Human DNA mismatch repair proteins

This application is a continuation-in-part of application Ser. No. 08/294,312, filed on Aug. 23, 1994, U.S. Pat. No. 6,380,369, which is a continuation-in-part of application Ser. No. 08/210,143, filed on Mar. 16, 1994, which is a continuation-in-part of application Ser. No. 08/187,757, filed on Jan. 27, 1994.

Part of the work performed during development of this invention utilized United States Government funds. Accordingly, the Government of the United States may have certain rights in this invention.

This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptides of the present invention are human homologs of the prokaryotic mutL4 gene and are hereinafter referred to as hMLH1, hMLH2 and hMLH3.

In both prolaryotes and eukaryotes, the DNA mismatch repair gene plays a prominent role in the correction of errors made during DNA replication and genetic recombination. The E.coli methyl-directed DNA mismatch repair system is the best understood DNA mismatch repair system to date. In E.coli , this repair pathway involves the products of the mutator genes mutS, mutL, mutH, and uvrD. Mutants of any one of these genes will reveal a mutator phenotype. MutS is a DNA mismatch-binding protein which initiates this repair process, uvrD is a DNA helicase and MutH is a latent endonuclease that incises at the unmethylated strands of a hemi-methylated GATC sequence. MutL protein is believed to recognize and bind to the mismatch-DNA-MutS-MutH complex to enhance the endonuclease activity of MutH protein. After the unmethylated DNA strand is cut by the MutH, single-stranded DNA-binding protein, DNA polymerase III, exonuclease I and DNA ligase are required to complete this repair process (Modrich P., Annu. Rev. Genetics, 25:229-53 (1991)).

Elements of the E.coli MutLHS system appears to be conserved during evolution in prokaryotes and eukaryotes. Genetic study analysis suggests that Saccharomyces cerevisiae has a mismatch repair system similar to the bacterial MutLHS system. In S. cerevisiae , at least two MutL homologs, PMS1 and MLH1, have been reported. Mutation of either one of them leads to a mitotic mutator phenotype (Prolla et al, Mol. Cell. Biol. 14:407-415 (1994)). At least three MutS homologs have been found in S.cerevisiae , namely MSH1, MSH2, and MSH3. Disruption of the MSH2 gene affects nuclear mutation rates. Mutants in S. cerevisae , MSH2, PMS1, and MLH1 have been found to exhibit increased rates of expansion and contraction of dinucleotide repeat sequences (Strand et al., Nature, 365:274-276 (1993)).

It has been reported that a number of human tumors such as lung cancer, prostate cancer, ovarian cancer, breast cancer, colon cancer and stomach cancer show instability of repeated DNA sequences (Han et al., Cancer, 53:5087-5089 (1993); Thibodeau et al., Science 260:816-819 (1993); Risinger et al., Cancer 53:5100-5103 (1993)). This phenomenon suggests that lack of the DNA mismatch repair is probably the cause of these tumors.

Little was known about the DNA mismatch repair system in humans until recently, the human homolog of the MutS gene was cloned and found to be responsible for hereditary nonpolyposis colon cancer (HNPCC), (Fishel et al., Cell, 75:1027-1038 (1993) and Leach et al., Cell, 75:1215-1225 (1993)). HNPCC was first linked to a locus at chromosome 2p16 which causes dinucleotide instability. It was then demonstrated that a DNA mismatch repair protein (MutS) homolog was located at this locus, and that C→T transitional mutations at several conserved regions were specifically observed in HNPCC patients. Hereditary nonpolyposis colorectal cancer is one of the most common hereditable diseases of man, affecting as many as one in two hundred individuals in the western world.

It has been demonstrated that hereditary colon cancer can result from mutations in several loci. Familial adenomatosis polyposis coli (APC), linked to a gene on chromosome 5, is responsible for a small minority of hereditary colon cancer. Hereditary colon cancer is also associated with Gardner's syndrome, Turcot's syndrome, Peutz-Jaeghers syndrome and juvenile polyposis coli. In addition, hereditary nonpolyposis colon cancer may be involved in 5% of all human colon cancer. All of the different types of familial colon cancer have been shown to be transmitted by a dominant autosomal mode of inheritance.

In addition to localization of HNPCC, to the short arm of chromosome 2, a second locus has been linked to a pre-disposition to HNPCC (Lindholm, et al., Nature Genetics, 5:279-282 (1993)). A strong linkage was demonstrated between a polymorphic marker on the short arm of chromosome 3 and the disease locus.

This finding suggests that mutations on various DNA mismatch repair proteins probably play crucial roles in the development of human hereditary diseases and cancers.

HNPCC is characterized clinically by an apparent autosomal dominantly inherited predisposition to cancer of the colon, endometrium and other organs. (Lynch, H. T. et al., Gastroenterology, 104:1535-1549 (1993)). The identification of markers at 2p16 and 3p21-22 which were linked to disease in selected HNPCC kindred unequivocally established its mendelian nature (Peltomaki, P. et al., Science, 260:810-812 (1993)). Tumors from HNPCC patients are characterized by widespread alterations of simple repeated sequences (microsatellites) (Aaltonen, L. A., et al., Science, 260:812-816 (1993)). This type of genetic instability was originally observed in a subset (12 to 18% of sporadic colorectal cancers (Id.). Studies in bacteria and yeast indicated that a defect in DNA mismatch repair genes can result in a similar instability of microsatellites (Levinson, G. and Gutman, G. A., Nuc. Acids Res., 15:5325-5338 (1987)), and it was hypothesized that deficiency in mismatched repair was responsible for HNPCC (Strand, M. et al., Nature, 365:274-276 (1993)). Analysis of extracts from HNPCC tumor cell lines showed mismatch repair was indeed deficient, adding definitive support to this conjecture (Parsons, R. P., et al., Cell, 75:1227-1236 (1993)). As not all HNPCC kindred can be linked to the same loci, and as at least three genes can produce a similar phenotype in yeast, it seems likely that other mismatch repair genes could play a role in some cases of HNPCC.

hMLH1 is most homologous to the yeast mutL-homolog yMLH1 while hMLH2 and hMLH3 have greater homology to the yeast mutL-homolog yPMS1 (hMLH2 and hMLH3 due to their homology to yeast PMS1 gene are sometimes referred to in the literature as hPMS1 and hPMS2). In addition to hMLH1, both the hMLH2 gene on chromosome 2q32 and the hMLH3 gene, on chromosome 7p22, were found to be mutated in the germ line of HNPCC patients. This doubles the number of genes implicated in HNPCC and may help explain the relatively high incidence of this disease.

In accordance with one aspect of the present invention, there are provided novel putative mature polypeptides which are hMLH1, hMLH2 and hMLH3, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof. The polypeptides of the present invention are of human origin.

In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding such polypeptides, including mRNAs, DNAs, cDNAs, genomic DNA as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.

In accordance with still another aspect of the present invention there are provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to hMLH1, hMLH2 and hMLH3 sequences.

In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptides by recombinant techniques which comprises culturing recombinant prokaryotic and/or eukaryotic host cells, containing an hMLH1, hMLH2 or hMLH3 nucleic acid sequence, under conditions promoting expression of said protein and subsequent recovery of said proteins.

In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptide, or polynucleotide encoding such polypeptide, for therapeutic purposes, for example, for the treatment of cancers.

In accordance with another aspect of the present invention there is provided a method of diagnosing a disease or a susceptibility to a disease related to a mutation in the hMLH1, hMLH2 or hMLH3 nucleic acid sequences and the proteins encoded by such nucleic acid sequences.

In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides, for in vitro purposes related to scientific research, synthesis of DNA and manufacture of DNA vectors.

These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.

The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.

FIGS. 1A-1F illustrate the cDNA sequence (SEQ ID NO:1) and corresponding deduced amino acid sequence for the human DNA repair protein HMLH1 (SEQ ID NO:2). The amino acids are represented by their standard one-letter abbreviations. Sequencing was performed using a 373 Automated DNA sequencer (Applied Biosystems, Inc.). Sequencing accuracy is predicted to be greater than 97% accurate.

FIGS 2 A- 2 H illustrate the cDNA sequence (SEQ ID NO:3) and corresponding deduced amino acid sequence of hMLH2(SEQ ID NO:4). The amino acids are represented by their standard one-letter abbreviations.

FIGS. 3A-3G illustrate the cDNA sequence (SEQ ID NO:5) and corresponding deduced amino acid sequence of hMLH3 (SEQ ID NO:6). The amino acids are represented by their standard one-letter abbreviations.

FIGS. 4A-4C Alignment of the predicted amino acid sequences of S. cerevisiae PMS1 (yPMS1), with the hMLH2 and hMLH3 amino acid sequences using MACAW (version 1.0) program. Amino acid in conserved blocks are capitalized and shaded on the mean of their pair-wise scores.

FIGS. 5A and 5B Mutational analysis of hMLH2. ( 5 A) IVSP analysis and mapping of the transcriptional stop mutation in HNPCC patient CW. Translation of codons 1 to 369 (lane 1), codons 1 to 290 (lane 2), and codons 1 to 214 (lane 3). CW is translated from the cDNA of patient CW, while NOR was translated from the cDNA of a normal individual. The arrowheads indicate the truncated polypeptide due to the potential stop mutation. The arrows indicate molecular weight markers in kilodaltons. ( 5 B) Sequence analysis of CW indicates a C to T transition at codon 233 (indicated by the arrow). Lanes 1 and 3 are sequence derived from control patients; lane 2 is sequence derived from genomic DNA of CW. The ddA mixes from each sequencing mix were loaded in adjacent lanes to facilitate comparison as were those for ddC, ddD, and ddT mixes.

FIGS. 6A and 6B Mutational analysis of hMLH3. ( 6 A) IVSP analysis of hMLH3 from patient GC. Lane GC is from fibroblasts-of individual GC; lane GCx is from the tumor of patient GC; lanes NOR1 and 2 are from normal control individuals. FL indicates full-length protein, and the arrowheads indicate the germ line truncated polypeptide. The arrows indicate molecular weight markers in kilodaltons ( 6 B) PCR analysis of DNA from a patient GC shows that the lesion in present in both hMLH3 alleles in tumor cells. Amplification was done using primers that amplify 5′, 3′, or within (MID) the region deleted in the cDNA. Lane 1, DNA derived from fibroblasts of patient GC; lane 2, DNA derived from tumor of patient GC; lane 3, DNA derived from a normal control patient; lane 4, reactions without DNA template. Arrows indicate molecular weight in base pairs.

In accordance with an aspect of the present invention, there are provided isolated nucleic acids (polynucleotides) which encode for the mature polypeptides having the deduced amino acid sequence of FIGS. 1, 2 and 3 (SEQ ID No. 2, 4 and 6) or for the mature polypeptides encoded by the cDNA of the clone deposited as ATCC Deposit No. 75649, 75651, 75650, deposited on Jan. 25, 1994.

ATCC Deposit No. 75649 is a cDNA clone which contains the full length sequence encoding, the human DNA repair protein referred to herein as hMLH1; ATCC Deposit No. 75651 is a cDNA clone containing the full length cDNA sequence encoding the human DNA repair protein referred to herein as hMLH2; ATCC Deposit No. 75650 is a cDNA clone containing the full length DNA sequence referred to herein as hMLH3.

The ATCC numbers referred to above are directed to biological deposits with the ATCC, 10801 University Boulevard, Manassas, Va. 20110-2209. The strains are being maintained under the terms of the Budapest treaty and will be made available to a patent office signatory to the Budapest Treaty.

Polynucleotides encoding the polypeptides of the present invention may be obtained from one or more libraries prepared from heart, lung, prostate, spleen, liver, gallbladder, fetal brain and testes tissues. The polynucleotides of hMLH1 were discovered from a human gallbladder cDNA library. In addition, six cDNA clones which are identical to the hMLH1 at the N-terminal ends were obtained from human cerebellum, eight-week embryo, fetal heart, HSC172 cells and Jurket cell cDNA libraries. The hMLH1 gene contains an open reading frame of 756 amino acids encoding for an 85 kD protein which exhibits homology to the bacterial and yeast mutL proteins. However, the 5′ non-translated region was obtained from the cDNA clone obtained from the fetal heart for the purpose of extending the non-translated region to design the oligonucleotides.

The hMLH2 gene was derived from a human T-cell lymphoma cDNA library. The hMLH2 cDNA clone identified an open reading frame of 2,796 base pairs flanked on both sides by in-frame termination codons. It is structurally related to the yeast PMS1 family. It contains an open reading frame encoding a protein of 932 amino acid residues. The protein exhibits the highest degree of homology to yeast PMS1 with 27% identity and 82% similarity over the entire protein.

A second region of significant homology among the three PMS related proteins is in the carboxyl terminus, between codons 800 to 900. This region shares a 22% and 47% homology between yeast PMS1 protein and hMLH2 and hMLH3 proteins, respectively, while very little homology of this region was observed between these proteins, and the other yeast mutL homolog, yMLH1.

The hMLH3 gene was derived from a human endometrial tumor cDNA library. The hMLH3 clone identified a 2,586 base pair open reading frame. It is structurally related to the yPMS2 protein family. It contains an open reading frame encoding a protein of 862 amino acid residues. The protein exhibits the highest degree of homology to yPMS2 with 32% identity and 66% similarity over the entire amino acid sequence.

It is significant with respect to a putative identification of hMLH1, hMLH2 and hMLH3 that the GFRGEAL domain which is conserved in mutL homologs derived from E. coli is conserved in the amino acid sequences of, hMLH1, hMLH2 and hMLH3.

The polynucleotides of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in FIGS. 1, 2 and 3 (SEQ ID No. 1) or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mature polypeptides as the DNA of FIGS. 1, 2 and 3 (SEQ ID No. 2, 4 and 6) or the deposited cDNA(s).

The polynucleotides which encode for the mature polypeptides of FIGS. 1, 2 and 3 (SEQ ID No. 2, 4 and 6) or for the mature polypeptides encoded by the deposited cDNAs may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5′ and/or 3′ of the coding sequence for the mature polypeptide.

Thus, the term “polynucleotide encoding a polypeptide” encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.

The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptides having the deduced amino acid sequences of FIGS. 1, 2 and 3 (SEQ ID No. 2, 4 and 6) or the polypeptides encoded by the cDNA of the deposited clones. The variants of the polynucleotides may be a naturally occurring allelic variant of the polynucleotides or a non-naturally occurring variant of the polynucleotides.

Thus, the present invention includes polynucleotides encoding the same mature polypeptides as shown in FIGS. 1, 2 and 3 (SEQ ID No. 2, 4 and 6) or the same mature polypeptides encoded by the cDNA of the deposited clones as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptides of FIGS. 1, 2 and 3 (SEQ ID No. 2, 4 and 6) or the polypeptides encoded by the cDNA of the deposited clones. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.

As hereinabove indicated, the polynucleotides may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in FIGS. 1, 2 and 3 (SEQ ID No. 1, 3 and 5) or of the coding sequence of the deposited clones. As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.

The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptides of the present invention. The marker sequence may be, for example, a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptides fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The term “gene” means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons).

Fragments of the full length gene of the present invention may be used as a hybridization probe for a cDNA library to isolate the full length cDNA and to isolate other cDNAs which have a high sequence similarity to the gene or similar biological activity. Probes of this type preferably have at least 30 bases and may contain, for example, 50 or more bases. The probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene including regulatory and promotor regions, exons, and introns. An example of a screen comprises isolating the coding region of the gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.

The present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 70%, preferably at least 90%, and more preferably at least 95% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides. As herein used, the term “stringent conditions” means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which either retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNAs of FIGS. 1, 2 and 3 (SEQ ID NO:l, 3 and 5) or the deposited cDNA(s).

Alternatively, the polynucleotide may have at least 20 bases, preferably 30 bases, and more preferably at least 50 bases which hybridize to a polynucleotide of the present invention and which has an identity thereto, as hereinabove described, and which may or may not retain activity. For example, such polynucleotides may be employed as probes for the polynucleotide of SEQ ID NO:1, 3 and 5 for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer.

Thus, the present invention is directed to polynucleotides having at least a 70% identity, preferably at least 90% and more preferably at least a 95% identity to a polynucleotide which encodes the polypeptide of SEQ ID NO:2, 4 and 6 as well as fragments thereof, which fragments have at least 30 bases and preferably at least 50 bases and to polypeptides encoded by such polynucleotides.

The deposit(s) referred to herein will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for purposes of Patent Procedure. These deposits are provided merely as convenience to those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. §112. The sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with any description of sequences herein. A license may be required to make, use or sell the deposited materials, and no such license is hereby granted.

The present invention further relates to polypeptides which have the deduced amino acid sequence of FIGS. 1, 2 and 3 (SEQ ID No. 2, 4 and 6) or which have the amino acid sequence encoded by the deposited cDNA(s), as well as fragments, analogs and derivatives of such polypeptides.

The terms “fragment,” “derivative” and “analog” when referring to the polypeptides of FIGS. 1, 2 and 3 (SEQ ID No. 2, 4 and 6) or that encoded by the deposited cDNA(s), means polypeptides which retain essentially the same biological function or activity as such polypeptides. Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.

The polypeptides of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptides of FIGS. 1, 2 and 3 (SEQ ID No. 2, 4 and 6) or that encoded by the deposited cDNAs may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol). Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

The term “isolated” means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the co-existing materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.

The polypeptides of the present invention include the polypeptide of SEQ ID NO:2, 4 and 6 (in particular the mature polypeptide) as well as polypeptides which have at least 70% similarity (preferably at least 70% identity) to the polypeptide of SEQ ID NO:2, 4 and 6 and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of SEQ ID NO:2, 4 and 6 and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the polypeptide of SEQ ID NO:2, 4 and 6 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.

As known in the art “similarity” between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.

Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.

The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the hMLH1, hMLH2 and hMLH3 genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques. Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host.

The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.

The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda P L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the proteins.

As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli , Streptomyces, Salmonella typhimurium ; fungal cells, such as yeast; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma; adenoviruses; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen, Inc.), pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid or vector may be used as long as they are replicable and viable in the host.

Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK232-8 and pCM7. Particular named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P R , P L and TRP. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cells containing the above-described constructs. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).

The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), α-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.

Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E.coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.

As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis., USA). These pBR322 “backbone” sections are combined with an appropriate promoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art.

Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.

The polypeptides can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.

The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated.

In accordance with a further aspect of the invention, there is provided a process for determining susceptibility to cancer, in particular, a hereditary cancer. Thus, a mutation in a human repair protein, which is a human homolog of mutL, and in particular those described herein, indicates a susceptibility to cancer, and the nucleic acid sequences encoding such human homologs may be employed in an assay for ascertaining such susceptibility. Thus, for example, the assay may be employed to determine a mutation in a human DNA repair protein as herein described, such as a deletion, truncation, insertion, frame shift, etc., with such mutation being indicative of a susceptibility to cancer.

A mutation may be ascertained for example, by a DNA sequencing assay. Tissue samples, including but not limited to blood samples are obtained from a human patient. The samples are processed by methods known in the art to capture the RNA. First strand cDNA is synthesized from the RNA samples by adding an oligonucleotide primer consisting of polythymidine residues which hybridize to the polyadenosine stretch present on the mRNA's. Reverse transcriptase and deoxynucleotides are added to allow synthesis of the first strand cDNA. Primer sequences are synthesized based on the DNA sequence of the DNA repair protein of the invention. The primer sequence is generally comprised of 15 to 30 and preferably from 18 to 25 consecutive bases of the human DNA repair gene. Table 1 sets forth an illustrative example of oligonucleotide primer sequences based on hMLH1. The primers are used in pairs (one “sense” strand and one “anti-sense”) to amplify the cDNA from the patients by the PCR method (Saiki et al., Nature, 324:163-166 (1986)) such that three overlapping fragments of the patient's cDNA's for such protein are generated. Table 1 also shows a list of preferred primer sequence pairs. The overlapping fragments are then subjected to dideoxynucleotide sequencing using a set of primer sequences synthesized to correspond to the base pairs of the cDNA's at a point approximately every 200 base pairs throughout the gene.

Preferred primer sequences pairs:

758, 1313

1319, 1320

660, 1909

725, 1995

1680, 2536

1727, 2610

The nucleotide sequences shown in Table 1 represent SEQ ID No. 7 through 19, respectively.

Table 2 lists representative examples of oligonucleotide primer sequences (sense and anti-sense) which may be used, and preferably the entire set of primer sequences are used for sequencing to determine where a mutation in the patient DNA repair protein may be. The primer sequences may be from 15 to 30 bases in length and are preferably between 18 and 25 bases in length. The sequence information determined from the patient is then compared to non-mutated sequences to determine if any mutations are present.

The nucleotide sequences shown in Table 2 represent SEQ ID No. 20 through 33, respectively.

In another embodiment, the primer sequences from Table 2 could be used in the PCR method to amplify a mutated region. The region could be sequenced and used as a diagnostic to predict a predisposition to such mutated genes.

Alternatively, the assay to detect mutations in the genes of the present invention may be performed by genetic testing based on DNA sequence differences achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al., Science, 230:1242 (1985)).

Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and S1 protection or the chemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)). Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.

Thus, the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, Western Blot analysis, direct DNA sequencing or the use of restriction enzymes, (e.g., Restriction Fragment Length Polymorphisms (RFLP)) and Southern blotting of genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.

The polypeptides may also be employed to treat cancers or to prevent cancers, by expression of such polypeptides in vivo, which is often referred to as “gene therapy.”

Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide. Such methods are well-known in the art. For example, cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding a polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedures known in the art. As known in the art, a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo. These and other methods for administering a polypeptide of the present invention by such method should be apparent to those skilled in the art from the teachings of the present invention. For example, the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.

Retroviruses from which the retroviral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus. In one embodiment, the retroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

The vector includes one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and β-actin promoters). Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.

The nucleic acid sequence encoding the polypeptide of the present invention is under the control of a suitable promoter. Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter; or hetorologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoters, such as the MMT promoter, the metallothionein promoter; heat shock promoters; the albumin promoter; the ApoAI promoter; human globin promoters; viral thymidine kinase promoters, such as the Herpes Simplex thymidine kinase promoter; retroviral LTRs (including the modified retroviral LTRs hereinabove described); the β-actin promoter; and human growth hormone promoters. The promoter also may be the native promoter which controls the gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, ψ-2, ψ-AM, PA12, T19-14X, VT-19-17-H2, ψCRE, ψCRIP, GP+E-86, GP+envAm12, and DAN cell lines as described in Miller, Human Gene Therapy , Vol. 1, pgs. 5-14 (1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaPO 4 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding the polypeptides. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide. Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.

Each of the cDNA sequences identified herein or a portion thereof can be used in numerous ways as polynucleotide reagents. The sequences can be used as diagnostic probes for the presence of a specific mRNA in a particular cell type. In addition, these sequences can be used as diagnostic probes suitable for use in genetic linkage analysis (polymorphisms).

The sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there is a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location. The mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3′ untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner. Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome-specific cDNA libraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with cDNA as short as 50 or 60 bases. For a review of this technique, see Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).

Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.

With current resolution of physical mapping and genetic mapping techniques, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb). hMLH2 has been localized using a genomic P1 clone (1670) which contained the 5′ region of the hMLH2 gene. Detailed analysis of human metaphase chromosome spreads, counterstained to reveal banding, indicated that the hMLH2 gene was located within bands 2q32. Likewise, hMLH3 was localized using a genomic P1 clone (2053) which contained the 3′ region of the hMLH3 gene. Detailed analysis of human metaphase chromosome spreads, counterstained to reveal banding, indicated that the hMLH3 gene was located within band 7p22, the most distal band on chromosome 7. Analysis with a variety of genomic clones showed that hMLH3 was a member of a subfamily of related genes, all on chromosome 7.

The polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.

Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention. Also, transgenic mice may be used to express humanized antibodies to immunogenic polypeptide products of this invention.

The present invention will be further described with reference to the following examples; however, it is to be understood that the present invention is not limited to such examples. All parts or amounts, unless otherwise specified, are by weight.

In order to facilitate understanding of the following examples certain frequently occurring methods and/or terms will be described.

“Plasmids” are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 μg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 μl of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37° C. are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res., 8:4057 (1980).

“Oligonucleotides” refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5′ phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.

“Ligation” refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units to T4 DNA ligase (“ligase”) per 0.5 μg of approximately equimolar amounts of the DNA fragments to be ligated.

Unless otherwise stated, transformation was performed as described in the method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973).

homo sapiens

2

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 Val 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

3

ggcacgagtg gctgcttgcg gctagtggat ggtaattgcc tgcctcgcgc tagcagcaag 60

ctgctctgtt aaaagcgaaa atg aaa caa ttg cct gcg gca aca gtt cga ctc 113

Met Lys Gln Leu Pro Ala Ala Thr Val Arg Leu

1 5 10

ctt tca agt tct cag atc atc act tcg gtg gtc agt gtt gta aaa gag 161

Leu Ser Ser Ser Gln Ile Ile Thr Ser Val Val Ser Val Val Lys Glu

15 20 25

ctt att gaa aac tcc ttg gat gct ggt gcc aca agc gta gat gtt aaa 209

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

30 35 40

ctg gag aac tat gga ttt gat aaa att gag gtg cga gat aac ggg gag 257

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

45 50 55

ggt atc aag gct gtt gat gca cct gta atg gca atg aag tac tac acc 305

Gly Ile Lys Ala Val Asp Ala Pro Val Met Ala Met Lys Tyr Tyr Thr

60 65 70 75

tca aaa ata aat agt cat gaa gat ctt gaa aat ttg aca act tac ggt 353

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

80 85 90

ttt cgt gga gaa gcc ttg ggg tca att tgt tgt ata gct gag gtt tta 401

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

95 100 105

att aca aca aga acg gct gct gat aat ttt agc acc cag tat gtt tta 449

Ile Thr Thr Arg Thr Ala Ala Asp Asn Phe Ser Thr Gln Tyr Val Leu

110 115 120

gat ggc agt ggc cac ata ctt tct cag aaa cct tca cat ctt ggt caa 497

Asp Gly Ser Gly His Ile Leu Ser Gln Lys Pro Ser His Leu Gly Gln

125 130 135

ggt aca act gta act gct tta aga tta ttt aag aat cta cct gta aga 545

Gly Thr Thr Val Thr Ala Leu Arg Leu Phe Lys Asn Leu Pro Val Arg

140 145 150 155

aag cag ttt tac tca act gca aaa aaa tgt aaa gat gaa ata aaa aag 593

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

160 165 170

atc caa gat ctc ctc atg agc ttt ggt atc ctt aaa cct gac tta agg 641

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

175 180 185

att gtc ttt gta cat aac aag gca gtt att tgg cag aaa agc aga gta 689

Ile Val Phe Val His Asn Lys Ala Val Ile Trp Gln Lys Ser Arg Val

190 195 200

tca gat cac aag atg gct ctc atg tca gtt ctg ggg act gct gtt atg 737

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

205 210 215

aac aat atg gaa tcc ttt cag tac cac tct gaa gaa tct cag att tat 785

Asn Asn Met Glu Ser Phe Gln Tyr His Ser Glu Glu Ser Gln Ile Tyr

220 225 230 235

ctc agt gga ttt ctt cca aag tgt gat gca gac cac tct ttc act agt 833

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

240 245 250

ctt tca aca cca gaa aga agt ttc atc ttc ata aac agt cga cca gta 881

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

255 260 265

cat caa aaa gat atc tta aag tta atc cga cat cat tac aat ctg aaa 929

His Gln Lys Asp Ile Leu Lys Leu Ile Arg His His Tyr Asn Leu Lys

270 275 280

tgc cta aag gaa tct act cgt ttg tat cct gtt ttc ttt ctg aaa atc 977

Cys Leu Lys Glu Ser Thr Arg Leu Tyr Pro Val Phe Phe Leu Lys Ile

285 290 295

gat gtt cct aca gct gat gtt gat gta aat tta aca cca gat aaa agc 1025

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

300 305 310 315

caa gta tta tta caa aat aag gaa tct gtt tta att gct ctt gaa aat 1073

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

320 325 330

ctg atg acg act tgt tat gga cca tta cct agt aca aat tct tat gaa 1121

Leu Met Thr Thr Cys Tyr Gly Pro Leu Pro Ser Thr Asn Ser Tyr Glu

335 340 345

aat aat aaa aca gat gtt tcc gca gct gac atc gtt ctt agt aaa aca 1169

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

350 355 360

gca gaa aca gat gtg ctt ttt aat aaa gtg gaa tca tct gga aag aat 1217

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

365 370 375

tat tca aat gtt gat act tca gtc att cca ttc caa aat gat atg cat 1265

Tyr Ser Asn Val Asp Thr Ser Val Ile Pro Phe Gln Asn Asp Met His

380 385 390 395

aat gat gaa tct gga aaa aac act gat gat tgt tta aat cac cag ata 1313

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

400 405 410

agt att ggt gac ttt ggt tat ggt cat tgt agt agt gaa att tct aac 1361

Ser Ile Gly Asp Phe Gly Tyr Gly His Cys Ser Ser Glu Ile Ser Asn

415 420 425

att gat aaa aac act aag aat gca ttt cag gac att tca atg agt aat 1409

Ile Asp Lys Asn Thr Lys Asn Ala Phe Gln Asp Ile Ser Met Ser Asn

430 435 440

gta tca tgg gag aac tct cag acg gaa tat agt aaa act tgt ttt ata 1457

Val Ser Trp Glu Asn Ser Gln Thr Glu Tyr Ser Lys Thr Cys Phe Ile

445 450 455

agt tcc gtt aag cac acc cag tca gaa aat ggc aat aaa gac cat ata 1505

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

460 465 470 475

gat gag agt ggg gaa aat gag gaa gaa gca ggt ctt gaa aac tct tcg 1553

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

480 485 490

gaa att tct gca gat gag tgg agc agg gga aat ata ctt aaa aat tca 1601

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

495 500 505

gtg gga gag aat att gaa cct gtg aaa att tta gtg cct gaa aaa agt 1649

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

510 515 520

tta cca tgt aaa gta agt aat aat aat tat cca atc cct gaa caa atg 1697

Leu Pro Cys Lys Val Ser Asn Asn Asn Tyr Pro Ile Pro Glu Gln Met

525 530 535

aat ctt aat gaa gat tca tgt aac aaa aaa tca aat gta ata gat aat 1745

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

540 545 550 555

aaa tct gga aaa gtt aca gct tat gat tta ctt agc aat cga gta atc 1793

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

560 565 570

aag aaa ccc atg tca gca agt gct ctt ttt gtt caa gat cat cgt cct 1841

Lys Lys Pro Met Ser Ala Ser Ala Leu Phe Val Gln Asp His Arg Pro

575 580 585

cag ttt ctc ata gaa aat cct aag act agt tta gag gat gca aca cta 1889

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

590 595 600

caa att gaa gaa ctg tgg aag aca ttg agt gaa gag gaa aaa ctg aaa 1937

Gln Ile Glu Glu Leu Trp Lys Thr Leu Ser Glu Glu Glu Lys Leu Lys

605 610 615

tat gaa gag aag gct act aaa gac ttg gaa cga tac aat agt caa atg 1985

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

620 625 630 635

aag aga gcc att gaa cag gag tca caa atg tca cta aaa gat ggc aga 2033

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

640 645 650

aaa aag ata aaa ccc acc agc gca tgg aat ttg gcc cag aag cac aag 2081

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

655 660 665

tta aaa acc tca tta tct aat caa cca aaa ctt gat gaa ctc ctt cag 2129

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

670 675 680

tcc caa att gaa aaa aga agg agt caa aat att aaa atg gta cag atc 2177

Ser Gln Ile Glu Lys Arg Arg Ser Gln Asn Ile Lys Met Val Gln Ile

685 690 695

ccc ttt tct atg aaa aac tta aaa ata aat ttt aag aaa caa aac aaa 2225

Pro Phe Ser Met Lys Asn Leu Lys Ile Asn Phe Lys Lys Gln Asn Lys

700 705 710 715

gtt gac tta gaa gag aag gat gaa cct tgc ttg atc cac aat ctc agg 2273

Val Asp Leu Glu Glu Lys Asp Glu Pro Cys Leu Ile His Asn Leu Arg

720 725 730

ttt cct gat gca tgg cta atg aca tcc aaa aca gag gta atg tta tta 2321

Phe Pro Asp Ala Trp Leu Met Thr Ser Lys Thr Glu Val Met Leu Leu

735 740 745

aat cca tat aga gta gaa gaa gcc ctg cta ttt aaa aga ctt ctt gag 2369

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

750 755 760

aat cat aaa ctt cct gca gag cca ctg gaa aag cca att atg tta aca 2417

Asn His Lys Leu Pro Ala Glu Pro Leu Glu Lys Pro Ile Met Leu Thr

765 770 775

gag agt ctt ttt aat gga tct cat tat tta gac gtt tta tat aaa atg 2465

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

780 785 790 795

aca gca gat gac caa aga tac agt gga tca act tac ctg tct gat cct 2513

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

800 805 810

cgt ctt aca gcg aat ggt ttc aag ata aaa ttg ata cca gga gtt tca 2561

Arg Leu Thr Ala Asn Gly Phe Lys Ile Lys Leu Ile Pro Gly Val Ser

815 820 825

att act gaa aat tac ttg gaa ata gaa gga atg gct aat tgt ctc cca 2609

Ile Thr Glu Asn Tyr Leu Glu Ile Glu Gly Met Ala Asn Cys Leu Pro

830 835 840

ttc tat gga gta gca gat tta aaa gaa att ctt aat gct ata tta aac 2657

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

845 850 855

aga aat gca aag gaa gtt tat gaa tgt aga cct cgc aaa gtg ata agt 2705

Arg Asn Ala Lys Glu Val Tyr Glu Cys Arg Pro Arg Lys Val Ile Ser

860 865 870 875

tat tta gag gga gaa gca gtg cgt cta tcc aga caa tta ccc atg tac 2753

Tyr Leu Glu Gly Glu Ala Val Arg Leu Ser Arg Gln Leu Pro Met Tyr

880 885 890

tta tca aaa gag gac atc caa gac att atc tac aga atg aag cac cag 2801

Leu Ser Lys Glu Asp Ile Gln Asp Ile Ile Tyr Arg Met Lys His Gln

895 900 905

ttt gga aat gaa att aaa gag tgt gtt cat ggt cgc cca ttt ttt cat 2849

Phe Gly Asn Glu Ile Lys Glu Cys Val His Gly Arg Pro Phe Phe His

910 915 920

cat tta acc tat ctt cca gaa act aca tga ttaaatatgt ttaagaagat 2899

His Leu Thr Tyr Leu Pro Glu Thr Thr

925 930

tagttaccat tgaaattggt tctgtcataa aacagcatga gtctggtttt aaattatctt 2959

tgtattatgt gtcacatggt tattttttaa atgaggattc actgacttgt ttttatattg 3019

aaaaaagttc cacgtattgt agaaaacgta aataaactaa taac 3063

4

932

homo sapiens

4

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

515 520 525

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

530 535 540

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

545 550 555 560

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

565 570 575

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

580 585 590

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

595 600 605

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

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

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

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

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

865 870 875 880

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

885 890 895

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

900 905 910

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

915 920 925

Pro Glu Thr Thr

930

5

cgaggcggat cgggtgttgc atcc atg gag cga gct gag agc tcg agt aca 51

Met Glu Arg Ala Glu Ser Ser Ser Thr

1 5

gaa cct gct aag gcc atc aaa cct att gat cgg aag tca gtc cat cag 99

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

10 15 20 25

att tgc tct ggg cag gtg gta ctg agt cta agc act gcg gta aag gag 147

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

30 35 40

tta gta gaa aac agt ctg gat gct ggt gcc act aat att gat cta aag 195

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

45 50 55

ctt aag gac tat gga gtg gat ctt att gaa gtt tca gac aat gga tgt 243

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

60 65 70

ggg gta gaa gaa gaa aac ttc gaa ggc tta act ctg aaa cat cac aca 291

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

75 80 85

tct aag att caa gag ttt gcc gac cta act cag gtt gaa act ttt ggc 339

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

90 95 100 105

ttt cgg ggg gaa gct ctg agc tca ctt tgt gca ctg agc gat gtc acc 387

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

110 115 120

att tct acc tgc cac gca tcg gcg aag gtt gga act cga ctg atg ttt 435

Ile Ser Thr Cys His Ala Ser Ala Lys Val Gly Thr Arg Leu Met Phe

125 130 135

gat cac aat ggg aaa att atc cag aaa acc ccc tac ccc cgc ccc aga 483

Asp His Asn Gly Lys Ile Ile Gln Lys Thr Pro Tyr Pro Arg Pro Arg

140 145 150

ggg acc aca gtc agc gtg cag cag tta ttt tcc aca cta cct gtg cgc 531

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

155 160 165

cat aag gaa ttt caa agg aat att aag aag gag tat gcc aaa atg gtc 579

His Lys Glu Phe Gln Arg Asn Ile Lys Lys Glu Tyr Ala Lys Met Val

170 175 180 185

cag gtc tta cat gca tac tgt atc att tca gca ggc atc cgt gta agt 627

Gln Val Leu His Ala Tyr Cys Ile Ile Ser Ala Gly Ile Arg Val Ser

190 195 200

tgc acc aat cag ctt gga caa gga aaa cga cag cct gtg gta tgc aca 675

Cys Thr Asn Gln Leu Gly Gln Gly Lys Arg Gln Pro Val Val Cys Thr

205 210 215

ggt gga agc ccc agc ata aag gaa aat atc ggc tct gtg ttt ggg cag 723

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

220 225 230

aag cag ttg caa agc ctc att cct ttt gtt cag ctg ccc cct agt gac 771

Lys Gln Leu Gln Ser Leu Ile Pro Phe Val Gln Leu Pro Pro Ser Asp

235 240 245

tcc gtg tgt gaa gag tac ggt ttg agc tgt tcg gat gct ctg cat aat 819

Ser Val Cys Glu Glu Tyr Gly Leu Ser Cys Ser Asp Ala Leu His Asn

250 255 260 265

ctt ttt tac atc tca ggt ttc att tca caa tgc acg cat gga gtt gga 867

Leu Phe Tyr Ile Ser Gly Phe Ile Ser Gln Cys Thr His Gly Val Gly

270 275 280

agg agt tca aca gac aga cag ttt ttc ttt atc aac cgg cgg cct tgt 915

Arg Ser Ser Thr Asp Arg Gln Phe Phe Phe Ile Asn Arg Arg Pro Cys

285 290 295

gac cca gca aag gtc tgc aga ctc gtg aat gag gtc tac cac atg tat 963

Asp Pro Ala Lys Val Cys Arg Leu Val Asn Glu Val Tyr His Met Tyr

300 305 310

aat cga cac cag tat cca ttt gtt gtt ctt aac att tct gtt gat tca 1011

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

315 320 325

gaa tgc gtt gat atc aat gtt act cca gat aaa agg caa att ttg cta 1059

Glu Cys Val Asp Ile Asn Val Thr Pro Asp Lys Arg Gln Ile Leu Leu

330 335 340 345

caa gag gaa aag ctt ttg ttg gca gtt tta aag acc tct ttg ata gga 1107

Gln Glu Glu Lys Leu Leu Leu Ala Val Leu Lys Thr Ser Leu Ile Gly

350 355 360

atg ttt gat agt gat gtc aac aag cta aat gtc agt cag cag cca ctg 1155

Met Phe Asp Ser Asp Val Asn Lys Leu Asn Val Ser Gln Gln Pro Leu

365 370 375

ctg gat gtt gaa ggt aac tta ata aaa atg cat gca gcg gat ttg gaa 1203

Leu Asp Val Glu Gly Asn Leu Ile Lys Met His Ala Ala Asp Leu Glu

380 385 390

aag ccc atg gta gaa aag cag gat caa tcc cct tca tta agg act gga 1251

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

395 400 405

gaa gaa aaa aaa gac gtg tcc att tcc aga ctg cga gag gcc ttt tct 1299

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

410 415 420 425

ctt cgt cac aca aca gag aac aag cct cac agc cca aag act cca gaa 1347

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

430 435 440

cca aga agg agc cct cta gga cag aaa agg ggt atg ctg tct tct agc 1395

Pro Arg Arg Ser Pro Leu Gly Gln Lys Arg Gly Met Leu Ser Ser Ser

445 450 455

act tca ggt gcc atc tct gac aaa ggc gtc ctg aga cct cag aaa gag 1443

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

460 465 470

gca gtg agt tcc agt cac gga ccc agt gac cct acg gac aga gcg gag 1491

Ala Val Ser Ser Ser His Gly Pro Ser Asp Pro Thr Asp Arg Ala Glu

475 480 485

gtg gag aag gac tcg ggg cac ggc agc act tcc gtg gat tct gag ggg 1539

Val Glu Lys Asp Ser Gly His Gly Ser Thr Ser Val Asp Ser Glu Gly

490 495 500 505

ttc agc atc cca gac acg ggc agt cac tgc agc agc gag tat gcg gcc 1587

Phe Ser Ile Pro Asp Thr Gly Ser His Cys Ser Ser Glu Tyr Ala Ala

510 515 520

agc tcc cca ggg gac agg ggc tcg cag gaa cat gtg gac tct cag gag 1635

Ser Ser Pro Gly Asp Arg Gly Ser Gln Glu His Val Asp Ser Gln Glu

525 530 535

aaa gcg cct gaa act gac gac tct ttt tca gat gtg gac tgc cat tca 1683

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

540 545 550

aac cag gaa gat acc gga tgt aaa ttt cga gtt ttg cct cag cca act 1731

Asn Gln Glu Asp Thr Gly Cys Lys Phe Arg Val Leu Pro Gln Pro Thr

555 560 565

aat ctc gca acc cca aac aca aag cgt ttt aaa aaa gaa gaa att ctt 1779

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

570 575 580 585

tcc agt tct gac att tgt caa aag tta gta aat act cag gac atg tca 1827

Ser Ser Ser Asp Ile Cys Gln Lys Leu Val Asn Thr Gln Asp Met Ser

590 595 600

gcc tct cag gtt gat gta gct gtg aaa att aat aag aaa gtt gtg ccc 1875

Ala Ser Gln Val Asp Val Ala Val Lys Ile Asn Lys Lys Val Val Pro

605 610 615

ctg gac ttt tct atg agt tct tta gct aaa cga ata aag cag tta cat 1923

Leu Asp Phe Ser Met Ser Ser Leu Ala Lys Arg Ile Lys Gln Leu His

620 625 630

cat gaa gca cag caa agt gaa ggg gaa cag aat tac agg aag ttt agg 1971

His Glu Ala Gln Gln Ser Glu Gly Glu Gln Asn Tyr Arg Lys Phe Arg

635 640 645

gca aag att tgt cct gga gaa aat caa gca gcc gaa gat gaa cta aga 2019

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

650 655 660 665

aaa gag ata agt aaa acg atg ttt gca gaa atg gaa atc att ggt cag 2067

Lys Glu Ile Ser Lys Thr Met Phe Ala Glu Met Glu Ile Ile Gly Gln

670 675 680

ttt aac ctg gga ttt ata ata acc aaa ctg aat gag gat atc ttc ata 2115

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

685 690 695

gtg gac cag cat gcc acg gac gag aag tat aac ttc gag atg ctg cag 2163

Val Asp Gln His Ala Thr Asp Glu Lys Tyr Asn Phe Glu Met Leu Gln

700 705 710

cag cac acc gtg ctc cag ggg cag agg ctc ata gca cct cag act ctc 2211

Gln His Thr Val Leu Gln Gly Gln Arg Leu Ile Ala Pro Gln Thr Leu

715 720 725

aac tta act gct gtt aat gaa gct gtt ctg ata gaa aat ctg gaa ata 2259

Asn Leu Thr Ala Val Asn Glu Ala Val Leu Ile Glu Asn Leu Glu Ile

730 735 740 745

ttt aga aag aat ggc ttt gat ttt gtt atc gat gaa aat gct cca gtc 2307

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

750 755 760

act gaa agg gct aaa ctg att tcc ttg cca act agt aaa aac tgg acc 2355

Thr Glu Arg Ala Lys Leu Ile Ser Leu Pro Thr Ser Lys Asn Trp Thr

765 770 775

ttc gga ccc cag gac gtc gat gaa ctg atc ttc atg ctg agc gac agc 2403

Phe Gly Pro Gln Asp Val Asp Glu Leu Ile Phe Met Leu Ser Asp Ser

780 785 790

cct ggg gtc atg tgc cgg cct tcc cga gtc aag cag atg ttt gcc tcc 2451

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

795 800 805

aga gcc tgc cgg aag tcg gtg atg att ggg act gct ctt aac aca agc 2499

Arg Ala Cys Arg Lys Ser Val Met Ile Gly Thr Ala Leu Asn Thr Ser

810 815 820 825

gag atg aag aaa ctg atc acc cac atg ggg gag atg gac cac ccc tgg 2547

Glu Met Lys Lys Leu Ile Thr His Met Gly Glu Met Asp His Pro Trp

830 835 840

aac tgt ccc cat gga agg cca acc atg aga cac atc gcc aac ctg ggt 2595

Asn Cys Pro His Gly Arg Pro Thr Met Arg His Ile Ala Asn Leu Gly

845 850 855

gtc att tct cag aac tga ccgtagtcac tgtatggaat aattggtttt 2643

Val Ile Ser Gln Asn

860

atcgcagatt tttatgtttt gaaagacaga gtcttcacta accttttttg ttttaaaatg 2703

aaacctgcta cttaaaaaaa atacacatca cacccattta aaagtgatct tgagaacctt 2763

ttcaaacc 2771

6

862

homo sapiens

6

Met Glu Arg Ala Glu Ser Ser Ser Thr Glu Pro Ala Lys Ala Ile Lys

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Leu Ser Cys Ser Asp Ala Leu His Asn Leu Phe Tyr Ile Ser Gly Phe

260 265 270

Ile Ser Gln Cys Thr His Gly Val Gly Arg Ser Ser Thr Asp Arg Gln

275 280 285

Phe Phe Phe Ile Asn Arg Arg Pro Cys Asp Pro Ala Lys Val Cys Arg

290 295 300

Leu Val Asn Glu Val Tyr His Met Tyr Asn Arg His Gln Tyr Pro Phe

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Ile Lys Met His Ala Ala Asp Leu Glu Lys Pro Met Val Glu Lys Gln

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Lys Gly Val Leu Arg Pro Gln Lys Glu Ala Val Ser Ser Ser His Gly

465 470 475 480

Pro Ser Asp Pro Thr Asp Arg Ala Glu Val Glu Lys Asp Ser Gly His

485 490 495

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

500 505 510

Ser His Cys Ser Ser Glu Tyr Ala Ala Ser Ser Pro Gly Asp Arg Gly

515 520 525

Ser Gln Glu His Val Asp Ser Gln Glu Lys Ala Pro Glu Thr Asp Asp

530 535 540

Ser Phe Ser Asp Val Asp Cys His Ser Asn Gln Glu Asp Thr Gly Cys

545 550 555 560

Lys Phe Arg Val Leu Pro Gln Pro Thr Asn Leu Ala Thr Pro Asn Thr

565 570 575

Lys Arg Phe Lys Lys Glu Glu Ile Leu Ser Ser Ser Asp Ile Cys Gln

580 585 590

Lys Leu Val Asn Thr Gln Asp Met Ser Ala Ser Gln Val Asp Val Ala

595 600 605

Val Lys Ile Asn Lys Lys Val Val Pro Leu Asp Phe Ser Met Ser Ser

610 615 620

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

625 630 635 640

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

645 650 655

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

660 665 670

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

675 680 685

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

690 695 700

Glu Lys Tyr Asn Phe Glu Met Leu Gln Gln His Thr Val Leu Gln Gly

705 710 715 720

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

725 730 735

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

740 745 750

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

755 760 765

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

770 775 780

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

785 790 795 800

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

805 810 815

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

820 825 830

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

835 840 845

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

850 855 860

7

20

Artificial Sequence

hMLH1 sense primer

7

gttgaacatc tagacgtctc 20

8

19

Artificial Sequence

hMLH1 sense primer

8

tcgtggcagg ggttattcg 19

9

19

Artificial Sequence

hMLH1 sense primer

9

ctacccaatg cctcaaccg 19

10

22

Artificial Sequence

hMLH1 sense primer

10

gagaactgat agaaattgga tg 22

11

18

Artificial Sequence

hMLH1 sense primer

11

gggacatgag gttctccg 18

12

19

Artificial Sequence

hMLH1 sense primer

12

gggctgtgtg aatcctcag 19

13

20

Artificial Sequence

hMLH1 antisense primer

13

cggttcacca ctgtctcgtc 20

14

18

Artificial Sequence

hMLH1 antisense primer

14

tccaggatgc tctcctcg 18

15

20

Artificial Sequence

hMLH1 antisense primer

15

caagtcctgg tagcaaagtc 20

16

19

Artificial Sequence

hMLH1 antisense primer

16

atggcaaggt caaagagcg 19

17

22

Artificial Sequence

hMLH1 antisense primer

17

caacaatgta ttcagnaagt cc 22

18

21

Artificial Sequence

hMLH1 antisense primer

18

ttgatacaac actttgtatc g 21

19

21

Artificial Sequence

hMLH1 antisense primer

19

ggaatactat cagaaggcaa g 21

20

21

Artificial Sequence

hMLH1 sense primer

20

acagagcaag ttactcagat g 21

21

20

Artificial Sequence

hMLH1 sense primer

21

gtacacaatg caggcattag 20

22

21

Artificial Sequence

hMLH1 sense primer

22

aatgtggatg ttaatgtgca c 21

23

18

Artificial Sequence

hMLH1 sense primer

23

ctgacctcgt cttcctac 18

24

19

Artificial Sequence

hMLH1 sense primer

24

cagcaagatg aggagatgc 19

25

21

Artificial Sequence

hMLH1 sense primer

25

ggaaatggtg gaagatgatt c 21

26

16

Artificial Sequence

hMLH1 sense primer

26

cttctcaaca ccaagc 16

27

21

Artificial Sequence

hMLH1 sense primer

27

gaaattgatg aggaagggaa c 21

28

22

Artificial Sequence

hMLH1 sense primer

28

cttctgattg acaactatgt gc 22

29

22

Artificial Sequence

hMLH1 sense primer

29

cacagaagat ggaaatatcc tg 22

30

20

Artificial Sequence

hMLH1 sense primer

30

gtgttggtag cacttaagac 20

31

20

Artificial Sequence

hMLH1 antisense primer

31

tttcccatat tcttcacttg 20

32

19

Artificial Sequence

hMLH1 antisense primer

32

gtaacatgag ccacatggc 19

33

19

Artificial Sequence

hMLH1 antisense primer

33

ccactgtctc gtccagccg 19

34

26

Artificial Sequence

hMLH1 5′ primer with BamHI restriction site

34

cgggatccat gtcgttcgtg gcaggg 26

35

26

Artificial Sequence

hMLH1 3′ primer with XbaI restriction site

35

gctctagatt aacacctctc aaagac 26

36

21

Artificial Sequence

hMLH1 primer useful for amplifying codons 1 to

394

36

gcatctagac gtttccttgg c 21

37

20

Artificial Sequence

primer useful for amplifying codons 1 to 394

of hMLH1

37

catccaagct tctgttcccg 20

38

19

Artificial Sequence

primer useful for amplifying codons 326 to 729

of hMLH1

38

ggggtgcagc agcacatcg 19

39

20

Artificial Sequence

primer useful for amplifying codons 326 to 729

of hMLH1

39

ggaggcagaa tgtgtgagcg 20

40

19

Artificial Sequence

primer useful for amplifying codons 602 to 756

plus 128 nucleotides of 3′ untranslated sequence of hMLH1

40

tcccaaagaa ggacttgct 19

41

22

Artificial Sequence

primer useful for amplifying codons 602 to 756

plus 128 nucleotides of 3′ untranslated sequence of hMLH1

41

agtataagtc ttaagtgcta cc 22

42

20

Artificial Sequence

primer useful for amplifying codons 578 to 632

of hMLH1

42

tttatggttt ctcacctgcc 20

43

19

Artificial Sequence

primer useful for amplifying codons 578 to 632

of hMLH1

43

gttatctgcc cacctcagc 19

44

59

Artificial Sequence

primer useful for amplifying codons 1 to 394

of hMLH1 wherein PCR product may be used for coupled

transcription-translation

44

ggatcctaat acgactcact atagggagac caccatggca tctagacgtt tcccttggc 59

45

20

Artificial Sequence

primer useful for amplifying codons 1 to 394

of hMLH1 wherein PCR product may be used for coupled

transcription-translation

45

catccaagct tctgttcccg 20

46

56

Artificial Sequence

primer useful for amplifying codons 326 to

729 of hMLH1 wherein PCR product may be used for coupled

transcription-translation

46

ggatcctaat acgactcact atagggagac caccatgggg gtgcagcagc acatcg 56

47

20

Artificial Sequence

primer useful for amplifying codons 326 to

729 of hMLH1 wherein PCR product may be used for coupled

transcription-translation

47

ggaggcagaa tgtgtgagcg 20

48

28

Artificial Sequence

hMLH2 5′ primer with a BamHI restriction site

48

cgggatccat gaaacaattg cctgcggc 28

49

26

Artificial Sequence

hMLH2 3′ primer with XbaI restriction site

49

gctctagacc agactcatgc tgtttt 26

50

26

Artificial Sequence

hMLH3 5′ primer with a BamHI restriction site

50

cgggatccat ggagcgagct gagagc 26

51

23

Artificial Sequence

hMLH3 3′ primer with XbaI restriction site

51

gctctagagt gaagactctg tct 23

52

20

Artificial Sequence

hMLH2 primer

52

aagctgctct gttaaaagcg 20

53

18

Artificial Sequence

hMLH2 primer

53

gcaccagcat ccaaggag 18

54

19

Artificial Sequence

hMLH3 primer

54

caaccatgag acacatcgc 19

55

20

Artificial Sequence

hMLH3 primer

55

aggttagtga agactctgtc 20

56

53

Artificial Sequence

primer useful for amplifying codons 1 to 500

of hMLH2

56

ggatcctaat acgactcact atagggagac caccatggaa caattgcctg cgg 53

57

18

Artificial Sequence

primer useful for amplifying codons 1 to 500

of hMLH2

57

cctgctccac tcatctgc 18

58

60

Artificial Sequence

primer useful for amplifying codons 270 to

755 of hMLH2

58

ggatcctaat acgactcact atagggagac caccatggaa gatatcttaa agttaatccg 60

59

21

Artificial Sequence

primer useful for amplifying codons 270 to

755 of hMLH2

59

ggcttcttct actctatatg g 21

60

58

Artificial Sequence

primer useful for amplifying from codon 485

to the translation termination site at codon 933 of hMLH2

60

ggatcctaat acgactcact atagggagac caccatggca ggtcttgaaa actcttcg 58

61

21

Artificial Sequence

primer useful for amplifying from codon

485 to the translation termination site at codon 933 of hMLH2

61

aaaacaagtc agtgaatcct c 21

62

20

Artificial Sequence

3′ primer useful for amplifying up to codon

369 of hMLH2

62

aagcacatct gtttctgctg 20

63

20

Artificial Sequence

3′ primer useful for amplifying up to codon

290 of hMLH2

63

acgagtagat tcctttaggc 20

64

19

Artificial Sequence

3′ primer useful for amplifying up to codon

214 of hMLH2

64

cagaactgac atgagagcc 19

65

52

Artificial Sequence

primer useful for amplifying codons 1 to

863 hMLH3

65

ggatcctaat acgactcact atagggagac caccatggag cgagctgaga gc 52

66

20

Artificial Sequence

primer useful for amplifying codons 1 to 863

hMLH3

66

aggttagtga agactctgtc 20

67

17

Artificial Sequence

primer useful for amplifying up to codon

472 of hMLH3

67

ctgaggtctc agcaggc 17

68

57

Artificial Sequence

primer useful for amplifying codons 415 to

863 of hMLH3

68

ggatcctaat acgactcact atagggagac caccatggtg tccatttcca gactgcg 57

69

20

Artificial Sequence

primer useful for amplifying codons 415 to

863 of hMLH3

69

aggttagtga agactctgtc 20

70

21

Artificial Sequence

primer useful for amplifying codons 195 to

233 of hMLH2

70

ttatttggca gaaaagcaga g 21

71

21

Artificial Sequence

primer useful for amplifying codons 195 to

233 of hMLH2

71

ttaaaagact aacctcttgc c 21

72

21

Artificial Sequence

sequencing primer useful for sequencing

codons 195 to 233 of hMLH2

72

ctgctgttat gaacaatatg g 21

73

19

Artificial Sequence

primer useful for amplifying codons 233 to

257 of hMLH3

73

cagaagcagt tgcaaagcc 19

74

20

Artificial Sequence

primer useful for amplifying codons 233 to

257 of hMLH3

74

aaaccgtact cttcacacac 20

75

20

Artificial Sequence

primer useful for amplifying codons 347 of

377 of hMLH3

75

gaggaaaagc ttttgttggc 20

76

18

Artificial Sequence

primer useful for amplifying codons 347 of

377 of hMLH3

76

cagtggctgc tgactgac 18

77

19

Artificial Sequence

primer useful for amplifying codons 439 to

472 of hMLH3

77

tccagaacca agaaggagc 19

78

16

Artificial Sequence

primer useful for amplifying codons 439 to

472 of hMLH3

78

tgaggtctca gcaggc 16