

catttatgat tttgccaact ttggtgttct gaggttatcg gaaccagcgc cactcttcga   1680 

cctggccatg ctggcttaga cagtcctgaa agtggctgga cagaggacga cggcccgaag   1740 

aagggcttgc agagtacatt gtcgagtttc tgaagagaag cgagatgctt gcagactatt   1800 

ctctgtgaga tcgatgagaa gggaacctga ttgattactc ttctgatgac agctatgtgc   1860 

cacctttgga gggactgcct atcttcattc ttcgactggc cactgaggtg aattgggtga   1920 

agaaaaggag tgttttgaaa gtctcagtaa agaatgtgct atgttttact ccattcggaa   1980 

gcagtatata ctggaggagt cgaccctctc aggccagcag agtgacatgc ctggctccac   2040 

gtcaaagccc tggaagtgga ctgtggagca cattatctat aaagccttcc gctcacacct   2100 

cctacctccg aagcatttca cagaagatgg caatgtcctg cagcttgcca acctgccaga   2160 

tctatacaaa gtctttgagc ggtgttaaat acaatcatag ccaccgtaga gactgcatga   2220 

ccatccaagg cgaagtgtat ggtactaatc tggaagccac agaataggac acttggtttc   2280 

agctccaggg ttttcagtgc tcactattct tgttctgtat cccagtattg gtgctgcaac   2340 

ttaatgtact tcacctgtgg attggctgca aataaactca cgtgtattgg aaaaaaggaa   2400 

ttcctgcagc ccgggggatc cactagttct agagcggccg ccaccggtgg agctccagct   2460 

tttgttccct ttagtgaggg ttaatttcga gcttggcgta atcatggtca tagctgtttc   2520 

ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataa      2577 

 
           
             136  
             728  
             PRT  
             Mus musculus  
           
            136 

Pro Ala Asn Ala Ile Lys Glu Met Ile Glu Asn Cys Leu Asp Ala Lys 
1               5                   10                  15 

Ser Thr Asn Ile Gln Val Val Val Lys Glu Gly Gly Leu Lys Leu Ile 
            20                  25                  30 

Gln Ile Gln Asp Asn Gly Thr Gly Ile Arg Lys Glu Asp Leu Asp Ile 
        35                  40                  45 

Val Cys Glu Arg Phe Thr Thr Ser Lys Leu Gln Thr Phe Glu Asp Leu 
    50                  55                  60 

Ala Ser Ile Ser Thr Tyr Gly Phe Arg Gly Glu His Leu Ala Ser Ile 
65                  70                  75                  80 

Ser His Val Ala His Val Thr Ile Thr Thr Lys Thr Ala Asp Gly Lys 
                85                  90                  95 

Cys Ala Tyr Arg Ala Ser Tyr Ser Asp Gly Lys Leu Gln Ala Pro Pro 
            100                 105                 110 

Lys Pro Cys Ala Gly Asn Gln Gly Thr Leu Ile Thr Val Glu Asp Leu 
        115                 120                 125 

Phe Tyr Asn Ile Ile Thr Arg Arg Lys Ala Leu Lys Asn Pro Ser Glu 
    130                 135                 140 

Glu Tyr Gly Lys Ile Leu Glu Val Val Gly Arg Tyr Ser Ile His Asn 
145                 150                 155                 160 

Ser Gly Ile Ser Ile Ser Val Lys Lys Gln Gly Glu Thr Val Ser Asp 
                165                 170                 175 

Val Arg Thr Leu Pro Asn Ala Thr Thr Val Asp Asn Ile Arg Ser Ile 
            180                 185                 190 

Phe Gly Asn Ala Val Ser Arg Glu Leu Ile Glu Val Gly Cys Glu Asp 
        195                 200                 205 

Lys Thr Leu Ala Phe Lys Met Asn Gly Tyr Ile Ser Asn Ala Lys Tyr 
    210                 215                 220 

Ser Val Lys Lys Cys Ile Phe Leu Leu Phe Ile Asn His Arg Leu Val 
225                 230                 235                 240 

Glu Ser Ala Ala Leu Arg Lys Ala Ile Glu Thr Val Tyr Ala Ala Tyr 
                245                 250                 255 

Leu Pro Lys Thr His Thr His Ser Cys Thr Ser Val Glx Asn Gln Pro 
            260                 265                 270 

Ser Glu Arg Asp Val Asn Val His Pro Thr Lys Thr Glu Val His Phe 
        275                 280                 285 

Leu His Glu Glu Ser Ile Leu Gln Arg Val Gln Gln His Ile Glu Ser 
    290                 295                 300 

Lys Leu Leu Gly Ser Asn Ser Ser Arg Met Val Phe His Pro Asp Leu 
305                 310                 315                 320 

Ala Ser Arg Thr Cys Trp Ala Ser Gly Glu Ala Ala Arg Pro Thr Thr 
                325                 330                 335 

Gly Val Ala Ser Ser Ser Thr Ser Gly Ser Gly Asp Lys Val Tyr Ala 
            340                 345                 350 

Tyr Gln Met Ser Arg Thr Asp Ser Arg Asp Gln Lys Leu Asp Ala Phe 
        355                 360                 365 

Leu Gln Pro Val Ser Ser Leu Val Pro Ser Gln Pro Gln Asp Pro Arg 
    370                 375                 380 

Pro Val Arg Gly Ala Arg Thr Glu Gly Ser Pro Glu Arg Ala Thr Arg 
385                 390                 395                 400 

Glu Asp Glu Glu Met Leu Ala Leu Pro Ala Pro Ala Glu Ala Ala Ala 
                405                 410                 415 

Glu Ser Glu Asn Leu Glu Arg Glu Ser Leu Met Glu Thr Ser Asp Ala 
            420                 425                 430 

Ala Gln Lys Ala Ala Pro Thr Ser Ser Pro Gly Ser Ser Arg Lys Ser 
        435                 440                 445 

His Arg Glu Asp Ser Asp Val Glu Met Val Glu Asn Ala Ser Gly Lys 
    450                 455                 460 

Glu Met Thr Ala Ala Cys Tyr Pro Arg Arg Arg Ile Ile Asn Leu Thr 
465                 470                 475                 480 

Ser Val Leu Ser Leu Gln Glu Glu Ile Ser Glu Arg Cys His Glu Thr 
                485                 490                 495 

Leu Arg Glu Ile Leu Arg Asn His Ser Phe Val Gly Cys Val Asn Pro 
            500                 505                 510 

Gln Trp Ala Leu Ala Gln His Gln Thr Lys Leu Tyr Leu Leu Asn Thr 
        515                 520                 525 

Thr Lys Leu Ser Glu Glu Leu Phe Tyr Gln Ile Leu Ile Tyr Asp Phe 
    530                 535                 540 

Ala Asn Phe Gly Val Leu Arg Leu Ser Glu Pro Ala Pro Leu Phe Asp 
545                 550                 555                 560 

Leu Ala Met Leu Ala Glx Thr Val Leu Lys Val Ala Gly Gln Arg Thr 
                565                 570                 575 

Thr Ala Arg Arg Arg Ala Cys Arg Val His Cys Arg Val Ser Glu Glu 
            580                 585                 590 

Lys Arg Asp Ala Cys Arg Leu Phe Ser Val Arg Ser Met Arg Arg Glu 
        595                 600                 605 

Pro Asp Glx Leu Leu Phe Glx Glx Gln Leu Cys Ala Thr Phe Gly Gly 
    610                 615                 620 

Thr Ala Tyr Leu His Ser Ser Thr Gly His Glx Gly Glu Leu Gly Glu 
625                 630                 635                 640 

Glu Lys Glu Cys Phe Glu Ser Leu Ser Lys Glu Cys Ala Met Phe Tyr 
                645                 650                 655 

Ser Ile Arg Lys Gln Tyr Ile Leu Glu Glu Ser Thr Leu Ser Gly Gln 
            660                 665                 670 

Gln Ser Asp Met Pro Gly Ser Thr Ser Lys Pro Trp Lys Trp Thr Val 
        675                 680                 685 

Glu His Ile Ile Tyr Lys Ala Phe Arg Ser His Leu Leu Pro Pro Lys 
    690                 695                 700 

His Phe Thr Glu Asp Gly Asn Val Leu Gln Leu Ala Asn Leu Pro Asp 
705                 710                 715                 720 

Leu Tyr Lys Val Phe Glu Arg Cys 
                725 

 
           
             137  
             3065  
             DNA  
             Mus musculus  
           
            137 

cggtgaaggt cctgaagaat ttccagattc ctgagtatca ttggaggaga cagataacct     60 

gtcgtcaggt aacgatggtg tatatgcaac agaaatgggt gttcctggag acgcgtcttt    120 

tcccgagagc ggcaccgcaa ctctcccgcg gtgactgtga ctggaggagt cctgcatcca    180 

tggagcaaac cgaaggcgtg agtacagaat gtgctaaggc catcaagcct attgatggga    240 

agtcagtcca tcaaatttgt tctgggcagg tgatactcag tttaagcacc gctgtgaagg    300 

agttgataga aaatagtgta gatgctggtg ctactactat tgatctaagg cttaaagact    360 

atggggtgga cctcattgaa gtttcagaca atggatgtgg ggtagaagaa gaaaactttg    420 

aaggtctagc tctgaaacat cacacatcta agattcaaga gtttgccgac ctcacgcagg    480 

ttgaaacttt cggctttcgg ggggaagctc tgagctctct gtgtgcacta agtgatgtca    540 

ctatatctac ctgccacggg tctgcaagcg ttgggactcg actggtgttt gaccataatg    600 

ggaaaatcac ccagaaaact ccctaccccc gacctaaagg aaccacagtc agtgtgcagc    660 

acttatttta tacactaccc gtgcgttaca aagagtttca gaggaacatt aaaaaggagt    720 

attccaaaat ggtgcaggtc ttacaggcgt actgtatcat ctcagcaggc gtccgtgtaa    780 

gctgcactaa tcagctcgga caggggaagc ggcacgctgt ggtgtgcaca agcggcacgt    840 

ctggcatgaa ggaaaatatc gggtctgtgt ttggccagaa gcagttgcaa agcctcattc    900 

cttttgttca gctgccccct agtgacgctg tgtgtgaaga gtacggcctg agcacttcag    960 

gacgccacaa aaccttttct acgttttcgg gcttcatttc acagtgcacg cacggcgccg   1020 

ggaggagtgc aacagacagg cagtttttct tcatcaatca gaggccctgt gacccagcaa   1080 

aggtctctaa gcttgtcaat gaggtttatc acatgtataa ccggcatcag tacccatttg   1140 

tcgtccttaa cgtttccgtt gactcagaat gtgtggatat taatgtaact ccagataaaa   1200 

ggcaaattct actacaagaa gagaagctat tgctggccgt tttaaagacc tccttgatag   1260 

gaatgtttga cagtgatgca aacaagctta atgtcaacca gcagccactg ctagatgttg   1320 

aaggtaactt agtaaagtcg catactgcag aactagaaaa gcctgtgcca ggaaagcaag   1380 

ataactctcc ttcactgaag agcacagcag acgagaaaag ggtagcatcc atctccaggc   1440 

tgagagaggc cttttctctt catcctacta aagagatcaa gtctaggggt ccagagactg   1500 

ctgaactgac acggagtttt ccaagtgaga aaaggggcgt gttatcctct tatccttcag   1560 

acgtcatctc ttacagaggc ctccgtggct cgcaggacaa attggtgagt cccacggaca   1620 

gccctggtga ctgtatggac agagagaaaa tagaaaaaga ctcagggctc agcagcacct   1680 

cagctggctc tgaggaagag ttcagcaccc cagaagtggc cagtagcttt agcagtgact   1740 

ataacgtgag ctccctagaa gacagacctt ctcaggaaac cataaactgt ggtgacctgc   1800 

tgccgtcctc caggtacagg acagtccttg aagccagaag accatggata tcaatgcaaa   1860 

gctctacctc tagctcgtct gtcacccaca aatgccaagc gcttcaagac agaggaagac   1920 

cctcaaatgt caacatatct caaagattgc ctggtcctca gagcacctca gcagctgagg   1980 

tcgatgtagc cataaaaatg aataagagat cgtgctcctc gagttctcta gctaagcgaa   2040 

tgaagcagtt acagcaccta aaggcgcaga acaaacatga actgagttac agaaaattta   2100 

gggccaagat ttgccctgga gaaaaccaag cagcagaaga tgaactcaga aaagagatta   2160 

gtaaatcgat gtttgcagag atggagatct tgggtcagtt taacctggga tttatagtaa   2220 

ccaaactgaa agaggacctc ttcctggtgg accagcatgc tgcggatgag aagtacaact   2280 

ttgagatgct gcagcagcac acggtgctcc aggcgcagag gctcatcacg tgggtgcaca   2340 

caggcttcag agttcccaga ccccagactc tgaacttaac tgctgtcaat gaagctgtac   2400 

tgatagaaaa tctggaaata ttcagaaaga atggctttga ctttgtcatt gatgaggatg   2460 

ctccagtcac tgaaagggct aaattgattt ccttaccaac tagtaaaaac tggacctttg   2520 

gaccccaaga tatagatgaa ctgatcttta tgttaagtga cagccctggg gtcatgtgcc   2580 

ggccctcacg agtcagacag atgtttgctt ccagagcctg tcggaagtca gtgatgattg   2640 

gaacggcgct caatgcgagc gagatgaaga agctcatcac ccacatgggt gagatggacc   2700 

acccctggaa ctgcccccac ggcaggccaa ccatgaggca cgttgccaat ctggatgtca   2760 

tctctcagaa ctgacacacc ccttgtagca tagagtttat tacagattgt tcggttcgca   2820 

aagagaaggt tttaagtaat ctgattatcg ttgtacaaaa attagcatgc tgctttaatg   2880 

tactggatcc atttaaaagc agtgttaagg caggcatgat ggagtgttcc tctagctcag   2940 

ctacttgggt gatccggtgg gagctcatgt gagcccagga ctttgagacc actccgagcc   3000 

acattcatga gactcaattc aaggacaaaa aaaaaaagat atttttgaag ccttttaaaa   3060 

aaaaa                                                               3065 

 
           
             138  
             864  
             PRT  
             Mus musculus  
           
            138 

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 Ser Gly Phe 
            260                 265                 270 

Ile Ser Gln Cys Thr His Gly Ala Gly Arg Ser Ala Thr Asp Arg Gln 
        275                 280                 285 

Phe Phe Phe Ile Asn Gln Arg Pro Cys Asp Pro Ala Lys Val Ser Lys 
    290                 295                 300 

Leu Val Asn Glu Val Tyr His Met Tyr Asn Arg His Gln Tyr Pro Phe 
305                 310                 315                 320 

Val Val Leu Asn 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 Ser 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 Leu Pro Ser Ser 
    530                 535                 540 

Arg Tyr Arg Thr Val Leu Glu Ala Arg Arg Pro Trp Ile Ser Met Gln 
545                 550                 555                 560 

Ser Ser Thr Ser Ser Ser Ser Val Thr His Lys Cys Gln Ala Leu Gln 
                565                 570                 575 

Asp Arg Gly Arg Pro Ser Asn Val Asn Ile Ser Gln Arg Leu Pro Gly 
            580                 585                 590 

Pro Gln Ser Thr Ser Ala Ala Glu Val Asp Val Ala Ile Lys Met Asn 
        595                 600                 605 

Lys Arg Ser Cys Ser Ser Ser Ser Leu Ala Lys Arg Met Lys Gln Leu 
    610                 615                 620 

Gln His Leu Lys Ala Gln Asn Lys His Glu Leu Ser Tyr Arg Lys Phe 
625                 630                 635                 640 

Arg Ala Lys Ile Cys Pro Gly Glu Asn Gln Ala Ala Glu Asp Glu Leu 
                645                 650                 655 

Arg Lys Glu Ile Ser Lys Ser Met Phe Ala Glu Met Glu Ile Leu Gly 
            660                 665                 670 

Gln Phe Asn Leu Gly Phe Ile Val Thr Lys Leu Lys Glu Asp Leu Phe 
        675                 680                 685 

Leu Val Asp Gln His Ala Ala Asp Glu Lys Tyr Asn Phe Glu Met Leu 
    690                 695                 700 

Gln Gln His Thr Val Leu Gln Ala Gln Arg Leu Ile Thr Trp Val His 
705                 710                 715                 720 

Thr Gly Phe Arg Val Pro Arg Pro Gln Thr Leu Asn Leu Thr Ala Val 
                725                 730                 735 

Asn Glu Ala Val Leu Ile Glu Asn Leu Glu Ile Phe Arg Lys Asn Gly 
            740                 745                 750 

Phe Asp Phe Val Ile Asp Glu Asp Ala Pro Val Thr Glu Arg Ala Lys 
        755                 760                 765 

Leu Ile Ser Leu Pro Thr Ser Lys Asn Trp Thr Phe Gly Pro Gln Asp 
    770                 775                 780 

Ile Asp Glu Leu Ile Phe Met Leu Ser Asp Ser Pro Gly Val Met Cys 
785                 790                 795                 800 

Arg Pro Ser Arg Val Arg Gln Met Phe Ala Ser Arg Ala Cys Arg Lys 
                805                 810                 815 

Ser Val Met Ile Gly Thr Ala Leu Asn Ala Ser Glu Met Lys Lys Leu 
            820                 825                 830 

Ile Thr His Met Gly Glu Met Asp His Pro Trp Asn Cys Pro His Gly 
        835                 840                 845 

Arg Pro Thr Met Arg His Val Ala Asn Leu Asp Val Ile Ser Gln Asn 
    850                 855                 860 

 
           
             139  
             29  
             DNA  
             Artificial  
             
               Artificial sequence is synthesized  
             
           
            139 

cttgattcta gagcytcncc nckraancc                                       29 

 
           
             140  
             29  
             DNA  
             Artificial  
             
               Artificial sequence is synthesized  
             
           
            140 

aggtcggagc tcaargaryt ngtnganaa                                       29 

 
           
             141  
             15  
             DNA  
             Homo sapiens  
           
            141 

acttgtggat tttgc                                                      15 

 
           
             142  
             15  
             DNA  
             Homo sapiens  
           
            142 

acttgtgaat tttgc                                                      15 

 
           
             143  
             22  
             DNA  
             Mus musculus  
           
            143 

ttcggtgaca gatttgtaaa tg                                              22 

 
           
             144  
             16  
             DNA  
             Mus musculus  
           
            144 

tttacggagc cctggc                                                     16 

 
           
             145  
             22  
             DNA  
             Mus musculus  
           
            145 

tcaccataaa aatagtttcc cg                                              22 

 
           
             146  
             22  
             DNA  
             Mus musculus  
           
            146 

tcctggatca tattttctga gc                                              22 

 
           
             147  
             22  
             DNA  
             Mus musculus  
           
            147 

tttcaggtat gtcctgttac cc                                              22 

 
           
             148  
             22  
             DNA  
             Mus musculus  
           
            148 

tgaggcagct tttaagaaac tc                                              22 

 
           
             149  
             2385  
             DNA  
             Homo sapiens  
           
            149 

gcacgagctc gtgccgttta gatgcaaaat ccacaagtat tcaagtgatt gttaaagagg     60 

gaggcctgaa gttgattcag atccaagaca atggcaccgg gatcaggaaa gaagatctgg    120 

atattgtatg tgaaaggttc actactagta aactgcagtc ctttgaggat ttagccagta    180 

tttctaccta tggctttcga ggtgaggctt tggccagcat aagccatgtg gctcatgtta    240 

ctattacaac gaaaacagct gatggaaagt gtgcatacag agcaagttac tcagatggaa    300 

aactgaaagc ccctcctaaa ccatgtgctg gcaatcaagg gacccagatc acggtggagg    360 

acctttttta caacatagcc acgaggagaa aagctttaaa aaatccaagt gaagaatatg    420 

ggaaaatttt ggaagttgtt ggcaggtatt cagtacacaa tgcaggcatt agtttctcag    480 

ttaaaaaaca aggagagaca gtagctgatg ttaggacact acccaatgcc tcaaccgtgg    540 

acaatattcg ctccatcttt ggaaatgctg ttagtcgaga actgatagaa attggatgtg    600 

aggataaaac cctagccttc aaaatgaatg gttacatatc caatgcaaac tactcagtga    660 

agaagtgcat cttcttactc ttcatcaacc atcgtctggt agaatcaact tccttgagaa    720 

aagccataga aacagtgtat gcagcctatt tgcccaaaaa cacacaccca ttcctgtacc    780 

tcagtttaga aatcagtccc cagaatgtgg atgttaatgt gcaccccaca aagcatgaag    840 

ttcacttcct gcacgaggag agcatcctgg agcgggtgca gcagcacatc gagagcaagc    900 

tcctgggctc caattcctcc aggatgtact tcacccagac tttgctacca ggacttgctg    960 

gcccctctgg ggagatggtt aaatccacaa caagtctgac ctcgtcttct acttctggaa   1020 

gtagtgataa ggtctatgcc caccagatgg ttcgtacaga ttcccgggaa cagaagcttg   1080 

atgcatttct gcagcctctg agcaaacccc tgtccagtca gccccaggcc attgtcacag   1140 

aggataagac agatatttct agtggcaggg ctaggcagca agatgaggag atgcttgaac   1200 

tcccagcccc tgctgaagtg gctgccaaaa atcagagctt ggcgggggat acaacaaagg   1260 

ggacttcaga aatgtcagag aagagaggac ctacttccag caaccccaga aagagacatc   1320 

gggaagattc ttgtggaaat ggtggaagat gatcccgaaa ggaaatgact gcagctgtcc   1380 

ccccggagaa ggatcattaa cctcactagt gttttgagtc tccaggaaga aattaatgag   1440 

cagggacatg aggttctccg ggagatgttg cataaccact ccttcgtggg ctgtgtgaat   1500 

cctcagtggg ccttggcaca gcatcaaacc aagttatacc ttctcaacac caccaagctt   1560 

agtgaagaac tgttctacca gatactcatt tatgattttg ccaattttgg tgttctcagg   1620 

ttatcggagc cagcaccgct ctttgacctt gccatgcttg ccttagatag tccagagagt   1680 

ggctggacag aggaagatgg tcccaaagaa ggacttgctg aatacattgt tgagtttctg   1740 

aagaagaagg ctgagatgct tgcagactat ttctctttgg aaattgatga ggaagggaac   1800 

ctgattggat taccccttct gattgacaac tatgtgcccc ctttggaggg actgcctatc   1860 

ttcattcttc gactagccac tgaggtgaat tgggacgaag aaaaggaatg ttttgaaagc   1920 

ctcagtaaag aatgcgctat gttctattcc atccggaagc agtacatatc tgaggagtcg   1980 

accctctcag gccagcagag tgaagtgcct ggctccattc caaactcctg gaagtggact   2040 

gtggaacaca ttgtctataa agccttgcgc tcacacattc tgcctcctaa catttcacag   2100 

aagatggaaa tatcctgcag cttgctaacc tgcctgatct atacaaagtc tttgagaggt   2160 

gtaaatatgg ttatttatgc actgtgggat gtgttcttct ttctctgtat tccgagacaa   2220 

agtgttgtat caaagtgtga tatacaaagt gtgccaacat aagtgttggt agcacttaag   2280 

acttatactt gccttctgat agtattcctt tatacacagt ggattgatta taaataaata   2340 

gatgtgtctt aacataaaaa aaaaaaaaaa agggggggcc cggta                   2385 

 
           
             150  
             821  
             DNA  
             Homo sapiens  
           
            150 

cccccctcga ggtcgacggt atcgataagc ttgatatcga attcctcgag gccacgaagg     60 

ccccatggag cgagctgaga gctcgagtac agaacctgct aaggccatca aacctattga    120 

tcggaagtca gtccatcaga tttgctctgg gccggtggta ccgagtctaa gcactgcggt    180 

gaaggagtta gtagaaaaca gtctggatgc tggtgccact aatattgatc taaagcttaa    240 

ggactatgga gtggatctca ttgaagtttc aggcaatgga tgtggggtag aagaagaaaa    300 

cttcgaaggc ttaactctga aacatcacac atctaagatt caagagtttg ccgacctacc    360 

tcaggttgaa acttttggct ttcgggggga agctctgagc tcactttgtg cactgagtga    420 

tgtcaccatt tctacctgcc atgtatcggc gaaggttggg actcgactgg tgtttgatca    480 

ctatgggaaa atcatccaga aaacccccta cccccacccc agagggatga cagtcagtgt    540 

gaagcagtta ttttctacgc tacctgtgca ccataaagaa tttcaaagga atattaagaa    600 

gaaacgtgcc tgcttccctt cgccttctgc cgtgattgtc agtttcctga ggcctcccca    660 

gccatgcttc ctgtacagcc tgcagaacta tggattcttg ctctgtcacc caggctggag    720 

tgcaatgcct cgatgtctgc tgactgcaac ctccgccctc ccaggtcaag tagttctcct    780 

gcctcggcct cctgagtagc tgggattaca ggcatgagca c                        821 

 
           
             151  
             651  
             DNA  
             Homo sapiens  
           
            151 

aaccgaggca aaaaaaaaat caattgaagg agttgccggg aaaaaaccca atgtgggaaa     60 

ttaaaaggcc aaattcccgg caaagaaaac ctcaatttta aacacccctc cccggggagg    120 

ggaaaaccct aaactcccaa gacctcttta ttgaacttgt cttgaaagaa gcctggaaaa    180 

ttaagaaaga tggccttgat ttgttatcgt gaaatgctca attcactgaa aggctaaact    240 

gattgccttg ccaactagtc aaaactgaac cttcgggacc ccaggacgtc gatgaactga    300 

tcttcatgct gagcgacagc cctggggtca tgtgccgacc ttcccgagtc aagcagatgt    360 

ttgcctccag agcctgccgg aagtcggtga tgattgggac tgctctcaac acaagcgaat    420 

gaagaaactg atcacccaca tgggggagat gggccacccc tggaactgtc cccatggaag    480 

gccaccatga gacacatcgc caacctgggt gtcatttctc agaactgacc gtagtcactg    540 

tatggaataa ttggttttat cgcagatttt tatgttttga aagacagagt cttcactaac    600 

cttttttgtt ttaaaatgaa acctgcggcc ctcgtggcct cgaggaattc c             651 

 
           
             152  
             177  
             DNA  
             Homo sapiens  
           
            152 

ctgtttagat gcaaaatcca caagtattca agtgattgtt aagagggagg cctgaagttg     60 

attcagatcc aagacaatgg caccgggatc aggaaagaag atctggatat tgtatgtgaa    120 

aggttcacta ctagtaaact gcagtccttt gaggatttag ccagtatttc tacctat       177 

 
           
             153  
             178  
             DNA  
             Homo sapiens  
           
            153 

cagtctggat gctggtgcca ctaatattga tctaaagctt aaggactatg gagtggatct     60 

tattgaagtt tcagacaatg gatgtggggt agaagaagaa aacttcgaag gcttaactct    120 

gaaacatcac acatctaaga ttcaagagtt tgccgaccta actcaggttg aaactttt      178