MXPA98005301A - Genes and products of genes related to the wer syndrome - Google Patents

Abstract

The present invention relates to nucleic acid molecules encoding products of WRN genes, expression vectors and suitable host cells to express said product

Description

GENES AND PRODUCTS OF GENES RELATED TO WERNER SYNDROME TECHNICAL FIELD The present invention relates generally to Werner Syndrome and more especially to methods and compositions suitable for use in the diagnosis and treatment of Werner Syndrome. BACKGROUND OF THE INVENTION Werner Syndrome (SW) is an autosomal recessive disorder with a complex phenotype. This disorder manifests itself in the premature presentation of diseases related to age and premature appearance of some of the physical aspects of normal aging. The presentation of symptoms usually occurs after adolescence. The disorder progresses during the course of life and normally patients have a shorter life expectancy with a modal age of death at 47 years. It is estimated that the prevalence of Werner Syndrome is for heterozygotes between 1-5 per 1,000 individuals and for homozygotes of 1-22 per 1,000,000 individuals. The clinical symptoms of Werner syndrome include both a prevalence of age-related diseases and the physical characteristics of aging. Such diseases include arteriosclerosis and heart disease, both benign and malignant neoplasms (usually sarcomas), diabetes mellitus, osteoporosis and ocular cataracts. The physical appearance of SW patients often manifests as short stature, premature callousness or hair loss, hypogonadism, altered skin pigmentation, hyperkeratosis, tight skin, bird-like facies, skin atrophy, skin ulcers on the leg and telangiectasia The majority of these diseases and characteristics occur in 40-90% of patients with SW. The diagnosis of SW is based mainly on the appearance of a certain number of these diseases and characteristics. A biochemical test, excessive excretion of hyaluronic acid in urine, can also be used to aid diagnosis. In addition to the observed signs and symptoms of aging, Werner's syndrome mimics normal aging as evidenced by the replicative potential of fibroblasts isolated from subjects with SW. The potential for fibroblast replication is reduced in these patients compared to fibroblasts isolated from controls of the same age and can be compared to the replicative potential of fibroblasts taken from larger subjects. In addition, an increased mutation regimen has been described in patients with SW. Such an abnormality manifests as chromosomal instability, such as inversions, reciprocal translocations, deletions and pseudodiploidy, and as a regime of increased mutation in the hypoxanthine phosphoribosyl transferase (HPRT) gene. Werner syndrome has been recognized as an autosomal recessive disorder. Goto et al. (Goto et al., Nature 355: 725-738, 1992) mapped the SW gene into the short arm of chromosome 8, using 21 affected Japanese families. The gene is located between the marker D8S87 and ankyrin (ANK1). More recently, the more refined mapping has signaled the SW gene to a region between marker D8S131 and D8S87, a range of 8.3 cM. The identification of the gene and the product of the gene should considerably increase the understanding of the basis of Werner Syndrome and allow biochemical and genetic approaches for diagnosis and treatment. The present invention provides a previously unidentified novel gene for Werner Syndrome and compositions for the diagnosis and treatment of SW and also provides other related advantages. COMPENDIUM OF THE INVENTION In summary, the present invention provides isolated nucleic acid molecules that encode the WRN gene, as well as portions thereof, representative of which is provided in the Figures. The protein that is encoded by the WRN gene is referred to hereinafter as the "WRN protein". Within other embodiments, nucleic acid molecules encoding a mutant WRN gene product are provided which increases the likelihood of Werner Syndrome (in a statistically significant way). Representative illustrations of said mutants are provided in Example 3. Within other aspects of the present invention, isolated nucleic acid molecules are provided, selected from the group consisting of (a) an isolated nucleic acid molecule as shown in the Figures, or complementary sequence thereof, (b) an isolated nucleic acid molecule that specifically hybridizes to the nucleic acid molecules of (a) under conditions of high restriction, and (c) an isolated nucleic acid encoding a product of the WRN gene (WRN protein). As used herein, it is to be understood that a nucleic acid molecule "specifically" hybridizes to a WRN gene (or related sequence) if it detectably hybridizes to said sequence, but does not significantly or detectably hybridize to the Bloom Syndrome gene (Ellis et al., Cell 83: 655-666, 1995). Within other aspects, expression vectors comprising a promoter operably linked to one of the nucleic acid molecules described above are provided. Representative examples of suitable promoters include tissue-specific promoters, as well as promoters such as the CMV I-E promoter, SV40 early promoter and MuLV LTR. Within the related aspects, viral vectors are provided which are capable of directing the expression to a nucleic acid molecule as described above. Representative examples of said viral vectors include herpes simplex vectors, adenovirus vectors, viral vectors associated with adenovirus and retroviral vectors. Host cells are also provided (eg, human cells, dogs, monkeys, rats or mice) having the vectors described above.

Within other aspects of the present invention, isolated proteins or polypeptides comprising a WRN gene product as well as peptides greater than 12, 13 or 200 amino acids are provided.

Within another modality, the protein is a mutant WRN gene product that increases the likelihood of Werner Syndrome. Within yet another aspect of the present invention, methods are provided for treating and avoiding Werner's Syndrome (as well as related diseases that are discussed in more detail below), comprising the step of administering to a patient a vector containing or expresses a nucleic acid molecule as described above, thereby reducing the likelihood or delay of the presentation of Werner Syndrome (or related disease) in the patient. Within a related aspect, methods are provided for treating or avoiding Werner Syndrome (and related diseases), comprising the step of administering a protein to a patient as described above, thereby reducing the likelihood or delay of the presentation of the Syndrome of Werner (or related disease) in the patient. Within certain embodiments, the above methods can be achieved by in vivo administration. Also provided by the present invention are pharmaceutical compositions comprising a nucleic acid molecule, vector, host cells, protein or antibody as described above, together with a pharmaceutically acceptable carrier or diluent.

Within other aspects of the present invention, antibodies are provided that specifically bind to a WRN protein or to unique peptides derived therefrom. As used herein, it should be understood that an antibody is specific for a WRN protein (or peptide) if it binds detectably and with a Kd of 10"7M or less (e.g., 10" 8M, 10). "9M, etc.), but does not bind detectably (or with an affinity greater than 10" 7M, (e.g., 10"SM, 10'5M, etc.) to an unrelated helicase (e.g., the Bloom Syndrome gene, supra) Hybridomas are also provided which are capable of producing said antibodies Within other aspects of the present invention, nucleic acid probes are provided which are capable of specifically hybridising (as defined below) to a WRN gene under conditions of high restriction Within a related aspect, said probes comprise at least a portion of the nucleotide sequence shown in the Figures or their complementary sequence, the probe being capable of specifically hybridizing to a gene. of mutant WRN under conditions of high restriction. of the present invention are generally at least 12 nucleotide bases in length, although they may be 14, 16, 18 bases or more. Initiator pairs capable of specifically amplifying a whole portion of any of the nucleic acid molecules described herein are also provided.

Within other aspects of the invention, methods are provided for diagnosing a patient who has an increased likelihood of contracting Werner Syndrome (or a related disease), comprising the steps of (a) obtaining from a patient a biological sample containing acid. nucleic acid, (b) incubating the nucleic acid with a probe that is capable of specifically hybridizing to a mutant WRN gene under conditions and for sufficient time to allow hybridization to occur, and (c) detecting the presence of the hybridized probe and thus determining that said patient has an increased likelihood of contracting Werner Syndrome (or related disease) Within another aspect, methods are provided comprising the steps of (a) obtaining from a patient a biological sample containing nucleic acid, (b) amplifying a selected nucleic acid sequence associated with a mutant WRN gene; and (c) detecting the presence of an amplified and thus determine that the patient has an increased likelihood of contracting Werner Syndrome (or a related disease). Suitable biological samples include nucleated cells obtained from peripheral blood smears or brain tissue., peptide vaccines are provided which comprise a portion of a mutant WRN gene product containing a mutation, in combination with a pharmaceutically acceptable carrier or diluent. Within yet another aspect, transgenic animals are provided whose germ cells and somatic cells contain a WRN gene (or lack thereof, ie, an "override") that is operably linked to an effective promoter for the expression of the gene, the gene being introduced into the animal, or an ancestor of the animal, into an embryonic stage Within one embodiment, the animal is a mouse, rat or dog. Within other embodiments, the WRN gene is expressed from a vector as described above. Within yet another embodiment, the WRN gene encodes a gene product of WRN These and other aspects of the present invention will be apparent by reference to the following detailed description and accompanying drawings. In addition, vain references are shown herein. ales describe in more detail certain procedures or compositions (v.gr, plasmids, etc.), and therefore are incorporated herein by reference in their entirety. BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTS Figure 1, is a genetic and physical map of the WRN region The genetic map (A) of the region is of the same sex with distances given in cM The polymorphic sites used (B) are PRAC sites of di-nucleotide and tri-nucleotide repeats The physical map presented (C) ) has determined approximate distances of overlapping non-chimeric CAL sizes and of genomic DNA sequence of overlapping P1 clones 2233, 2253, 3833, 2236 and 3101 The order of the markers was determined from the content of tagged sites of sequences (SES) of CAL, P1 clones and cosmid clones and of genomic DNA sequence of P1 clones. CALs presented (D) present the minimum slope and are the CALs used for the cDNA selection experiments. The P1 and cosmid clones needed for the minimum cloning path are shown in (E). The clones shown are clones P1 except for 8C11, which is a cosmid clone. The order of the cosmids was established by the SES content. Figures 2A and 2B are the DNA sequences (SEQ ID No. 70) and predicted amino acids (SEQ ID No. 71) of the transcription of the WRN gene. The amino acid code of a letter is used in Figure 2B. Figures 3A-3C are the DNA and predicted amino acid sequence of an alternate WRN gene transcript (SEQ ID Nos. 72 and 73). Figures 4A-4G, are the alignment of the product of the WRN gene (SEQ ID No. 74) with known helicases of S. pombe (SEQ ID No. 76), E. coli (SEQ ID No. 75), human (FIG. SEQ ID No. 77) and the Bloom Syndrome gene "BLM" (SEQ ID No.78). Figures 5A-5U are the genomic DNA sequence of the region containing the WRN gene (SEQ ID No. 79). Figure 6 presents a cDNA sequence of the mouse WRN gene (SEQ ID Nos. 205 and 206). Figure 7 is a genomic DNA sequence of the mouse WRN gene (SEQ ID Nos. 207-209).

Figure 8 is a diagram of the WRN gene product with location of mutations, A, WRN cDNA. Top-down numbering refers to the numbered cDNA sequence in GenBank L76937 B, WRN gene product. Helicase domain is designated as "DH", reasons are indicated from I to VI C, location of mutations The numbering through the bottom refers to mutations * Mutation meaningless? frame change mutation caused by the suppression of a single base Gray lines frame change mutations causing suppression of exons D, predicted proteins The lines represent the different predicted truncated proteins produced from the mutations in the WRN gene Figures 9A, 9B and 9C, are photomecographs showing the location of the WRN gene product by tinsing fluorescent antibodies (panel A), nuclei (panel B) and the cell field (panel C) expressing the WRN gene Figure 10, shows the alignment of the WRN gene products of mice and humans DETAILED DESCRIPTION OF THE INVENTION Definitions Before exhibiting in detail the invention, it may be helpful for the understanding of the same to display definitions of certain terms and to list and define the abbreviations that will be used further ahead "Genetic marker" is any segment of a chromosome that is distinctly unique in the genome, and polymorphic in the genome. to population in a way that provides information about the inheritance of linked DNA sequences, genes and / or other markers "Vector" refers to a set that is capable of directing the expression of a WRN gene, as well as any additional sequence or gene of interest The vector should include transcourse promoter elements that are operably linked to the genes of interest The vector can be composed of deoxyrpbonucleic acids ("DNA"), ribonucleic acids ("RNA") or a combination of the two (v gr , a chimeric DNA-RNA) Optionally, the vector may include a polyadenylation sequence, one or more restriction sites, as well as one or more selectable markers such as neomycin phosphotransferase or hygromycin phosphotransferase. Additionally, depending on the host cell chosen and the vector employee, other genetic elements can also be incorporated such as an origin of replication, nucleic acid restriction sites ad proteins, enhancers, sequences that confer transcription mducibility and selectable markers, in the vectors described in the present CAL abbreviations, artificial yeast chromosome; ESE, expressed sequence label, PCR, polymerase chain reaction, RCP-TI PCR process in which RNA is first transcribed to DNA in the first step using reverse transcpptase (TI); CDNA, any DNA made by copying an RNA sequence in the form of DNA. As noted above, the present invention provides methods and compositions for the detection and treatment of Werner Syndrome, as well as related diseases. These methods and compositions include a family of genes related to Werner's Syndrome and the proteins encoded by it, which have been implicated in the presentation of Werner's Syndrome. These genes and proteins, including genetic markers, nucleic acid sequences and clones, are also useful in the creation of in vitro and animal models and screening useful tests for the study of Werner Syndrome, including the possible identification of other genes involved in Werner Syndrome. The present invention also provides vector constructs, genetic markers, nucleic acid sequences, clones, diagnostic tests and compositions and methods for the identification of individuals who tend to suffer from Werner Syndrome. Genes and Products of Genes Related to Werner Syndrome The present invention provides isolated nucleic acid molecules comprising a portion of the gene that is involved in the presentation of SW. In summary, as can be seen in Figure 4, this gene encodes a protein that is similar in amino acid sequence to several DNA helicases that depend on known ATP (enzymes that unwind in double DNA). It is less similar to the known heiicases of RNA-DNA. Helicases are involved in DNA replication, often joining the origin of replication, and / or the replication complex. In addition, single-stranded DNA that is involved in recombination can be generated by DNA helicases. Although the Figures show various aspects of the WRN gene (or portions thereof), it should be understood that within the context of the present invention, references to one or more of these genes include derivatives of genes that are substantially similar to the genes ( and, where appropriate, the proteins including peptides and polypeptides that are encoded by the genes and their derivatives). As used herein, a nucleotide sequence is considered to be "substantially similar" if: (a) the nucleotide sequence is derived from the coding region of the described genes and includes, for example, portions of the sequence or Allelic variations of the sequences treated above, or alternatively, encode a helicase-like activity (Bjornson et al., Biochem 3307: 14306-14316, 1994); (b) the nucleotide sequence is capable of hybridizing to nucleotide sequences of the present invention under high or very high restriction (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, NY , 1989); or (c) the nucleic acid sequences degenerate as a result of the genetic code of the nucleic acid sequences defined in (a) or (b). In addition, the nucleic acid molecule described herein includes both complementary and non-complementary sequences, so long as the sequences in some manner meet the criteria set forth herein. Within the context of the present invention, high restriction means normal hybridization conditions. (v gr, 5x SSPE, 0 5% SDS at 65 ° C, or the equivalent) while very high restriction means hybridization conditions such that the nucleotide sequence can selectively hybridize to a single allele of the SW-related gene. WRN can be isolated from genomic DNA or cDNA Genomic DNA libraries constructed from chromosomal vectors, such as CAL (yeast artificial chromosomes), bacteriophage vectors, such as? EMBL3,? Gt10, cosmids or plasmids are suitable for use. CDNAs constructed in plasmid or other bacteriophage vectors are suitable for screening These banks can be build using methods and techniques known in the art (see Sambrook et al., Molecular Cloning Laboratory Manual, Cold Spring Harbor Press, 1989) or purchased from commercial sources (v. gr, Clontech, Palo Alto, CA) Within one modality, the gene of WRN is isolated by PCR performed on genomic DNA or cDNA or DNA from banks, or is isolated by probe hybridization of genomic DNA libraries or cDNAs. PCR primers and probes for hybridization screening can be designed based on the DNA sequence of the WRN presented herein The DNA sequence of a portion of the WRN gene and the entire coding sequence is presented in the Figures. The primers for PCR should be derived from the sequence in the 5 'and 3 untranslated region with the purpose of isolate a full-length cDNA. The primers must not have self-complementary sequences or have complementary sequences at their 3 'end and (to avoid mitordor-dimer formation) preferably, the initiators have a GC content of approximately 50% and contain restriction sites. The primers are attached to the cDNA and sufficient cycles of PCR are carried out to give a product that is easily visualized by gel electrophoresis and tinsion. The amplified fragment is purified and inserted into a vector, such as? Gt10 or pBS (M13 + ), and it spreads. An oligonucleotide hybridization probe suitable for screening genomic DNA or cDNA libraries can be designed based on the sequence provided herein. Preferably, the oligonucleotide is 20-30 base pairs long. Said oligonucleotide can be synthesized by automatic synthesis. The oligonucleotide can be conveniently labeled at the 5 'end and with a reporter molecule, such as a radionuclide, (e.g., 32 P) or biotin. The bank is laminated as colonies or phages, depending on the vector and the recombinant DNA is transferred to nylon or nitrocellulose membranes. After denaturation, neutralization and DNA fixation to the membrane, the membranes are hybridized with the labeled probe. The membrane is washed and the reporter molecule is detected. The hybridization or phage colonies are isolated and propagated. Candidate clones or amplified fragments of PCR can be verified by containing WRN DNA by any of several means. For example, candidate clones can be hybridized with a second non-overlapping probe or subjected to DNA sequence analysis. In this way the clones containing the WRN gene, which are suitable for use in the present invention are isolated. The structure of the proteins encoded for the nucleic acid molecules described herein can be predicted from the primary translation products using hydrophobicity graphics function, for example, P / C Gene, Lasergen System, DNA STAR, Madison, Wisconsin, or according to the methods described by Kyte and Doolittle (J. Mol. Biol. 157: 105-132, 1982). The WRN proteins of the present invention can be prepared in the form of acidic or basic salts or in neutral form. In addition, the individual amino acid residues can be modified by oxidation or reduction. In addition, various substitutions, deletions or additions to the amino acid or nucleic acid sequences can be made, the network effect of which is to retain or increase or decrease the biological activity of the wild-type mutant protein. In addition, due to the degeneracy in the genetic code, for example, there may be considerable variation in the nucleotide sequences encoding the same amino acid sequence.

Other derivatives of the WRN proteins described herein include conjugates of the proteins together with other proteins or polypeptides. This can be achieved, for example, by the synthesis of N-terminal or C-terminal fusion proteins that can be added to facilitate the purification or identification of WRN proteins (see for example, U.S. Patent No. 4,851,341; see also, Hopp et al., Bio / Technology 6: 1204, 1988). Alternatively, fusion proteins such as WRN-β-galactosidase protein or WRN-luciferase protein can be constructed in order to aid in the identification, expression and analysis of WRN proteins. The WRN proteins of the present invention can be constructed using a wide variety of techniques described herein. In addition, mutations can be introduced in particular by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites that allow binding to fragments of the native sequence. After binding, the resulting reconstructed sequence encodes a derivative having the insertion, substitution or deletion of desired amino acids. Alternatively, site-specific mutagenesis procedures for oligonucleotide (or segment-specific) sites can be employed to provide an altered gene having particular codons altered in accordance with the required substitution, deletion or insertion. Illustrative methods for forming the alterations discussed above are described by Walder et al. (Gene 42: 133,1986); Bauer and others. (Gene 37:73, 1985); Craik (Bio Techniques, January 1985, 12-19); Smith et al., (Genetic Engineering: Principies and Methods, Plenum Press, 1981); and Sambrook and others. (Supra). Suppression or truncation derivatives of the WRN proteins (e.g., a soluble extracellular portion) can also be constructed using convenient restriction endonuclease sites adjacent to the desired deletion. Subsequent to the restriction, the pendants can be filled and the DNA re-ligated. Illustrative methods for forming the alterations shown above are described by Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, 1989). The mutations of the present invention preferably preserve the reading frame of the coding sequences. In addition, the mutations will preferably not create complementary regions that could hybridize to produce secondary mRNA structures, such as loops or pins, that could adversely affect translation of the mRNA. Although a mutation site can be predetermined, it is not necessary that the nature of the mutation itself is predetermined. For example, in order to select the optimal characteristics of mutants at a given site, random mutagenesis in the target codon can be carried out and the expressed mutants can be screened for biological indicator activity. Alternatively, mutations can be introduced into particular sites by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites that allow binding to fragments of the native sequence. After binding, the resulting reconstructed sequence encodes a derivative having the desired insertion, substitution or deletion of amino acids. WRN proteins can also be constructed using PCR mutagenesis techniques, chemical mutagenesis (Drinkwater and Klinedinst, PNAS 83: 3402-3406, 1986), for lack of enforced nucleotide incorporation (eg, Liao and Wise Gene 88). : 107-111, 1990), or by the use of randomly mutagenized oligonucleotides (Horwitz et al., Genome 3: 112-117, 1989). The proteins can be isolated, among other methods by cultivating suitable host systems and vectors to produce the recombinant translation products of the present invention. Supernatants of said cell lines or inclusions of proteins or cells in which the protein is not excreted in the supernatant, they can be treated by a variety of purification procedures in order to isolate the desired proteins. For example, the supernatant can be concentrated first using commercially available protein concentration filters, such as an Amicon or Millipore Pellicon ultrafiltration unit. After concentration, the concentrate can be applied to a suitable purification matrix such as, for example, an anti-protein antibody bound to a suitable support. Alternatively, anion or cation exchange resins can be used in order to purify the protein. As a further alternative, one or more reverse phase high performance liquid chromatography steps (CLAR-Fl) may be employed. Other methods for isolating the proteins of the present invention are also well known in the experience of the art. A protein is considered "isolated" within the context of the present invention if no other (undesired) protein is detected according to the analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie blue tinsion. Within other embodiments, the desired protein can be isolated so that no other (undesired) protein is detected according to the SDS-PAGE analysis followed by silver staining. Expression of a WRN gene The present invention also provides for the manipulation and expression of the genes described above by culturing host cells containing a vector capable of expressing the genes described above. Such vectors or vector constructs include synthetic nucleic acid or cDNA-derived nucleic acid molecules encoding WRN proteins, which are operably linked to suitable translational or translational regulatory elements. Suitable regulatory elements can be derived from a variety of sources, including bacterial, fungal, viral, mammalian, insect or plant genes. The selection of appropriate regulatory elements depends on the chosen host cell and can be easily achieved by someone skilled in the art.

Examples of regulatory elements include: a transcriptional promoter and enhancer or RNA polymerase linker sequence, a transcriptional terminator and a ribosomal binding sequence, including a transduction initiation signal. The nucleic acid molecules encoding any of the WRN proteins described above can be readily expressed by a wide variety of prokaryotic and eukaryotic host cells, including bacterial, mammalian, yeast or other fungal, viral, insect or plant cells. Methods for transforming or transfecting said cells in order to express foreign DNA are well known in the art (see, for example, U.S. Patent Nos. 4,704,362; Hinnen et al., Proc. Nati. Acad. Sci USA 75: 1929- 1933, 1978, Murray et al, U.S. Patent No. 4,801,542, Upshalll et al, U.S. Patent No. 4,935,349, Hagen et al, U.S. Patent No. 4,784,950, Axel et al., U.S. Pat. No. 4,399,216; others, U.S. Patent No. 4,766,075, and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989; for plant cells see Czako and Marton, Plant Physiol., 104: 1067-1071. , 1994, and Paszkowski et al., Biotech, 24: 387-392, 1992). Bacterial host cells suitable for carrying out the present invention include E. coli, B. subtilis, Salmonella tiphymurium, and several species within the genus of Pseudomonas, Streptomyces and Staphylococcus, as well as many other bacterial species well known to someone with experience in The matter. Representative examples of bacterial host cells include DH5 (Stratagene, La Jolla, California). The bacterial expression vectors preferably comprise a promoter that functions in the host cell, one or more selectable phenotypic markers and a bacterial origin of replication. Representative promoters include β-lactamase (penicillinase) and lactose promoter system (see Chang et al, Nature 275: 615, 1978), the T7 RNA polymerase promoter (Studier et al., Meth. In Enzymol. 89, 1990), the lambda promoter (Elvin et al., Gene 87: 123-126, 1990), the trp promoter (Nichols and Yanofsky, Meth. In Enzymology 101: 155, 1983) and the tac promoter (Russell et al. Gene 20: 231, 1982). Representative selectable markers include various antibiotic resistance markers such as kanamycin or ampicillin resistance genes. Many plasmids suitable for transforming host cells are well known in the art, including among others, pBR322 (see Bolivar et al., Gene 2:95, 1977), the plasmids of pUC, pUC18, pUC19, pUC118, pUC119 (see Messing, Meth. In Enzymology 101: 20-77, 1983 and Vieira and Messing, Gene 19: 259-268, 1982) and pNH8A,? NH16a, pNH18a, and Bluescript M13 (Stratagene, La Jolla, Calif.). Yeast host cells and fungi suitable for carrying out the invention include, among others, Saccharomyces pombe, Saccharomyces cerevisiae, the genus Pichia or Kluyveromyces and various species of the genus Aspergillus (McKnight et al., U.S. Patent No. 4,935,349). Suitable expression vectors for yeast and fungi include, among others, YCp50 (ATCC No. 37419) for yeast, and the cloning vector of amdS pV3 (Turnbull, Biol / Technology 7: 169, 1989), YRp7 (Struhl et al. , Proc. Nati, Acad. Sci. USA 76: 1035-1039, 1978), YEp13 (Broach et al., Gene 8: 121-133, 1979), pJDB249 and pJDB219 (Beggs, Nature 275: 104-108, 1978) and derivatives thereof. Preferred promoters for use in yeast include promoters of yeast glycolytic genes (Hitzeman et al., J. Mol Appl. Genet, 1: 419-434, 1982) or alcohol dehydrogenase genes (Young et al., In Genetic Engineering of Microorganisms for Chemicals, Hollaender et al. (eds.) p.355, Plenum, New York, 1982; Ammerer, Meth. Enzymol. 101: 192-201, 1983). Examples of useful promoters for fungal vectors include those derived from glycolytic genes of Aspergillus nidulans, such as the adh3 promoter (McKnight et al., EMBO J. 4: 2093-2099, 1985). The expression units may also include a transcriptional terminator. An example of a suitable terminator is the adh3 terminator (McKnigh et al., Ibid., 1985). Like bacterial vectors, yeast vectors will generally include a selectable marker, which can be one of any number of genes that exhibits a dominant phenotype for which a phenotypic analysis exists in order to allow them to be selected from the transformants. Preferred selectable markers are those that complement the auxotrophy of host cells, provide antibiotic resistance or allow a cell to use specific carbon sources, and include Ieu2 (Broach et al., Ibid.), Ura3 (Botstein et al., Gene 8:17 , 1979), or his3 (Struhl et al., Ibid.). Another suitable selectable marker is the cat gene, which confers chloramphenicol resistance on yeast cells. Techniques for transforming fungi are well known in the literature and have been described, for example, by Beggs (ibid.), Hinnen et al. (Proc. Nati. Acad. Sci. USA 75: 1929-1933, 1978), Yelton and others (Proc. Nati, Acad. Sci. USA 81: 1740-1747, 1984), and Russell (Nature 301: 167-169, 1983). The genotype of the host cell may contain a genetic defect that is complemented by the selectable marker present in the expression vector. The choice of a particular guest and selectable marker is within the level of ordinary experience in the subject. The protocols for yeast transformation are also well known to those of ordinary skill in the art. For example, transformation can be easily accomplished by preparation of yeast spheroplasts with DNA (see Hinnen et al., PNAS USA 75: 1919, 1978) or by treatment with alkali salts such as LiCl (see Itoh et al., J. Bacteriology 153: 163, 1983). The transformation of fungi can also be carried out using polyethylene glycol as described by Cullen et al., (Bio / Technology 5: 369, 1987).

Viral vectors include those that comprise a promoter that directs the expression of an isolated nucleic acid molecule encoding a WRN protein as described above. A wide variety of promoters can be used within the context of the present invention, including for example, promoters such as MoMLV LTR, RSV LTR, Firend MuLV LTR, adenoviral promoter (Ohno et al., Science 265: 781-784, 1994), promoter / neomycin phosphotransferase enhancer, late parvovirus promoter (Koering et al., Hum The Gene Therap., 5: 457-463, 1994), Herpes TK promoter, SV40 promoter, metallothioninol gene enhancer / promoter, immediate early promoter of cytomegalovirus and the immediate late promoter of cytomegalovirus. Within the particularly preferred embodiments of the invention, the promoter is a tissue-specific promoter (see, e.g., WO 91/02805, EP 0,415,731, and WO 90/07936). Representative examples of suitable tissue-specific promoters include neural specific enolase promoter, platelet-derived growth factor-beta promoter, bone morpho-genetic protein promoter, human alfal-chimerin promoter, synapsin I promoter and promoter. of synapsin II. In addition, other viral-specific promoters (e.g., retroviral promoters (including those observed before, as well as others such as HIV promoters), hepatitis, herpes (e.g., EBV) can be used from the promoters observed above. and specific bacterial, fungal or parasite promoters (eg, of malaria), in order to target a specific cell or tissue that is infected with a virus, bacterium, fungus or parasite. WRNs of the present invention can be expressed from a variety of viral vectors, including for example, viral herpes vectors (e.g., US Patent No. 5,288,641), adenoviral vectors (e.g., WO 94/26914). , WO 93/9191; Kolls et al., PNAS 91 (1): 215-219, 1994; Kass-Eisler et al., PNAS 90 (24) 11498-502, 1993; Guzman et al., Circulation 88 (6): 2838 -48, 1993; Guzman et al., Cir. Res. 73 (6) 1202-1207, 1993; Zabner et al., Cell 75 (2) 207-216, 1993; Li et al., Hum Gene Ther. 4 (4) : 403-409, 1993, Caillaud et al., Eur. J. Neurosci. 5 (10: 1287-1291, 1993; Vicent et al., Nat. Genet. 5 (2): 130-134, 1993; Jaffe et al., Nat Genet. 1 (5) 372-378, 1992; and Levrero et al. , Gene 101 (2): 195-201, 1991), adeno-associated viral vectors (WO 95/13365: Flotte et al., PNAS 90 (22): 10613-10617, 1993), baculovirus vectors, parvovirus vectors ( Koering et al., Hum, Gene Therap, 5457-463, 1994), pox virus vectors (Panicali and Paoletti, PNAS 79: 4927-4931, 1982, and Ozaki et al., Biochem. Biophys. Res. Comm. 193 (2 ) 653-660, 1993), and retroviruses (e.g., EP 0,415,731, WO 90/07936, WO 91/0285, WO 94/03622, WO 93/25698, WO 93/25234, US Patent No. 5,219,740; WO 93/11230; WO 93/10218. Viral vectors may likewise be constructed to contain a mixture of different elements (eg, promoters, envelope sequences and the like) of different viruses or non-viral sources. of various modalities, either the viral vector itself or a viral particle containing the viral vector can be used in the methods and compositions described above. Mammalian cells suitable for carrying out the present invention include, among others: PC12 (ATCC No. CRL1721), neuroblastoma N1E-115, neuroblastoma SK-N-BE (2) C, adrenergic neuroblastoma SHSY5, murine cholinergic cell lines NS20Y and NG108-15, or rat F2 dorsal root ganglia lines, COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g., ATCC No. CRL 6281; BHK 570 cell line (deposited in the American Type Culture Collection under accession number CRL 10314), CHO (ATCC No. CCL 61), HeLa (eg, ATCC No. CCL2), 293 (ATCC No. 1573, Graham et al. J. Gen. Virol 36: 59-72, 1977) and NS-1 cells Other mammalian cell lines are used within the present invention including Rat Hep I (ATCC No. CRL 1600), Hep II of Rata (ATCC No. CRL 1548), TCMK (ATCC No. CCL 139) Human Lung (ATOC No. CCL 75.1), Human hepatoma (ATCC No. HTB-52), Hep G2 (ATCC No. HB 8065 ), mouse liver (ATCC No. CCL 29.1), NCTC 14690 (ATCC No. CCL 9.1), SP2 / 0-Ag14 (ATCC No. 1581), HIT-T15 (ATCC No. CRL 1777), and RINm 5AHT2B ( Orskov and Nielson, FEBS 229 (1): 175-178, 1988). Mammalian expression vectors to be used for the purpose of carrying out the present invention include a promoter capable of directing the transcription of a cloned gene or cDNA. Preferred promoters include viral promoters and cellular promoters. Viral promoters include the cytomegalovirus immediate early promoter (Boshart et al., Cell 41: 521-530, 1985), the cytomegalovirus immediate late promoter, SV40 promoter (Subramani et al., Mol. Cell. Biol. 1: 854- 864, 1981), MMTV LTR, RSV LTR, metallothionine-1, adenovirus E1a. Cell promoters include the mouse metallothionin-1 promoter (Palmiter et al., U.S. Patent No. 4,579,821) a mouse Vk promoter (Bergman et al., Proc. Nati, Acad. Sci. USA 81: 7041-7045, 1983; Grant and others, Nucí. Acids Res. 15: 5496, 1987) and a mouse VH promoter (Loh et al., Cell 33: 85-93, 1983). The choice of the promoter will depend, at least in part, on the level of expression desired or on the recipient cell line that will be transfected. Such expression vectors may also contain a group of RNA derivation sites located downstream of the promoter and upstream of the DNA sequence encoding the peptide or protein of interest. Preferred RNA derivation sites can be obtained from adenovirus genes and / or immunoglobulin genes. A polyadenylation signal located downstream of the coding sequence of interest is also contained in the expression vectors. Suitable polyadenylation signals include the early or late polyadenylation signals of SV40 (Kaufman and Sharp, ibid.), The polyadenylation signal of the adenovirus 5 E1B region and the terminator of the human growth hormone gene (DeNoto et al. , Nuc Acids Res. 9: 3719-3730, 1981). The expression vectors can include a viral leader sequence without coding, such as the tripartite leader of Adenovirus 2, located between the promoter and the RNA derivation sites. Preferred vectors may also include enhancer sequences, such as the SV40 enhancer. Expression vectors may also include sequences encoding VA adenovirus RNAs. Suitable expression vectors can be obtained from commercial sources (e.g., Stratagene, La Jolla, Calif.). Constructs of vectors comprising cloned DNA sequences can be introduced into cells of cultured mammals, for example by calcium phosphate-mediated transfection (Wigler et al., Cell 14: 725, 1978; Corsaro and Pearson, Somatic Cell. Genetics 7: 603, 1981; Graham and van der Eb, Virology 42: 456, 1973), electroporation (Neumann et al., EMBO J. 1: 841-845, 1982), or transfection mediated by DEAE-dextran (Ausubel et al., (Eds. ), Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987). In order to identify cells that have stably integrated the cloned DNA, a selectable marker is generally introduced into the cells together with the gene or cDNA of interest. Preferred selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin and methotrexate. The selectable marker can be an amplifiable selectable marker. The selectable amplifiable markers are the DHFR gene and the neomycin resistance gene. Selectable markers are reviewed by Th M ly (Mamalian Cell Technology, Butterworth Publishers, Stoneham MA, which is incorporated herein by reference). Mammalian cells containing a suitable vector are allowed to develop for a time, typically 1-2 days, to begin expressing the DNA sequences of interest. The selection of the drug is then applied to the selection of the growth of cells that are expressing the selectable marker in a stable form. For cells that have been transfected with an amplifiable, selectable marker, the drug concentration can be increased in a stepwise fashion to select the increased copy number of the cloned sequences, thus increasing expression levels. Cells expressing introduced sequences are screened and screened for production of the protein of interest in the desired form or at the desired level. The cells that satisfy these criteria can then be cloned and their production increased. Protocols for the transfection of mammalian cells are well known to those of ordinary skill in the art. Representative methods include retroviral, adenoviral and protoplast fusion-mediated transfection (see Sambrook et al., Supra). Constructs of pure vectors can also be collected by muscle cells or other suitable cells subsequent to injection into the muscle of a mammal (or other animals).

Numerous insect host cells known in the art may also be useful within the present invention in view of the present specification. For example, the use of baculoviruses as vectors for expressing heterologous DNA sequences in insect cells has been reviewed by Atkinson et al. (Pestic, Sci. 28: 215-224, 1990). Numerous host cells of plants known in the art may also be useful within the present invention, in view of the present specification. For example, the use of Agrobacterium rizogenes as vectors for expressing genes in plant cells has been reviewed by Sinkar et al., (J. Biosci. (Bangalore) 11: 47-58, 1987). The WRN proteins can be prepared by developing (usually by culturing) the host / vector systems described above, in order to express the recombinant WRN proteins. The recombinantly produced WRN proteins can be further purified as described in more detail below. Within the related aspects of the present invention, the WRN proteins can be expressed in a transgenic animal whose germ cells and somatic cells contain an WRN ungen which is operably linked to an effective promoter for gene expression. Alternatively, in a similar manner the transgenic animals can be prepared in such a way that they lack the WRN gene (e.g., "knocked out" mice). Such transgenics can be prepared in a variety of non-human animals, including mice, rats, rabbits, sheep, dogs, goats and pigs (see Hammer et al., Nature 315680-683, 1985, Almiter et al., Science 222,809 -814, 1983, Brinster and others Proc Nati Acad Sci USA 824438-4442, 1985 Paimiter and Bpnster Cell 41 343-345, 1985 and Patents of E UA Nos. 5,175,383, 5,087,571, 4,736,866, 5,387,742, 5,347,075, 5,221,778, and 5,175,384) In summary, an expression vector, including a nucleic acid molecule that will be expressed together with properly positioned expression control sequences, is introduced into the pronucleus of fertilized eggs, eg, by microinjection. The integration of the injected DNA is detected by analysis of DNA spots of tissue samples. It is preferred that the introduced DNA be incorporated into the germ line of the animal so that it is passed on to the progeny of the animal. Tissue specific expression can be achieved by the use of a tissue-specific promoter or by the use of an inducible promoter, such as metallothionine gene promoter (Palmiter et al., 1983, ibid), which allows regulated expression of the transgene. The vectors of the present invention can contain or express a wide variety of additional nucleic acid molecules in place of or in addition to a WRN protein as described above, either from one or several separate promoters for example, the viral vector can express a lymphokine or lymphokine receptor, antisense sequence or ribozyme or toxins. Representative examples of lymphokines include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, f L-12, IL-13, IL-14, I L-15, GM-CSF, G-CSF, M-CSF, interferon alpha, interferon beta, interferon gamma and tumor necrosis factors, as well as their respective receptors. Representative examples of antisense sequences include antisense sequences that block the expression of WRN protein mutants. Representative examples of toxins include: ricin, abrin, diphtheria toxin, cholera toxin, saporin, gelonin, scab antiviral protein, tritin, Shigella toxin, and Pseudomonas exotoxin A. Within other aspects of the invention, the molecules of constrictor oligonucleotides are provided, which specifically inhibit the expression of mutant WRN nucleic acid sequences (see generally, Hirashima et al., In Molecular Biology of RNA: New Perspectives (M. Inouye and BS Dudock, eds., 1987 Ácademic Press, San Diego, p, 401); Oligonuceotides: Antisense Inhibitors of Gene Expression (J.S. Cohen, by: 1989 MacMillan Press, London); Stein and Cheng, Science 261: 1004-1012 (1993); WO 95/10607, E.U.A. 5,359,051; WO 92/06693; and EP-A2-612844). In summary, said molecules are constructed in a manner that is complementary to, and allow to form Watson-Crick base pairs with a transcribed WRN mutant mRNA sequence region containing a WRN mutation. The resulting double-stranded nucleic acid infers with the subsequent processing of the mRNA, thus preventing protein synthesis.

Within other related aspects of the invention, the ribosomal molecules are provided wherein a sequence of constrictor oligonucleotides is incorporated into a ribosome that can specifically separate the mRNA molecules transcribed from a mutant WRN gene (see generally, Kim et al. , Proc. Nati, Acad. Sci. USA 84: 8788 (1987), Haseloff, et al., Nature 234: 585 (1988), Cech, JAMA 260: 3030 (1988), Jeffries, et al., Nucleic Acids Res. : 1371 (1989), US 4,093,246, US 5,354,855: US 5,144,855, US 5,272,262, US 5,254,678, and US 4,987,071). In accordance with this aspect of the invention, the antisense sequence that is incorporated into a ribosome includes a sequence complementary to, and which can form Watson-Crick base pairs with, a region of the transcribed mutant WRN mRNA containing a mutation. of WRN. The α-sense sequence thus becomes a target agent for delivering the catalytic ribosime activity specifically to the mutant WRN mRNA, wherein said catalytic activity separates the mRNA to render it incapable of being subsequently processed for translation of WRN proteins. Host Cells As described above, the nucleic acid molecules encoding the WRN proteins of the present invention (or vectors containing and / or expressing related mutants) can be easily introduced into a wide variety of host cells. Representative examples of such host cells include plant cells, eukaryotic cells and prokaryotic cells. Within preferred embodiments, nucleic acid molecules are introduced into cells from a vertebrate or warm-blooded animal, such as a human, macaque cell. , dog, cow, horse, pig, sheep, rat, hamster, mouse or fish, or any hybrid thereof. Preferred prokaryotic host cells for use within the present invention include E. coli, Salmonella, Bacillus, Shigella. Pseudomonas, Streptomyces and other genera. Techniques for transforming these hosts and expressing foreign DNA sequences cloned in the present are well known in the art (see, for example, Maniatis et al., Molecular Cloning: A. Laboratory Manual, Cold Spring Harbor Laboratory, 1982, which are incorporated herein). here by reference, or Sambrook et al., supra). Vectors used to express cloned DNA sequences in bacterial hosts will generally contain a selectable marker, such as a gene for resistance to antibiotics and a promoter that functions in the host cell. Suitable promoters include the trp promoter systems (Nichols and Yanofsky, Meth, Enzymol 101: 155-164, 1983), lac (Casadaban et al., J. Bacteriol., 143: 971-980, 1980), and phage? (Queen, J. Mol, Appl. Genet, 2: 1-10, 1983). Plasmids useful for transforming bacteria include pUC plasmids (Messing, Meth, Enzymol 101: 20-78, 1983, Vieira and Messing, Gene 19: 259-268, 1982), pBR322 (Bolivar et al., Gene 2:95). -113, 1977), pCQV2 (Queen, ibid.), And derivatives thereof. The plasmids can contain both viral and bacterial elements. Preferred eukaryotic cells include cultured mammalian cell lines (e.g., rodent or animal cell lines) and fungal cells, including yeast species (e.g., Saccharomy ssp, particularly S. cerevisiae, Schizosaccharomyces ssp, or Kluyveromyces ssp) or filamentous fungi (e.g., Aspergillus ssp, Neurospora ssp.). Strains of the yeast Saccharomyces cerevisiae are particularly preferred. Methods for producing recombinant proteins in a variety of prokaryotic and eukaryotic host cells are generally known in the art (see, "Gene Expression Technology," Methods in Enzymology, Vol. 185, Goeddel (ed.) Academic Press, San Diego, Cal. if., 1990 see also, "Guide to Yeast Genetics and Molecular Biology," Methods in Enzymology, Guthrie and Fink (eds.). Academic Press, San Diego, Calif., 1991). In general, a host cell will be selected on the basis of its ability to produce the protein of interest at a high level or its ability to carry out at least some of the processing steps necessary for the biological activity of the protein. In this manner, the number of cloned DNA sequences that must be introduced into the host cell can be minimized and the overall production of the biologically active protein can be maximized.

Nucleic acid molecules (or vectors) can be introduced into host cells by a wide variety of mechanisms, including for example calcium phosphate mediated transfection (Wigler et al., Cell 14: 725, 1978), lipofection; gene triggering (Corsaro and Pearson, Somatic Cell Gen. 7: 603, 1981; Graham and Van der Eb, Virology 52: 456, 1993), electroporation (Neumann et al., EMBO J. 1: 841-845, 1982), retroviral, adenovirus, mediated by protoplast fusion or transfection mediated by DEAE-dextran (Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, NY, 1987). The host cells containing vector constructs of the present invention were then cultured to express a DNA molecule as described above. The cells are cultured according to the normal methods in a culture medium containing nutrients required for the growth of the chosen host cells. A variety of suitable media are known in the art and generally include a source of carbon, a source of nitrogen, essential amino acids, vitamins and minerals, as well as other components eg, growth factors or serum, which may be required by the particular host cells. The growth medium will generally be selected for cells that contain the DNA constructs for example, by drug selection or deficiency in an essential nutrient that is complemented by the selectable marker in the DNA construct or co-transfected with the DNA construct . Suitable growth conditions for yeast cells, for example, include culturing in a chemically defined medium, comprising a source of nitrogen, which may be a nitrogen source without amino acids or a yeast extract, inorganic salts, vitamins and supplements. essential amino acids at a temperature between 4 ° C and 37 ° C, with 30 ° C being particularly preferred. The pH of the medium is preferably maintained at a pH greater than 2 and less than 8, more preferably pH 5-6. Methods to maintain a stable pH include regularization of pH and constant pH control. Preferred agents for pH control include sodium hydroxide. Preferred pH regulating agents include succinic acid and Bis-Tris (Sigma Chemical Co., St., Louis, Mo.). Because of the tendency of yeast host cells to hyperglycolisate heterologous proteins, it may be preferable to express the nucleic acid molecules of the present invention in yeast cells having a defect in a gene required for glycosylation linked to asparagine. Said cells preferably develop in a medium containing an osmotic stabilizer. A preferred osmotic stabilizer is sorbitol supplemented in the medium at a concentration between 0.1 M and 1.5 M, preferably at 0.5 M or 1.0 M. Cultured mammalian cells are generally cultured in commercially available serum-free or serum-free media. The selection of a suitable growth medium and conditions for the particular cell line used is well within the level of ordinary skill in the art. Antibodies Antibodies to the WRN proteins discussed above can be easily prepared given the description provided herein. Such antibodies, within certain embodiments, can specifically recognize the wild-type WRN protein in place of a mutant WRN protein, the mutant WRN protein in place of the wild-type WRN protein, or equally recognize both protein forms. mutants and wild type of the WRN protein. Antibodies can be used to isolate the protein, establish the intracellular localization of the WRN protein, inhibit the activity of the protein (antagonist), or increase the activity of the protein (agonist). Recognition of intracellular location - the product of the WRN gene may be abnormal in patients with WRN mutations, thus enabling the development of a rapid sieving analysis. Likewise, analyzes for small molecules that interact with the product of the WRN gene will be facilitated by the development of antibodies and localization studies. Within the context of the present invention, the antibodies are understood to include monoclonal antibodies, polyclonal antibodies, anti-iodotypic antibodies, fragments of antibodies (e.g., Fab, and F (ab ') 2, variable regions of F, or Complementary terminator regions) As discussed above, it is understood that the antibodies are specific against a WRN protein if it binds with a Kd of greater than or equal to 10"7M, preferably more than or equal to 10" dM Affinity of a monoclonal antibody or binding partner can be readily determined by one of ordinary skill in the art (see Scatchard, Ann, NY Acad Sci 51 660-672, 1949). In summary, polyclonal antibodies can be easily generated by someone with ordinary experience. in the matter of a variety of warm-blooded animals such as horses, cows, various birds, rabbits, mice or rats. Typically, a WRN protein or single peptide thereof of 13-20 amino acids (preferred preferably conjugated to the hemocyanin of the orifice fungus by entanglement with glutaraldehyde) is used to immunize the animal by intrapeptoneal, intramuscular, infraocular or subcutaneous injections, an auxiliary such as Freund's complete or incomplete adjuvant. As an example, a peptide corresponding to the residues 1375, 1387 of the WRN polypeptide sequence is used to raise a rabbit antiserum of rabbits After several booster immunizations, the serum samples are collected and tested for reactivity with the WRN protein or peptide. Particularly preferred polyclonal antisera they will give a signal in one of these analyzes that is at least three times greater than the antecedent. Once the titration of the animal has reached a platform in terms of its reactivity to the protein, large quantities of antiserum can easily be obtained either by weekly bleeding or exsanguinating the animal. Monoclonal antibodies can be easily generated using conventional techniques (see U.S. Patent Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993 which are incorporated herein by reference; see also Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyzes, Plenum Press, Kennett, McKearn, and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press , 1988, which are also incorporated herein by reference). Briefly, within an embodiment an animal such as a rat or mouse is injected with a WRN protein or portion thereof as described above. The protein can be mixed with an auxiliary such as Freund's complete or incomplete adjuvant in order to increase the resulting immune response. Between one and three weeks after the initial immunization the animal can be re-immunized against booster immunization and tested for its protein reactivity using the assays described above. Once the animal has reached a platform in this reactivity to the injected protein, it is sacrificed, and organs containing large numbers of B cells such as the spleen and lymph nodes are cultured. Cells obtained from the immunized animal can be immortalized by transfection with a virus such as Epstein-Barr virus (EBV) (see Glasky and Reading, Hybridoma 8 (4): 377-389, 1989).

Alternatively, within a preferred embodiment, suspensions of cultured spleen and / or lymph node cells are fused with the appropriate myeloma cell in order to create a "hybridoma" that secretes monoclonal antibodies. Suitable myeloma lines include, for example, NS-1 (ATCC No. TIB 18), and PeX63-Ag 8.653 (ATCC No. CRL 1580). After fusion, the cells can be placed in culture dishes containing a suitable medium, such as RPMI 1640, or DMEM (Dulbecco's Modified Eagles Medium) (JRH Biosciences, Lenexa, Kansas), as well as additional ingredients, such as serum of fetal bovine (SBF, ie, Hyclone, Logan, Utah, or JRH Biosciences). Additionally, the medium should contain a reagent that selectively allows a growth of fused spleen and rneomyoma cells such as HAT (hypoxanthine, aminopterin, and thymidine) (Sigma Chemical Co., St. Louis, Missouri). After about seven days, the resulting fused cells or hybridomas can be screened for the presence of antibodies that are reactive against a WRN protein. A wide variety of assays can be used to determine the presence of antibodies that are reactive against the proteins of the present invention, including for example immuno-electrophoresis radioimmunoassay, radioimmunoprecipitations, enzyme-linked immunosorbent assays (ELISA), spot spot analysis , Western analysis, immunoprecipitation, Inhibition or Competition Analysis and sandwich analysis (see US Patent Nos. 4,376,110 and 4,486,530, see also Antibodies A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988) Following several clonal dilutions and some analyzes, a hibpdome can be isolated which produces antibodies reactive against the WRN protein. other techniques can be used to construct monoclonal antibodies, (see William D Huse et al., "Generation of a Large Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambda," Science 246 1275-1281, December 1989, see also L Sastry et al., "Cloning of the Immunological Repertoire m Eschenchia coll for Generation of Monoclonal Catalytic Antibodies Construction of a Heavy Chain Variable Region-Specific cDNA Library", Proc Nati Acad Sci USA 865728-5732, August 1989 see also Michelle Alting-Mees et al., "Monoclonal Antibody Expression Librarles A Rapid Alternative to Hibpdomas "Strategies m Molecular Biology 3 1-9, January 1990, these references describe a system In short, the mRNA is isolated from a population of B cells and used to create cDNA expression banks of heavy chain immunoglobulins. and light in the vectors lmmunoZap (H) and? lmmunoZap (L) These vectors can be screened individually or co-expressed to form fragments of Fab or antibodies (see Huse et al., supra, see also Sastry and others supra). positive can subsequently be converted to non-lithic plasmids that allow the expression of high levels of E coll monoclonal antibody fragments. Similarly, portions or fragments, such as fragments of Fab and Fv antibodies can also be constructed using DNA digestion techniques. enzymatic or recombinant to incorporate the variable regions of a gene encoding a specific binding antibody Den In one embodiment, the genes encoding the variable region of a hibpdoma that produces a monoclonal antibody of interest are "amplified using nucleotide primers for the variable region. These primers can be synthesized by someone of ordinary skill in the art or can be purchased from commercially available sources Stratacite (La Jolla, Cahf) sells primers for variable regions of mice and humans including, among others, primers for VHa, VHb, HCV, VHd, Cm, and V and C regions. These primers can be used to amplify heavy or light chain variable regions that can be inserted into vectors such as lmmunoZAP ™ H or lmmunoZAP ™ L (Stratacyte), respectively These vectors can then be introduced into systems based on E coli yeast or mammals for their expression. Using these techniques, large quantities of a single chain protein containing a fusion of the VH and V domains can be produced. (See Bird et al., Sciences 242423-426, 1988) In addition, such techniques can be used for a "mupnos" antibody to a "human" antibody, without altering the binding specificity of the antibody. have obtained, can be isolated or purified by many techniques well known to those of ordinary experience in the field (see Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Har Bor Laboratory Press, 1988). Suitable techniques include peptide or protein affinity columns, CLAR or CLAR-FI, purification on protein A or protein G columns or any combination of these techniques. Analysis Useful analyzes within the context of the present invention include those assays for detecting agonists or antagonists of WRN protein activity. Other analyzes are useful for screening the bank of peptide molecules or organic molecules. Still other analyzes are useful for the identification and / or isolation of nucleic acid molecules and / or peptides within the present invention, the identification of proteins that interact or bind to the WRN protein, for the diagnosis of a patient with a Increased probability of contracting Werner syndrome, or for the diagnosis of a patient with susceptibility to, or the manifestation of, a disease related to WRN. Diagnostic Tests Based on Nucleic Acids In summary, another aspect of the present invention provides probes and primers for detecting WRN genes and / or mutants thereof. In one embodiment of this aspect, probes are provided which are capable of specifically hybridizing to DNA or RNA of the WRN genes. For the purposes of the present invention, the probes are "capable of hybridizing" to DNA or RNA of the WRN gene if they hybridize to a WRN gene under conditions of high or moderate restriction (see Sambrook et al., supra) but not meaningfully or detectably to an unrelated helicase gene such as the Bloom Syndrome gene (Ellis et al., Cell 83655- 666, 1995). Preferably, the probe hybridizes to suitable nucleotide sequences under conditions of high restriction, such as hybridization in 5x SSPE, 1x Denhardt's solution, 0.1% SDS at 65 ° C, and at least one wash to remove the nonhybridized probe in presence of 02x SSC, 1x Denhardt's solution 0 1% SDS at 65 ° C Except as provided in some manner herein, the probe sequences are designed to allow hybridization to WRN genes, but not to DNA sequences or RNA from other genes. For example, probes are used to hybridize nucleic acids that are present in a biological sample isolated from a patient. The hybridized probe is then detected thus indicating the presence of the desired cellular nucleic acid. Preferably, the cellular nucleic acid is subjected to to an amplification procedure, such as PCR, before hybridization Alternatively, the WRN gene can be amplified and the amplified product subjected to sequencing n DNA The WRN mutants can be detected by DNA sequence analysis or hybridization with specific oligonucleotide probes and alleles under conditions and for a time sufficient to allow hybridization to specific allele. Typically, the buffer solution of hybridization and washing will contain tetramethyl ammonium chloride or the like (see Sambrook et al., Supra). The nucleic acid probes of the present invention may be composed of deoxyribonucleic acids (DNA), ribonucleic acids (RNA), nucleic acid analogs (e.g., peptide nucleic acids), or any combination thereof, and may be as few as about 12 nucleotides in length, usually about 14 to 18 nucleotides in length and possibly as large as the entire sequence of a WRN gene. The selection of probe size depends somewhat on the use of the probe and is within the experience of the subject. Proper probes can be constructed and labeled using techniques that are well known in the art. Shorter probes of, for example, 12 bases can be synthetically generated and labeled with 3 P using T4 polynucleotide kinase. Longer probes of approximately 75 bases or less than 1.5 kb are preferably generated for example, by PCR amplification in the presence of labeled precursors such as [α-32P] dCTP, digoxigenin-dUTP, or biotin-dATP. Probes larger than 1.5 kb are usually amplified more easily by transfecting a cell with a plasmid containing the relevant probe. developing the transfected cell in large quantities and purifying the relevant sequence of the transfected cells (see Sambrook et al. supra). The probe can be labeled by a variety of markers, including for example, radioactive labels, fluorescent labels, enzymatic labels and chromogenic markers. Use of 32P is particularly preferred for marking a particular probe. It is a feature of this aspect of the invention that the probes can be used to detect the presence of WRN mRNA or DNA within a sample. However, if the relevant sample is present in only a limited number can be beneficial to amplify the relevant sequence so that it can be detected or obtained more easily A variety of methods can be used in order to amplify a selected sequence, including, for example, RNA amplification (see Lizardi et al. , Bio / Technology 6 1197-1202, 1988, Kramer et al., Natu re 339401-402, 1989, Lomeh et al., Clmical Chem, 35 (9) 1826-1821, 1989, U.S. Patent No. 4,786,600), and DNA amplification using the LCR or polymerase chain reaction ('PCR') ( see, U.S. Patent Nos. 4,683,195, 4,683,202 and 4,800 159) (see also U.S. Patent Nos. 4,876,187 and 5,011,769 which describe an alternative detection / amplification system comprising the use of cleavable linkages) or other methods of nucleic acid amplification that they are well within the level of ordinary experience in the field. With respect to CPR, for example, the method can be modified as is known in the art. The transcriptional increase in PCR can be achieved by incorporating bacteriophage T7 RNA polymerase promoter sequences in one of the primary oligonucleotides and immunoenzymatic detection of the increased emitter products can be performed using anti-RNA: DNA antibodies (Blais, Appl. Environ, Microbiol., 60: 348-352, 1994). PCR can also be used in combination with reverse dot spot hybridization (Lida et al., FEMS Microbiol, Lett 114: 167-172, 1993). The PCR products can be analyzed quantitatively and by the incorporation of dUTP (Duplaa et al., Anal. Biochem. 212: 229-236, 1993) and the samples can be sampled filters for detection of PCR probes (Bej et al. Appll. Environ Microbiol. 57: 3529-3534, 1991). Within a particularly preferred embodiment, the PCR amplification is used to detect WRN DNA. In summary, as described in more detail below, a DNA sample is denatured at 95 ° C in order to generate DNA from a single strand. The DNA sample can be a cDNA generated from RNA. Specific primers are used attached to single-stranded DNA at 37 ° C to 70 ° C, depending on the proportion of AT / GC in the primers. The primers are extended at 72 ° C with Taq DNA polymerase or other stable DNA polymerase in order to generate the opposite strand of the standard. These steps constitute a cycle, which can be repeated in order to amplify the selected sequence. For greater specificity, nested PCR can be performed. In nested PCR, a second amplification is carried out using a second set of primers derived from sequences within the first amplified product. The entire WRN coding region can be amplified from the cDNA using three sets of primers to generate lengths of fragments that are conveniently sized to determine their sequence. In a preferred embodiment, the nested RCP is performed. Within an alternative preferred embodiment, the LCR amplification is used for amplification. The LCR primers are synthesized so that the 5 'base of the upstream primer is capable of hybridizing to a single base pair in a desired gene to specifically detect a WRN gene. Within a preferred embodiment, the probes are used in an automated non-isotopic strategy wherein the target nucleic acid sequences are amplified by PCR and then the desired products are determined by a binding analysis of colorimetric oligonucleotides (AUO) (Nickerson et al. , Proc. Nati, Acad. Sci. USA 81: 8923-89278, 1990). The primers for the amplification of a selected sequence should be selected from the sequences that are highly specific for WRN (and not, eg, in Bloom Syndrome, supra) and stable duplexes with the target sequence. The primers must also be non-complementary, especially at the 3 'end, nor must they form dimers with themselves or other primers, and they must not form secondary or duplex structures with other DNA regions. In general, the primers of about 18 to 20 nucleotides are ios preferred, and can be easily synthesized using techniques well known in the art. The products of PCR and other nucleic acid amplification products can be quantified using techniques known in the art (Duplaa et al., Anal Biochem 212229-236, 1993, Higuchi et al., Bio / Technology 11 1026-1030 Within a modality of the invention; nucleic acid diagnostics can be developed by being able to detect the presence of Werner Syndrome or of several related diseases that can be caused by Werner Syndrome. In summary, severe mutations in the WRN gene can lead to Werner Syndrome, as well as to a host of related diseases, including for example, increased frequency of some benign or malign neoplasms (especially sarcomas) cataracts, cardiovascular disease, osteoporosis, type I and type II diabetes, cataracts, skin changes similar to sclerodoma and hyperkeratosis Mutations Less severe of the gene may lead to the presentation of the same group of diseases, but at a higher age In addition, many of the related diseases may be associated with mutations in the WRN gene. For example, diabetes and osteoporosis are often associated with aging. older and individuals with these diseases (or others) are screened for mutations in WRN. Any of the assays described herein can be used. Especially preferred is RCP-TI in conjunction with DNA sequence determination. To correlate a mutation or polymorphism with disease, preferred subjects are offspring pairs. in which a descendant has the disease Once the mutation is identified, other suitable screening assays can be used to analyze particular nucleotide changes Because the sequences of the two gene copies of the individuals not affected with the disease Werner, can be correlated with medical histories of these patients to define these correspondences, these alleles can therefore be used as a diagnosis for susceptibilities of these diseases, once the relationship is defined Certain non-null form of the gene, for example in the homozygous or heterozygous state can significantly affect the pr Openness of carriers to develop, for example cancer These propensities can be assessed by examining the gene sequences (both copies) in a statistically significant sample of cancer patients Other diseases (see above) can be examined similarly for significant correlations with certain alleles To detect certain casual relationship can be used the Xl-square test or other statistical test, to examine the significance of any correlation between appropriate genotypes and disease status as recorded in medical records, using normal good practices of medical epidemiology The sequences that define each of the alleles are valuable diagnostic indicators for an increased susceptibility of the disease. Therefore, from the nucleic acid sequences provided herein, a wide variety of diseases related to Werner Syndrome can be easily detected. Another cellular phenotype of Werner's patient cells is the increased frequency of deletion mutation in the cells. Clearly, defective helicase in these cells leads to a specific mutant phenotype, while not returning hypertensive cells to a variety of chemical or physical mutants that damage DNA, such as ionizing radiation. The disease states, or sensitivities that result from a high frequency of suppression, can therefore be controlled, in part, by alterations of the Werner gene and some alleles therefore can be diagnoses of this kind of medical conditions. Analysis for agonists and antagonists Also by the present invention are provided agonists or antagonists of the WRN gene product comprising a protein, peptide, chemical or peptide mimic that binds to the product of the WRN gene or interacts with a protein that binds to the product of the WRN gene so that the binding of the agonist or antagonist affects the activity of the WRN gene product. An agonist will activate or increase the activity of the WRN gene product. An antagonist will inhibit or decrease the activity of the WRN gene product. The activity of the WRN gene product can be measured in an analysis, such as a helicase analysis or other analysis that measures a product activity of the WRN gene. Other analyzes measure the binding of protein that interacts with WRN and is necessary for its activity. The agonists and antagonists of the WRN gene product can be used to increase the activity or inhibit the activity of the gene product. Such agonists and antagonists can be identified by a variety of methods. For example, proteins that bind and activate WRN can be identified using a 2-yeast hybrid detection system. In this system, the WRN gene is fused to a DNA binding domain or an activation domain of a yeast gene such as GAL4. A cDNA library is constructed in a vector such that the inserts are fused to one of the domains. The vectors are co-transfected into yeast and selected for the transcriptional activation of a reporter gene (Fields and Song, Nature 340: 245, 1989). The proteins that bind to WRN are candidate agonists. Three different proteins that bind to WRN have been identified in an initial screening using the 2-hybrid system. When the binding site on the WRN gene product is determined, the molecules that bind and activate the WRN protein can be designated and evaluated. For example, computer modeling of the union site can be designed imitations of that union. Antibodies to the binding site can be generated and analogs of the native binding proteins can also be generated. Any of these molecules is tested for the activity of agonists or antagonists by a functional analysis of the product of the WRN gene. For example, to test antagonist activity, yeasts are co-transfected with WRN and each binding protein is fused to a DNA binding domain or an activation domain. The test molecule is administered and the administration is monitored. An antagonist will inhibit the activation of the reporter gene by at least 50%. Similarly, the agonist activity can be measured by increasing the activity of WRN in a 2-hybrid system of yeast or by coupling the test compound to a DNA binding domain or activation and monitoring the activity of the reporter gene. The WRN proteins, nucleic acid molecules encoding said proteins, anti-WRN protein antibodies and agonists or antagonists, as described before and below, are labeled with a variety of molecules, including, for example, fluorescent molecules, toxins and radionuclides. Representative examples of fluorescent molecules include fluorescein, Phycobili proteins, such as phycoerythrin, rhodamine, Texas red and luciferase. Representative examples of toxins include, recin, abrin, diphtheria toxin of abrin, cholera toxin, gelonin, antiviral protein of grana, tritin, toxin of Shigella and exotoxin A of Pseudomonas. Representative examples of radionuclides include Cu-64, Ga-67, Ga-68, Zr-89, Ru-97, Tc-99m, Rh-105, Pd-109, In-111, 1-123, 1-125, 1-131, Re-186, Re-188, Au-199, Pb-203, At-211, Pb-212, and B? -212 In addition, the antibodies described above can also be labeled or conjugated to a pair of a ligand binding pair Representative examples include avidin-biot and riboflavin-riboflavin binding protein The methods for conjugating or labeling WRN proteins, nucleic acid molecules encoding said proteins, anti-WRN protein antibodies and agonists or antagonists, such as it was treated before, with the representative tags exhibited before can be easily achieved by someone with ordinary skill in the art (see Tpchothecene Antibody conjugate, US Patent No. 4,744,981, Antibody Conjugate, US Patent No. 5,106,951, Fluorogenic Materials and Labeling Techpiques, USA No 4,018,884, Metal Radionuclide Labeled Pro Teins for Diagnosis and Therapy, US Patent No. 4,897,255, and Metal Radionuch of Cheiating compounds for Improved Chelation Kinetics Patent No. E A No. 4, 988,496, see also, Method In Enzymology, Vol 34, Affimty Techmques, Enzyme Punfication Part B, Jakovy and Wilchek (eds, Academic Press, New York, p 30, 1974, see also Wilchek and Bayer "The Avidm-Biotin complex in Bioanalytical Applications ", Anal Biochem 171 1-32, 1988) Pharmaceutical Compositions As noted above, the present invention also provides a variety of pharmaceutical compositions, comprising one of the WRN proteins described above, nucleic acid molecules, vectors, antibodies, host cells, agonists or antagonists, together with a pharmaceutically or physiologically acceptable carrier, excipients or diluents. Generally, such vehicles should not be toxic to the recipients at the doses and concentrations employed. Ordinarily, the preparation of said compositions comprises combining the therapeutic agent with buffer solutions, antioxidants such as ascorbic acid, low molecular weight polypeptides (less than about 10 residues), proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, agents chelators such as EDTA, glutathione and other stabilizers and excipients. Saline with neutral regulated pH or saline mixed with nonspecific serum albumin are illustrative suitable diluents. In addition, the pharmaceutical compositions of the present invention can be prepared for the administration of a variety of different routes. In addition, the pharmaceutical compositions of the present invention can be placed into a container, along with packaging material which provides instructions regarding the use of said pharmaceutical compositions. Generally, such instructions will include a tangible expression describing the concentration of reagents, as well as within certain embodiments, the relative amounts of ingredients or diluents of excipients, (e.g., water, saline or PBS) that may be necessary to reconstitute the pharmaceutical composition.

Methods for Treating or Avoiding Werner Syndrome The present invention also provides methods for treating or avoiding Werner Syndrome (or related diseases), comprising the step of administering a vector to a patient (eg expression vector, viral vector, or viral particle containing a vector) or nucleic acid molecule alone as described above. thus reducing the likelihood or delay of the presentation of Werner Syndrome (or related disease). Similarly, therapeutic peptides, peptide mimics or small molecules can be used to delay the presentation of Werner Syndrome, decrease symptoms or interrupt or slow the progression of the disease. Such a therapy can be tested in a transgenic animal model expressing mutant protein, wild-type and mutant protein, or in an in vitro analysis system (e.g., a helicase assay such as that described by Bjornson et al., Biochem, 3307: 14306-14316, 1994).

As noted above, the present invention provides methods for treating or avoiding Werner's Syndrome by administering to a patient a therapeutically effective amount of an antagonist or pharmaceutical composition as described herein. These patients can be identified by clinical diagnosis based on the classic symptoms of Werner Syndrome. As will be evident to someone with experience in the field, the amount and frequency of administration will depend of course. of such factors as the nature and severity of the indication being treated, the desired response, the condition of the patient and so on. Typically, the compositions can be administered by a variety of techniques as noted above. Within other embodiments of the invention, the vectors that contain or express the nucleic acid molecules encoding the WRN proteins described above, or even the nucleic acid molecules themselves can be administered by a variety of alternative techniques, including for example the administration of asiaioosomocoid (ASOR) conjugated with complexes of poly-L-lysine DNA (Cpstano et al., PNAS 92122-92126 1993), DNA linked to killed adenovirus (Cupel et al., Hum Gene Ther 3 (2) 147-154, 1992), introduction mediated by cytofectin (DMRIE-DOPE, Vical, Calif), direct DNA injection (Acsadi et al., Nature 352815-818, 1991), DNA ligand (Wu and other J Of Biol Chem 264 16985- 16987, 1989), lipofection (Felgner et al. Proc Nati Acad Sci USA 847413-7417, 1989), liposomes (Pickepng et al., Circ 89 (1) 13-21, 1994 and Wang et al. PNAS 847851-7855, 1987), bombardment of microprojectiles (Williams et al., PNAS 882726-2730, 1991), and direct delivery of nucleic acids encoding the WRN protein alone (Vile and Hart Cancer Res 53 3860-3864, 1993), or using PEG-acid complexes The following examples are offered by way of illustration, and not by way of limitation EXAMPLES EXAMPLE 1 CLONING OF THE WRN GENE OF CHROMOSOME 8 The SW site (WRN) was initially located at 8p12 by conventional mapping methods (Goto et al. Nature 355735-738, 1992) and genetic position was refined using meiotic and homozygous mapping (Schellenberg et al., 1992, Nakura, et al., Genomics 23: 600-608, 1994; Thomas, Genomics 16685-690, 1993) The latter is possible given that many subjects with SW are descendants of consanguineous marriages (Table 1) The initial mapping work (Nakura, et al., Genomics 23600-608, 1994, Oshima et al., Genomics 23 100-113, 1994) placed the WRN site in a range of 8 3 cM flanked by D8S137 and D8S87 (Figure 1) D8S339, a marker within this range, was the closest site tested (q = 0 001, Zmax = 15 93) Multiple point analysis placed WRN within 06 cM of D8S339, although the region between D8S87 and FGFR could not be excluded Subsequently, the short random repeat polymorphism (PRAC) markers in glutathione reductase (GRS) ) and D8S339 were found to be in unbalanced binding with SW in Japanese SW subjects (Yu, American Journal of Human Genetics 55 356-364, 1994). To clone the WRN gene, they generated an artificial yeast chromosome (CAL) P1, and an contiguous cosmid was generated starting in the GRS / D8S339 region and extended by methods of Progress to cover approximately 3 Mb Additional markers of 16 PRACs were identified in the contiguous CAL (Figure 1B) were identified to define recombinants and delineate the boundaries of the binding disequilibrium region For the ordering of markers and identification of genes, the cosmids and P1 clones were also isolated and used to construct a small contiguous partial clone of the region (Figure 1E) The WRN region was defined by obligate recombinants in C41C3S3 excluding the telomepca region at this marker and in y896R9 excluding the centromeopic region to this Thus, the region of C41C3S2 to y896R9, which is approximately 1 2 Mb (Figure 1C), is considered the minimum region of WRN The genes of the region n of WRN were identified by trapping exons using the pSL3 vector (Buckler et al., Proc Nati Acad Sci USA 884005-4009 1991, Church et al., Nat Genet 698-105, 1994), hybridization of immobilized CAL cDNA libraries ( Papmoo et al., Proc Nati Acad Sci USA 873166-3169, 1991) and comparison of the genomic sequence databases of the DNA sequences using BLAST (Altschul et al., J Mol Biol 215-403-410, 1990) and the program to find exons GRAIL (Uberbacher and Mural, Proc Nati Acad Sci USA 88 1261, 1991) The genomic sequence was determined for region defined by clones of P1 2233, 2253, 3833, 2236, 2237, 2932 6738 and 2934 and the clone of cosmids 176 C6 Each method identifies short segments of expressed sequences, which are then used to screen a fibroblast cDNA library arranged to identify longer cDNA clones. This bank was selected because the SW fibroblasts have a phenotype of premature senescence in vitro, indicating that the WRN gene is probably expressed in this type of cells. The genes identified by this process were screened for WRN mutations using polymerase-reverse transcriptase chain reaction (RCP-TI). Seven subjects were screened initially for mutations; five 'subjects of WRN (2 Caucasians and 3 Japanese) and 2 control subjects (1 Caucasian and 1 Japanese). Prior to the identification of the WRN gene, the following genes in the region were screened for mutations; GSR, PP2AB, TFIIEB and genes that correspond to other sites labeled with expressed sequence (SESe). The candidate WRN site gene was detected initially using the genomic sequence of the P1 2934 clone to search the SES database. A single ESA of 245 bp R58879 was detected, which is homologous to 3 segments of the genomic sequence separated by the intronic budget sequence. The sequence of R58879 was used to identify longer cDNA clones from a normal fibroblast cDNA library. An initial 2.1 kb cDNA clone containing ESE R58879, corresponding to the 3 'end of the gene, was obtained by screening an array of clones by PCR using primers A and B, (see below). Initiators A and B are derived from the sequence of R58879 and produce a 145 bp fragment after amplification. Longer clones were identified by PCR screening with the 5EA and 5EB primers, which were derived from sequences within a predicted exon located at p2934 and 5 'for sequences contained in the initial 2.1 kb clone. Six additional clones were identified. 8 clones were obtained by plaque hybridization. The longest clone is 4.0 kb in length. The additional sequence was obtained by the RAGE method using the 5EA primer to initiate cDNA synthesis of the first strand. A 2.5 kb product was obtained containing an additional 1.-4 kb of sequence. There is evidence that R58879 is expressed by Northern blot analysis, in which 6.5 kg and 8 kb of transcripts were detected in a variety of tissues including heart, placenta, muscle and pancreas, transcripts were also detected by PCR products. TI of fibroblasts of lymphoblastoid cell line. EXAMPLE 2 CLONING OF THE WRN GENE OF SUBJECTS The WRN gene can be isolated from patients and mutations or polymorphisms determined by sequence analysis. Peripheral blood cells are obtained by venous puncture and hypotonic lysis of erythrocytes. DNA or RNA is isolated from these cells and the WRN gene is isolated by amplification. The gene sequence can be obtained by amplifications of the exons of genomic DNA or by PCR-TI, followed by the determination of DNA sequence. Suitable primers for determining the DNA sequence and for performing RCP-TI are listed below (primers A-R are SEQ ID Nos. 1-18 respectively, and primers 5EA-5EG are SEQ ID Nos. 19-15 respectively). Two cDNAs were identified and shown in Figures 2 and 3. There is some uncertainty regarding the identity of a few bases in the 5 'untranslated region in Figure 2. Two PCR-TI reactions were used to obtain the gene of different tissues. The synthesis of the first cDNA strand was carried out according to normal procedures (eg, with a Stratagene Stratagene kit). The cDNA was subjected to a pair of nested PCR identifications, the first with primers I and J (SEQ ID Nos 9 and 10), followed by primers K and L (SEQ ID Nos. 11 and 12) and the second with 5ED primers. and P (SEQ ID Nos. 22 and 16) followed by initiators 5EE and B (SEQ ID Nos 23 and 2). These fragments were isolated and used for sequencing in order to identify differences in the gene sequence or division pattern. Initiators AH (SEQ ID Nos. 1-8) and KR (SEQ ID Nos 11-18) were used to sequence the first PCR-TI fragment Initiators B, 5EA, 53B, 53C, 533, 53F and 5EG (SEQ. ID Nos 2 19, 20, 21, 23, 24 and 25, respectively) were used to sequence the second PCR-TI fragment. Sequencing is performed on ABI373A using sequencing equipment from Applied Biosystems Division of Perkin-Elmer FS according to the manufacturer's instructions.

A 5 '- CTGGCAAGGATCAAACAGAGAG B 5' - 5'-TGGCAAATTGGTAGAAGCTAGG CTTTATGAAGCCMTTTCTACCC C D 5 '-AAATAACTATGCTTTCTTACATTTAC E 5' -CTCCCGTCAACTCAGATATGAG F 5 '- 5'-G CTGTTTGTAAATGTAAGAAAGCATAG GAGCTATGATGACACCACTGC H 5' -ACTGAGCAACAGAGTGAGACC I 5 '-GGATCTGGTCTCACTCTGTTGC J 5'- TTGCCTAGTGCAApGGTCTCC K 5 '- AGTGCAGTGGTGTCATCATAGC L 5' - CCTATTTAATGGCACCCAAAATGC M 5 '- 5'-N C AGTCTATGGCCATCACATACTC ACCGCTTGGGATAAGTGCATGC O 5' -GAGAAGAAGTCTAACTTGGAGAAG P 5 '-TTCTGGTGACTGTACCATGATAC Q 5' - CCAAAGGAAGTGATACCAGCAAG R 5 '-ACAGCAAGAAACATAATTGTTCTGG 5 EA 5' - GAACTTTGAAGTCCATCACGACC 5EB 5 ' -GCATTAATAAAGCTGACATTCGCC 5EC 5 '- CApACGGTGCTCCTAAGGACATG 5ED 5' - GATGGATTTGAAGATGGAGTAGAAG 5EE 5 '- TGAAAGAGAATATGGAAAGAGCTTG 5EF 5'-GTAGAACCAACTCATTCTAAATGCT 5EG 5' -AATTTGCGTGTCATCCpGCGCA The exons of the 5 'end of the WRN gene can be amplified for DNA samples using the primers listed below (primers E1A-E13B are SEQ ID Nos. 26-57, respectively). The DNA sequence was determined using the same primers and an ABI373A automatic sequencer using sequencing equipment from Applied Biosystems Division of Perkin-Elmer FS according to the manufacturer's instructions. E1A 5 '-TCCTAGTCACCCATCTGAAGTC E1B 5' -CATGAAACpGCTTCTAGGACAC E2A 5'-CCCAGGAGTTCGAGACCATCC E2B 5'-TTACAATCGGCCACApCATCAC E2C 5'-TGTMTCCCAACACTTTGGGAGG E2D 5'-AGTGGAAGAATTCATAGTGGATGG E3A 5 '-TAGCTpATGAAGCCAATTTCTACC E3B d' -AATCCAAAGAATCAATAGACAAGTC E3C 5 '-GCTTGAAGGATGAGGCTCTGAG E3D 5' -TGTTCAGAATGAGCACGATGGG E4A 5 '-CTTGTGAGAGGCCTATAAACTGG E4B 5' -GGTAAACAGTGTAGGAGTCTGC E5A 5 '-GCCA? TpCTCTTTAATTGGAAAGG E5B 5' -ATCTTATTCATCTTTCTGAGAATGG E6A 5 '-TGAAATAGCCCAACATCTGACAG E6B 5' -GATTAATTTGACAGCTTGATTAGGC E7A 5 '-TGAAATATAAACTCAGACTCTTAGC E7B 5' -GTACTGATTTGGAAAGACATTCTC E8A 5 '-GATGTGACAGTGGAAGCTATGG E8B d' -GGAAAAATGTGGTATCTGAAGCTC E9A 5 '-AAGTGAGCAAATGpGCTTCTGG E9B 5'-TCATTAGGAAGCTGAACATCAGC E10A 5'-GTTGGAGGAAATTGATCCCAAGTC ElOB 5'-TGpGCTTATGGGTTTAACTTGTG El1A 5'-TAAAGGATTAATGCTGTTAACAGTG E12A E11B TCACACTGAGCATTTACTACCTG 5'-5' 5'-CTTTGGCAACCTTCCACCTTCC -GTAATCATATCAGAATTCATAACAG E12b E12c 5'-GCAAAGGAAATGTAGCACATAGAG E12D 5 ' -TO GGCTATAGGCATTTGAAAGAGG E13A 5'-GTAGGCTCCCAGAAGACCCAG E13B 5'-GAAAGGATGGGTGTGTATTCAGG EXAMPLE 3 Identification of M allele Alleles The sequence of A D N c (Fig. 2) was linked to genomic analysis to identify the structure of exons and synthesizers synthesized for PCR amplification of each exon. The DNA sequence of the 13 exons was determined for 5 patients and two unaffected individuals. In 4 out of 5 patients, changes of a single base pair led to separation effects or retention codons in the open reading frame of the gene. In the fifth patient, a single change in base pair resulted in a transition from system to arginine, which may alter the function of the gene. Each of the exons was also sequenced in 96 unaffected control individuals (48 Caucasians and 48 Japanese) and none of the mutations was found in any of the control individuals. The first mutation is a mutation in a separation acceptor site. In the next sequence, the sequence of GGTAGAAA starts at nucleotide 2030 (Figure 2). The change from g to c results in a deletion of 95 bp. The preparation of DNA for mutational analyzes of RCP-TI revealed that for a subject, the amplification product was shorter than that observed in products of other subject with SW and control. DNA sequence analysis of the PCR-TI product revealed that 95 bp was missing compared to other samples. The missing sequence corresponds to a single exon. This exon and genomic flanking segments were sequenced from the subject with SW and controls and a single base change (G? C) at the site of dividing donors was detected. The subject was descended from a marriage of first cousins and as was expected homozygous for this mutation the same mutation was found in a total of 18 out of 300 subjects with Japanese with SW and, therefore it is the most common Japanese mutation with SW. The deletion of this exon results in a change in the expected open reading frame and a premature detection codon. This mutation was not observed in 46 Japanese controls and 46 Caucasian controls between the mutation vehicles, 12/16 had 141 bp alleles in GRS2-PRAC. wild type: ttttaatagGGTAGAAA (SEQ ID No. 58) Werners: ttttaatacGGTAGAAA (SEQ ID No. 59) The second mutation changes a C to T in nucleotide 2384 (Figure 2) by changing a glutamine to a detection codon, which gives as result a predicted truncated protein. This mutation was observed in a single subject. The E11A and E11B primers flank this sequence and amplify a 360 bp fragment. wild type gln: GAAGCTAGGCAGAAACAT (SEQ ID No. 60) Werners: GAAGCTAGGTAGAAACAT (SEQ ID No. 61) ter The third mutation changes a C to T at nucleotide 2804 (Figure 2), which alters an arginine codon to a codon of arrest resulting in a predicted truncated protein. Four Japanese subjects with SW and one Caucasian subject with W5 had this mutation. The E8A and E8B primers flanked this sequence and amplified a product of 267 bp. arg wild type: TTGGAGCGAGCA (SEQ ID No. 62) Werners: TTGGAGTGAGCA (SEQ ID No. 63) ter The fourth mutation is a deletion of 4 bp through a separation junction. The sequence of exons shown below begins at nucleotide 2579 (Figure 2). This mutation was identified in a W5 Sirius congener. The E4A and E4B primers flank this mutation and amplify a fragment of 267 bp. wild type: ctgtagACAGACACCTC (SEQ ID No. 68) Werners: ctgt AGACACCTC (SEQ ID No. 69) The fifth mutation is a nonsense mutation. An A T is altered to a G at nucleotide 2113 (Figure 2), changing the wild-type phe codon to a leu codon. This change is a polymorphism with each allele present at a frequency of approximately 0.5 and that does not seem to correlate with SW. Wild type phe: AAGAAGTTTCTTCTG (SEQ ID No. 64) Werners: AAGAAGTTGCTTCTG (SEQ ID No. 65) leu The sixth mutation is a nonsense mutation by changing a T to a C at nucleotide 2990 (Figure 2) and codon cys to a codon of arg. Wild type cys: CCTTCATGTGAT (SEQ ID No. 66) Werners: CCTTCACGTGAT (SEQ ID No. 67) arg These point mutations can also be identified by PCR using primers that contain the wild type or mutant nucleotide in the 3 'base. Two separate reactions are carried out using one of these primers and a second common initiator. The amplification is detected in the reaction containing the matched primer. EXAMPLE 4 CHARACTERIZATION OF THE WRN GENE AND GENE PRODUCT The 2 kb WRN cDNA hybridizes to a 6.5 kb RNA and a less abundant 8 kb RNA on a Northern graph. , suggesting that a full length coding region is approximately 5.2 kb long. An overlapping cDNA clone has been isolated which extends the sequence by 2 kb. The insert of this clone is used to test cDNA libraries in order to identify other clones that contain the 5 'end of the cDNA or full length sequence. The alternative separation events are detected by sequencing the complete cDNA sequence from a number of different tissues, including fully differentiated cells and support cells and the full scale transcripts of genes identified by sequence comparison. Additional exons are identified as above by additional genomic sequencing and GRAIL analysis. The predicted amino acid sequence is shown in the Figures 2B and 3. Figure 2 shows cDNA and predicted amino acid sequences of the WRN gene. Figure 3 presents cDNA and predicted amino acid sequences from a less abundant transcription of the WRN gene. The longest open reading frame is shown from the first methionine in that frame. The predicted WRN protein consists of 1,432 amino acids divided into three regions: an N-terminal region, a central region containing 7 motifs (I, la, II, III, IV, V and VI) characteristic of the helicase DNA and RNA superfamily. (Gorbalenya et al., Nucleic Acid Res. 17: 4713, 1989) and a C-terminal region (Figure 8). Unlike the central region, the N-terminal and C-terminal domains of the predicted protein show no amino acid identity to other helicases or to any previously described protein. Because many heiicasas function as part of a multi-protein complex, the N-terminal and / or C-terminal domain may contain interaction sites for these other proteins while the central helicase domain functions in the actual duplex enzymatic unwinding of DNA or RNA. The N-terminal region, encompassing approximately codons 1 to 539, is acidic; there are 109 aspartate or glutamate residues, including an extension of 14 acid residues in a sequence of 19 amino acids (codons 507-526). The extensions of acid residues are found in a helicase of group B complementation of Xeroderma pigmentosum (XP), the helicase of Bloom Syndrome and the helicase of the mental retardation syndrome α-thalassemia linked to the X chromosome. In the WRN gene, this region also contains a random duplication of 27 amino acids in which each copy is encoded by a single exon. Because this duplication is exact at the nucleotide level and because the flanking intron sequences for the two exons encoding the duplication are also highly similar, it is assumed that this duplication is the result of a relatively recent event. The duplicated regions are also highly acidic with 8 glutamate or aspartate residues of 27 amino acids and only 2 basic amino acids (a histidine residue and a lysine residue). The central region of the -WRN gene, extending approximately codons 540-963, is highly homologous to other helicases from a wide range of organisms including the ReqQ gene of E. coli, the SGS1 gene of S. cerevisiae, a predicted helicase ( F18C5C) of C. elegans, and several human helicases. Therefore, by sequence similarity, the WRN gene is a member of a DNA superfamily of DExH-box and RNA helicases. The observed sequence principle consists of 7 motifs found in other helicases. These motifs include a predicted nucleotide binding site (motif I) and an Mg2 + binding site (DEAH sequences, il motif). Some or all of the 7 motifs are supposed to form an active enzymatic site for the unwinding of DNA / RNA. The presence of the DEAH sequence and an ATP binding motif further suggests that the product of the WRN gene is a functional helicase. The C-terminal end of the WRN gene, from codons 964 to 1432, has limited identity to other genes. The only identity identified is a loss of similarity of E. coli from the ReqQ gene and C. elegans gene F18C5.2. EXAMPLE 5 I DENTIFICATION AND DETECTION OF MUTATION IS IN THE WRN GENE WRN mutations or polymorphisms can be identified by several methods, including sequence analysis. Although any cell (different erythrocytes) can be used to isolate nucleic acids, peripheral blood mononuclear cells (PBMC) are preferred. Peripheral blood mononuclear cells are obtained by venous puncture and subsequent hypotonic lysis of erythrocytes. The RNA is island and the cDNA synthesis of the first strand is carried out using a Strat-script computer of PCR-TI according to the instructions of the manufacturer (Stratagene, La Jolla, part numbers 200347 and 200420). Three fragments of RCP-TI were amplified using a RCP LA ver device. 2, using pH buffer solution containing 1.5 mM Mg + 2 (TaKaRa Shuzo Co., Ltd., Japan, part number RR013A). Nested CPR is performed. In this reaction a second PCR is carried out using a pair of primers within the sequence amplified by the first PCR reaction. The cyclization conditions for each amplification are: 10 minutes at 95 ° C, 35 cycles of 1 minute at 60 ° C, 1 minute at 72 ° C and 1 minute at 95 ° C, followed by 7 minutes at 72 ° C on a Perkin-Elmer 9600 PCR machine. The amplified fragments are purified using 96-well plate rotating columns (Wang et al., Anal. Biochem. 226: 85-90, 1995). The A DN sequence was determined using an FS dye terminator sequencing device (Applied Biosystems Division of Perkin Elmer) and the specific primers described below. An automatic Applied Biosystems ABI373A DNA sequencer is used to determine the sequence. The amplified fragments and appropriate primers are listed in Table 1, and the sequences of the primer are listed in Table 2. The DNA sequences are aligned with the known sequence (Figure 2A) using the program Sequencer (Gene Codes). , Michigan) to identify any discrepancies between the patient samples and the reference sequence. Table 1 PCR and sequence primers Table 2 Sequence of Initiators B 5 '-CTTTATGAAGCCAATTTCTACCC (SEQ ID No.2) J 5 '-TTGCCTAGTGCAATTGGTCTCC (SEQ ID No.10) L 5' -CCTATTTAATGGCACCCAAAATGC (SEQ ID No.12) M 5 '-CAGTCTATGGCCATCACATACTC (SEQ ID No.13) N 5 '-ACCGCTTGGGATAAGTGCATGC (SEQ ID No.14) O 5 '-GAGAAGAAGTCTAACTTGGAGAAG (SEQ ID No.15) P 5 '-TTCTGGTGACTGTACCATGATAC (SEQ ID No.16) Q 5 '-CCAAAGGAAGTGATACCAGCAAG (SEQ ID No.17) R 5 '-ACAGCAAGAAACATAATTGTTCTGG (SEQ ID No.18) 5EA 5 '-GAACTTTGAAGTCCATCACGACC • (SEQ ID No.19) 5EB 5 '-GCATTAATAAAGCTGACATTCGCC (SEQ ID No.20) 5EC 5' -CATTACGGTGCTCCTAAGGACATG (SEQ ID No.21) 5ED 5 '-GATGGATTTGAAGATGGAGTAGAAG (SEQ ID No.22) 5EE 5 '-TGAAAGAGAATATGGAAAGAGCTTG (SEQ ID No.23) 5EH 5 '-CATTGGGAGATAAATGCTCAGTAGA (SEQ ID No.80) 5EJ 5 '-AGATGTACTTTGGCCATTCCAG (SEQ ID No.81) 5EK 5 '-GCCATGACAGCAACATTATCTC (SEQ ID No.82) 5EL 5 '-CTTACTGCTACTGCAAGTTCTTC (SEQ ID No.83) 5EM 5 '-TCGATCAAAACCAGTACAGGTG (SEQ ID No.84) 5EN 5 '-GCAGATGTAGGAGACAAATCATC (SEQ ID No.85) 5EO 5 * -TCATCCAAAATCTCTAAATTTCGG (SEQ ID No.86) 5EP 5 '-CTGAGGACCAGAAACTGTATGC (SEQ ID No.87) 5ES 5 '-GCTGATTTGGTGTCTAGCCTGG (SEQ ID No.88) 5ET 5 '-TGCCTGGGTTGCAGGCCTGC (SEQ ID No.89) 5EX 5 '-TTGGAAACAACTGCACAGCAGC (SEQ ID No.90) 5E1 5'-GATCCAGTGAATTCTAAGAAGGG (SEQ ID No.91) EJ EM PLO 6 ISOLATION OF GENOMIC ICO Q U E CON T E N SYN D RO ME OF WERN ER To facilitate the mutational analysis of the WRN gene, the structure of introns-exons was determined. The WRN gene is located in the genomic sequence of the clone of P1 2934. However, this clone only contains the 3 'end of the gene (exons 21 to 35). Genomic clones containing the 5 'end are obtained from a specific cosmid bank for 8 chromosomes LA08NC01 (Wood et al., Cytogenet, Cell Genet, 59: 243, 1992) by screening clones adjacent to clone P1 2934. In summary, this bank it is arranged for PCR screening as described in Amemiya et al. (Nucí Acids Res. 20: 2559, 1992). Cosmids containing WRN are identified using groups of primers 5E6 / 5EY, 5ED / 5E12 and CD-A / C DB (Table 3), which are derived from the WRN DNA sequence (Figure 1; Gen Bank Access No. L76937). Four advance steps produced cosmids 193B5, 1 14D2, 78D8 and 194C3, which contained the remaining exons. The primers derived from WRN cDNA were used for the initial sequence analysis of the cosmid clones. The resulting sequence (Figure 5) is compared to the cDNA sequence to identify introns-exon boundaries. The sequencing primers are then designed from the introns sequences to obtain sequences in the reverse direction and to obtain the second limiting factor that defines the binding of introns-exons. This strategy is used to define the exons not present in clone 2934 of P 1.

Table 3. Initiator sequence and PCR conditions for WRN analysis Region Sequence of Initiator Size of g + 2 pH Product (mM) (bp) ~ domain-N 5E6 d'-GATATTGTTTTGTATTTACCCATGAAGAC (SEQ ID No.164) 106 15 8 ~ 3 ~ 5EY 5 ^ TCCGCTGCTGTGCAGTTGTTTCC (SEQ ID No.165) 5ED domain 5'-GATGGATTTGAAGATGGAGTAGAAG (SEQ ID No.22) 158 2.0 8.3 central 53125'-TCAGTAGATTTATAAGCAATATCAC (SEQ ID No.166) clominio-C CD-A 5'-CTGGCAAGGATCAAACAGAGAG ( SEQ ID No.167) 144 2.0 8.3 CD-B 5'-CTTTATGAAGCCAATTTCTACCC (SEQ ID No.168) The Fixed temperature was 160 ° C for all groups of initiators. Table 4 presents a summary of the structure of the genomic WRN gene. The first column identifies the exon, the second column indicates the base numbers of the cDNA that is derived from the exon, the third column denotes the size of the exon in bp, the fourth column shows the sequence of the borders with sequences of introns in the letters of lower squares and sequences of exons in the letters of the upper squares, the fifth column shows notable features of the exons. Table 4 Structure of Intron-Exons of the WRN Gene Exon Location Size of Intron-Exon Limit Sequences Characteristics of Exon cDNA (pb) exons I 1-155 >; 155 5'UTR TTCTCGGggtaaagtgtc (SEQ ID No 169) 156-327 172 tacctctcagTTTTCTTT AAAGAAAGgtatgttgtt codond'UTR ATG (SEQ ID No 170) 328-440 113 taaactcaagGCATGTGT GATATTAGgtaagtgatt (SEQ ID No 171) 441-586 146 ctcactttagCATGAGTC CATGTCAGgttggtatct (SEQ ID No 172) 587-735 149 aatgttacagTTTTTCCCATAAAAAGgtaaaagcaa (SEQ ID No. 173) 736-885 150 tcatttctagCTGAAATG ATGCTATgtacgtgctt (SEQ ID No. 174) 886-955 70 ttttttatagGCTGGTTT AAATAAAGgtatgttaag (SEQ ID No. 175) 956-1076 115 ttccccctagAGGAAGAA CCACGGAGgttaaatatt (SEQ ID No. 176) 1071-430 ttttttttagGGTTTCTA CTACTGAGgtactaaaat 1500 (SEQ ID No. 177) 10 1501-81 ttttttaaagCATTTATC TGCTTAAGggtatgttta duplicate exon 1581 (SEQ ID No. 178) 11 1582-81 ttttttaaagCATTTATC TGCTTAAGggtatgttta duplicate exon (SEQ ID No. 179) 1662 2 1663- 145 aaactttcagTCTTTAGA TGATAAGGgtaagcactg (SEQ ID No 180) 1807 3 1808-76 ttatttccagACI '1 i IG TTTAAACCgtgagtataa (SEQ ID No 181) 1883 4 1884- 68 caccttcaagAGTTCAGT GGCAAC "rGgtaagttgta Reason for hel icasa (SEQ ID No. 182) 1951 (5 'end) 5 1952- 109 tcatttcaagGATATGGA CAGCTTAAgtaagtcatg Helicase motif (SEQ ID No. 183) 2060 (3' end) and 6 661-69 cttcttatagAATGTCCA ATTAAATTgtgagtaatt (SEQ ID No. 184) 2129 Exon Location Size of Intron-Exon Limit Sequences Characteristics of Exon cDNA (pb) exons 17 2130-83 gtttttacagAGGTAAAT TGATATTGgtaagtgata 2212 (SEQ ID No. 185) 18 2213- 107 ttttttacagGTATCACG TGCCAATGgtaagctttg Elicase Reason 2319 (SEQ ID No. 186) II 19 2320- 185 catcattcagGTTCCAAT AAAACAAGgtaaggattt Reason for helicase (SEQ ID No. 187) 2504 llf 20 2505- 175 ttttctttagTTCCCACT AAATTCAGgtatgaggat Reason for helicase 2679 (SEQ ID No. 188) IV 21 2680- 182 ttgttctcagTGTGTCAT TTAAATAGgtaaaaaaaa Reason for helicase (SEQ ID No. 189) 2861 V and I 22 2862- 102 taatcgacagGCACCTTC AGGAGACAgtatgtatta 2963 (SEQ ID No. 190) 23 2964-93 tcttgggtagAATCATCT AGGTCCAGgtaaagattt 3056 (SEQ ID No. 191) 24 3057- 142 ttttatttagATTGGATC GAGGATCTgtaagtatat 3198 (SEQ ID No. 192) 25 3199- 171 ctaatttcagAATTCTCA CGAAAAAGgtaaacagtg (SEQ ID No. 193) 3369 26 3370-95 cttttaatagGGTAGAAA CTGCCTAGgttcattttt 3464 (SEQ ID No. 194) 27 3565-76 tattttttagTTCGAAAA AGAAGAAGgtttgtttta (SEQ ID No. 195) 3540 28 3541-74 ttaaatgcagTCTAACTT AAAAAAAGgtacagagtt (SEQ ID No. 196) 3614 29 76 aatattttagTATCATGG 3615- AGACTCAGgtaaggcttt (SEQ ID No. 197) 3690 30 3691- 113 ttttgttcagATTGTGTT AAAATGAGgtaaactatc (SEQ ID No. 198) 3803 31 3804- 115 ttaaacacagACCAACTA GTGTTCAGgtaaaatact (SEQ ID No. 199) 3918 Exon Location Size Limits Sequences of Introns-exons Characteristics of cDNAs from Exon exons (Pb) 32 3919- 132 aattctgtagACAGACCT .... TGCCTTTGgtaagtgtga 4050 (SEQ ID No. 200) 33 4051- 163 ctttctctagAAGAGCAT .... CAACTCAGgtgagaggca 4213 (SEQ ID No. 201) 34 4214- 209 tcgtttacagATATGAGT .... ATACTGAGgtattaatta (SEQ ID No. 202) 4422 35 4423- 768 tttcctacagACTTCATC .... Codon TAA 3 '(SEQ ID No. 203) 5190 Note. The exons are in the upper box and the sequences of introns are in the letters in the lower box. As shown before, WRN contains a total of 35 exons ranging in size from 68 bp (exon 14) to 768 bp (exon 35). The coding region begins in the second exon (Table 2). As previously observed, there is a region duplicated in the WRN cDNA sequence that is 27 amino acids in length. This duplication was exactly maintained at the level of nucleotides in the cDNA. At the genomic level, the duplicated sequences were present as two exons (exons 10 and 11), each exon containing only the duplicated nucleotides. The intron sequences adjacent to these 2 exons are also highly conserved, suggesting that a relatively recent duplication event responds to these repeated exons. In addition, since intron and surrounding sequences were conserved, it was not possible to design primers that could specifically amplify exons 10 and 11. The helicase region of the WRN gene is contained in exons 14-21. The helicase motif 1 is the division between exons 14 and 15 while the remaining motifs are each in an individual exon (Table 4). This region, from codon 569 to 859, has sequence similarity for the helicase motifs of 7 assignments. In addition, although the sequences between the motifs are not conserved, the separation is very similar-in the genes of a wide scale of species. For example, helicase domains in RecQ gene of E. coli are in an extension of 288 amino acids purchased with 291 amino acids for the WRN gene. EXAMPLE 7 IDENTIFICATION OF MUTATIONS Initially, 4 different mutations in the C-terminal domain of WRN were identified. These mutations counted more than 80% of the patients with Japanese with SW examined. The 4 mutations are in the region of the C-terminal domain of WRN and the resulting predicted protein contained in the intact Helicase domain. Subjects with additional SW were screened to identify additional mutations. The genomic structure information is used to design PCR primers to amplify each exon, which is subjected to DNA sequence analysis. Five additional mutations of WRN are described; 2 are located in the consensual helicase motifs and another 2 are predicted to produce truncated proteins without the helicase domains. These mutations suggest that at least some subjects with SW, the enzymatic helicase activity is destroyed and supports that the complete loss of the function of the product of the WRN gene causes the Werner Syndrome. Although any cell can be used to isolate DNA, CMSP is preferred. As before, PBMC are obtained by venous puncture and subsequent hypotonic lysis of erythrocytes. CMSP is smoothed by the addition of detergent, using 0.5% - NP-40, 0.5% Triton-X100, or 0.5% SDS. A non-ionic detergent is used in a non-ionic detergent, no further purification of DNA is necessary, but the treatment of proteinase K and the subsequent heat death of the enzyme (95 ° C for 10 minutes) is required. The genomic DNA is amplified according to the PCR conditions recited above using the primers listed in Table 5. Exons 9 and 10 are contained in a DNA region that duplicates. The primer pair of exons 9 and 10 is fixed to the sequences outside of the duplication. The amplified product is analyzed by DNA sequence determination, hybridization with allele-specific probe or other mutation detection method. When the DNA sequences are determined, the sequence of the amplified exon is aligned with the known sequence (Figure 2A) and any discrepancies between the patient samples and the reference sequence are identified. Table 5 Fragment Sequence of Initiators Size of Mg + 2 pH of CPR product (bp) (mM) exon 1 583 A 5'-AGGGCCTCCACGCATGACGC 1.5 8.3 (SEQ ID No. 92) B 5'-AGTCTGTTTTTCCAGAATCTCCC (SEQ ID No. 93) exon 2 A 5'-CCTATGCTTGGACCTAGGTGTC 339 1.5 8.3 (SEQ ID No. 94) B 5'- GAAGTTTACAAGTAACAACTGACTC (SEQ ID No. 95) exon 3 A d-ACTATAAATTGAATGCpCAGTGAAC 316 1.5 8.3 (SEQ ID No. 96) B 5'-GAACACACCTCACCTGTAAAACTC (SEQ ID No. 97) exon 4 E S'-GGTAAACCACCATACCTGGCC 691 1.5 8.3 (SEQ ID No. 98) F 5 - GTACATATCCTGGTCATTTAGCC (SEQ ID No. 99) exon 5 B 5'-ATTCAGATAGAAAGTACATTCTGTG 369 1.5 8.3 (SEQ ID No. 100) E 5'-GTTAAGAAATACTCAAGGTCAATGTG (SEQ ID No. 101) exon 6 A 5'-GGTtGTATTTTGGTATAACATTTCC 374 1.5 8.3 (SEQ ID No. 102) B 5'-ATATpTGGTAGAGTTTCTGCCAC (SEQ ID No. 103) exon 7 A 5 - CTCTTCGATTpTCTGAAGATGGG 291 1.5 8.3 (SEQ ID No. 104) B d' CCCTAATAGTCAGGAGTGTTCAG (SEQ ID No. 105) exon 8 AS ^ -GGAAAGAAAATGAAAATGTGATCCC 316 4.0 8.3 (SEQ ID No. 106) B 5'-CAGCCTTAATGAATAGTApcpCAC (SEQ ID No. 107) C 5'-ATTGATCTTTTAAGTGAAGGTCAGC exon 9 (SEQ ID No. 108) 668 1.5 8.3 D 5'-CTGCAACAGAGACTGTATGTCCC (SEQ ID No. 109) Fragment Sequence of Initiators Size of Mg + 2 pH. CPR product (bp) (mM) exon 12 A 5'-GCpTCGACAA TTGTAGGCCC 377 1.5 9.0 (SEQ ID No. 110) B 5 '-CCAAACCATCCAAAACTGGATCC (SEQ ID No. 111) exon 13 A 5'-TAACCCATGGTAGCTGTCACTG 285 1.5 8.3 ( SEQ ID No. 112) B 5'-CTGpGCTGTTAAGCAGACAGG (SEQ ID No. 113) exon 14 C 5'-TTGAATGGGACATTGGTCAAATGG 348 1.5 8.3 (SEQ ID No. 114) F 5'-GTAGpGCATTTGTATTTTGAGAGT (SEQ ID No. 115) exon 15 C S'-GTAAAAAGAAATGAAAGCATCAAAGG 246 4.0 8.3 (SEQ ID No. 116) D 5'-TCACCCACAGAAGAAAAAAAGAGG (SEQ ID No. 117) exon 16 A 5'-CAAAAAAGAAAATTGCAAAGAACAGG (SEQ ID No. 118) 282 4.0 8.3 B 5'-CAGCAACATGTAATTCACCCACG (SEQ ID No. 119) exon 17 5 '-GAAGAGACTGGAATTGGGTTTGG 532 1.5 8.3 (SEQ ID No. 120) 5' -ATAGAGTATCATGGGATAAGATAGG (SEQ ID No. 121) exon 18 A 5'-TTCTCCpTGGAGATGTAGATGAG 273 4.0 10 (SEQ ID No. 122) B 5'-TCpCAGCpCTTTACCACTCCCCA (SEQ ID No. 123) exon 19 A 5'-CATGGTGTTTGACAACAGGATGG (SEQ ID No. 124) 396 4.0 9.0 B S'-GTTAAATATGCATTAGAAGGAAAtCG (SEQ ID No. 125) A 5'-ATAAAACCAAACGGGTCTGAAGC exon 20 (SEQ ID No. 126) 342 4.0 8.3 B 5'- AAAAGAAGTATTCAATAAAGATCTGG (SEQ ID No. 127) Fragment Sequence of Initiators Size of Mg + 2 pH CPR product (bp) (mM) exon 21 A 5 '-AATTCCACTTTGTGCCAGGGACT 397 1.5 9.0 (SEQ ID No. 128) B d'-ACTTGGGATACTGGAAATAGCCT (SEQ ID No. 129) exon 22 A 5'-TTTTTATCTTGATGGGGTGTGGG 356 1.5 9.0 (SEQ ID No. 130) B 5'- AAATTCAGCACACATGTAACAGCA (SEQ ID No. 131) exon 23 A 5'-CTGAAGTCAAATAATGAAGTCCCA 360 4.0 8.3 (SEQ ID No. 132) B 5'-GTTTGCTTTCTCATATCTAAACACA (SEQ ID No. 133) exon 24 A 5'-CTTGTGAGAGGCCTATAAACTGG 267 1.5 8.3 (SEQ ID No. 134) B 5'- GGTAAACAGTGTAGGAGTCTGC (SEQ ID No. 135) exon 25 C 5'-GCTTGAAGGATGAGGCTCTGAG 461 1.5 8.3 (SEQ ID No. 136) D 5'-TGTTCAGAATGAGCACGATGGG (SEQ ID No. 137) exon 26 A 5'-CTTGTGAGAGGCCTATAAACTGG 267 1.5 8.3 (SEQ ID No. 138) B 5'-GGTAAACAGTGTAGGAGTCTGC (SEQ ID No. 139) exon 27 A 5 '-GCCAppCTCTpAApGGAAAGG 274 1.5 8.3 (SEQ ID No. 140) B 5 '-ATCTTApCATCTpCTGAGAATGG (SEQ ID No. 141) exon 28 A 5' -TGAAATAGCCCAACATCTGACAG 291 1.5 8.3 (SEQ ID No. 142) B 5'-GATTAATTTGACAGCTTGApAGGC (SEQ ID No. 143) exon 29 A 5 '-TGAAATATAAACTCAGACTCpAGC 303 15 8.3 (SEQ ID No. 144) B 5'-GTACTGATpGGAAAGACApCTC (SEQ ID No. 145) Fragment Initiator Sequence Mg Size + 2 pH of CPR product (bp) (mM) exon 30 A 5'-GATGTGACAGTGGAAGCTATGG 307 1.5 8.3 (SEQ ID No. 146) B S'-GGAAAAATGTGGTATCTGAAGCTC (SEQ ID No. 147) exon 31 A 5'-AAGTGAGCAAATGpGCpCTGG 304 1.5 8.3 (SEQ ID No. 148) B 5"-TCApAGGAAGCTGAACATCAGC (SEQ ID No. 149) exon 32 A 5 '-GpGGAGGAAApGATCCCAAGTC 351 (SEQ ID No. 150) 1.5 8.3 B-TGpGCpATGGGpTAACpGTG (SEQ ID No. 151) exon 33 A 5'-TAAAGGATTAATGCTGTTAACAGTG 360 1.5 8.3 (SEQ ID No. 152) B 5'-TCACACTGAGCATpACTACCTG (SEQ ID No. 153) exon 34 C 5"-GCAAAGGAAATGTAGCACATAGAG (SEQ ID No. 154) 491 1.5 8.3 D S'- AGGCTATAGGCATpGAAAGAGG (SEQ ID No. 155) exon 35 A 5'-GTAGGCTCCCAGAAGACCCAG 406 1.5 8.3 (SEQ ID No. 156) B 5'-GAAAGGATGGGTGTGTApCAGG (SEQ ID No. 157) mutation 7 GD A 5'-ACAGGCCATAGTpGCCAACCC 426 (SEQ ID No. 158) 1.5 9.0 GD D 5'-TGGTApAGAATpCCCpTCpCC (SEQ ID No. 159) DJG RCP-TI 5EE d'-TGAAAGAGAATATGGAAAGAGGCpG (SEQ ID No. 160) 2002 1.5 8.3 B 5 '- CpTATGAAGCCAApTCTACCC (SEQ ID No. 161) A 5'-TCAAAATCAGTCGCCTCATCCC P2934AT1 (SEQ 10 No. 162) 168 2.0 8.3 B 5' -CAATGTATCAGTCAGGGpCACC (SEQ ID No. 163) The fixing temperature was 60 ° C for all initiator groups. Mutations were detected by amplifying WRN exons of the genomic DNA and cycle sequencing directly from the PCR products by sequencing of terminator-dye cycles (Perkin Elmer) and an AB1373 automatic DNA sequencer. Before sequencing, fragments of exons amplified by PCR were purified using a QIAquick 8 PCR purification kit (Qiagen). The resulting sequences were aligned by FASTA (GCG) analysis. The nucleotide differences between SW and controls were subsequently confirmed by sequencing the reverse strand. The reverse transcriptase RCP-based methods (PCR-TI) used to identify some mutations (mutations 1-4 and 9, Table 6) and to confirm the expected consequences of separation-union mutations. The PCR-TI products were synthesized from the mRNA isolated from the lymphoblastoid cell lines (Qiagen Oligotex, Qiagen). Large genomic deletion was detected in genomic DNA using long-scale PCR (Expand Long Stamp PCR System, Boehringer Mannheim). Diagnostic Criteria The patients with SW were from a International Registry of subjects with Werner Syndrome. The diagnostic criteria are based on the following signs and symptoms (Nakura et al., 1994). The cardinal signs are: 1) bilateral characters; 2) dermatological pathology characteristic of a tense skin, atrophic skin, pigmentary alterations, ulcer, hyperkeratosis, regional subcutaneous atrophy) and characteristic features ("bird" face); 3) short stature; 4) paternal consanguinity (3rd or greater cousin) or affected offspring; 5) premature blunting and / or thinning of the scalp; 6) positive urinary hyaluronic acid test at 24 hours, (when available). Additional criteria are: 1) diabetes mellitus; 2) hypogonadism (secondary sexual underdevelopment, decreased fertility, testicular or ovarian atrophy); 3) osteoporosis; 4) osteosclerosis of distal phalanges of fingers of the hands and / or toes (diagnosis with x-rays); 5) soft tissue calcification; 6) evidence of premature arteriosclerosis (eg, history of myocardial infarction); 7) mesenchymal neoplasms, rare neoplasms or multiple neoplasms, 8) voice changes (loud, shrill, or hoarse voice); 9) flat foot. The diagnostic ratings are as follows: "Defined", all the cardinal signs (# 6 when available) and any other 2; "Probable", the first 3 cardinal signs and any other 2; "Possible", cataracts or dermatological alterations and other 4 any; "Excluded", presentation of signs and symptoms before adolescence (it is inappropriate except short stature due to current data on preadolescent growth patterns) or a negative hyaluronic acid test. The family designations are as previously used (Nakura et al., 1994, Goddard et al., 1996, Yu et al., 1996). Mutations in Subjects with SW. The initial screening of the WRN gene was based on the sequence of only the 3 'end to the helicase domains. In this mutation screening, the primers amplify exons 2-35 together with approximately 80 bp of the intron flanking sequence (Table 5). Initially, 9 SW subjects (Caucasian subjects DJG, EKL and FES, and Japanese subjects IB, KO, OW, KUN, WKH and WSF) were screened for mutations. These subjects were selected based on the haplotype analysis that suggested that each subject had a different mutation (Yu et al., 1994. Goddard et al., 1996). A total of 30 Japanese subjects and 36 Caucasian subjects were finally screened for each mutation by DNA sequence analysis of the appropriate exon. Table 6. Summary of WRN Mutations Codon Exon Mutation Commentary Sequence Type Mutation Length Nucleotides The Predicted Protein None 1432 1 1165 30 substitution CAG (Gln) to TAG nonsense 1164 (terminator) 2 1305 33 substitution CGA (Arg) to TGA nonsense 1034 3 1230 32 4 bp gtag-ACAG to gt- 4 bp deletion to 1247 deletion AG the splice-donor site 1047-24 substitution tag-GGT to tac-GGT substitution to 1060 1078 splice-donor site 369 9 substitution CGA (Arg) to TGA nonsense 368 (terminator) 22 substitution CGA (Arg) to TGA nonsense 888 (terminator) 759- 20 substitution CAG-gta to CAG-tta substitution to site 760 816 splice-receiver 389 1 pb AGAG (Arg) to GAG change of frame 391 suppression (Glu) ) 697- 19- deletion deletion 1186 942 23 (> 15 kb) genomic Table 7 Mutation Status of Subjects with SW1 The diagnostic classification is as previously described (Nakura et al., 1994). Diagnostic Categories: DDefinida, pProbable; "Possible; 'Insufficient Data The country of origin (ethnic group) of the non-Japanese subjects are: aG00780, USA (Caucasian), AG04103, USA (Caucasian), CTA, England (India, East Africa, Asia), CP3, France (Caucasian), DJG, Germany (German), EKL, Switzerland (German), FES, Germany (German), NF, France (Caucasian), SUG, USA (Caucasian), SYR, Syria (Syrian), AG04103 and AG00780 were obtained as cell lines of Aging Cell Replacement (Camden, New Jersey). Bowl new mutations of SW were detected in the WRN gene (designated 5-9)., Table 6). Two of the mutations (5 and 6) were single-base substitutions creating nonsense codons. Mutation 5 results in a transition from C? T by changing an Arg to a stop codon (Table 6, Fig. 6). The predicted protein is truncated at 368 amino acids, excluding the helicase region, which begins at codon 569. Three Japanese subjects and 3 Caucasians were homozygous and 1 Japanese and 4 Caucasians were heterozygous for this mutation (Table 7). Mutation 6 is also a transition from C? T by changing an Arg to a nonsense codon. One Caucasian SW subject was homozygous for this mutation one second was a compound heterozygote. The expected protein product is 888 amino acids. A third substitution mutation (mutation 7) was a change of G? T at a division-receptor site, generating a truncated mRNA without exon 20 and a WRN protein prematurely terminated at amino acid 760. A single Japanese subject with WS He was homozygous for this mutation. Two deletions were observed. One (mutation 8) is a deletion of 1 bp at codon 389 resulting in a frame change and an ionality of 391 of the predicted truncated protein. This mutation is found in a Caucasian patient as a heterozygote. The second (mutation 9) is a much larger deletion. Two deletions were observed. One (mutation 8) is a deletion of 1 bp at codon 389 resulting in a frame change and a predicted truncated protein of 391 amino acids in length. This mutation is found in a Caucasian patient as a heterozygote. The second (mutation 9) is a much larger deletion. This deletion was first observed in PCR-TI experiments when two different PCR-TI products were obtained from RNA prepared from the DJG subject. The PCR-TI products produced by the 5EE and B primers (Table 5) produced two different products, one with the expected size of 2009 bp and a second shorter product of about 700 bp smaller. The DNA sequence of the shorter product revealed that exons 19 to 23 were missing. To further establish the nature of this mutation, the primers (exon 18A and exon 24A, Table-5) derived from the exons flanking this potential coarse suppression (exons). 18 and 24) were used to amplify genomic DNA from the DJG subject using a large-scale PCR protocol. A single 5 kb fragment was observed corresponding to the shorter PCR-TI product (The normal fragment was not observed, which is calculated to be >20 kb) The complete DNA sequence of this 5 kb fragment was determined and contained the expected 3 'and 5' ends of exons 18 and 24, respectively. The exonic sequences were separated by Itronic sequences adjacent to the 3 'and 5' ends of exons 18 and 24, respectively. No sequences of exons 19-23 were found in the 5 kb fragment. In other subjects and controls, the intron sequence in the 3 'intron to exon 18 contained 531 bp of the single sequence followed by a repeat element of Alu of 241 bp. Likewise, for the region 5 'to exon 24, there is an Alu repeat element separated from exon 24 by 3,460 bp of unique sequence. The 4938 bp fragment of subject DJG contained these intron flanking sequences of single exons separated by a single Alu element. Therefore, presumably this deletion occurred due to a recombination error in 2 highly homologous Alu elements within the WRN gene. A group of primers, GD-A and GD-D (Table 5) was designed to specifically amplify a short fragment (426 bp) through this junction. It was shown that only one additional Caucasian SW patient, SUG, contains this genomic deletion. The additional PCR amplification of the exons within this suppressed region demonstrated that both DJG and SUG are heterozygous for this mutation. Origins of WRN Mutations. Because multiple subjects have the same mutation and because the same mutation was observed in different ethnic groups, at least some of the mutations probably originated in common founders. Evidence from a common founder was examined using 2 short random repeat polymorphisms (PRAC) within the WRN gene. These PRAC, D8S2162 and p2934AT1, were isolated from the same clone of P1 (p2934) and are within 17.5 kb of each. While D8S2162 is not particularly polymorphic (heterozygosity = 54% in Japanese and 70% in Caucasians) and is mainly a 2-allele system (alleles of 140 and 142 bp), p2934AT1 is highly polymorphic (heterosigosity = 78% in both Japanese populations). as of Caucasians). For mutation 4, which has only been observed in Japanese subjects, all but 1 subject had the haplotype of D8S2164 / p2934AT1 of 140-148 (Table 8). The only exception, JO2, had the 140-150 haplotype, with the allele of p2934AT1 being 2 bp different from the 148 bp allele observed in other subjects with mutation 4. This difference of 2 bp could be the result of a 2bp mutation , as was commonly observed at the PRAC dinucleotide repeat site (Weber and Wong, 1993). The haplotype data agree with a common Japanese founder and agree with the ligation imbalance observed in the same Japanese subjects for other markers in the WRN region (Yu et al., 1994; Goodard et al., 1996). For mutations 2 and 5, in the Japanese, the 896R18-p2934AT1 haplotype for the small number of available subjects, agree with common founders for each mutation. However, non-Japanese subjects with mutations 2 and 5 have discordant p2934AT1 genotypes when compared to Japanese subjects with the same mutations. These results do not support a common founder for Japanese and non-Japanese subjects with mutations 2 and 5. Within non-Japanese subjects, for mutation 5, there may be as many as 3 different founders since in both cases, different subjects with mutation 5 do not agree with p2934AT1 (eg, compare AG00780 for EKL). It should be noted that the absence of evidence for a common founder does not necessarily exclude the possibility of a single mutational event of origin. The recombination and / or intragenic mutations creating new alleles at 2 PRAC sites, over time, could obscure the origins of the different WRN mutations. Table 8. PRAC genotypes in the WRN1 gene 1 Genotype data for HH, SK were not available. ST, TH, TK and ZM. For y896R18, alleles in bp (frequency for Caucasians, frequency for Japanese) were as follows: 136 (0.030, 0.025), 138 (0.020, 0.010); 140 (0.460, 0.576); 142 (0.337, 0359), 144 (0.084, 0.010); 146 (0.010); 148 (0.009, 0.010); 150 (0 059, 0). For p2934AT1, alleles in bp (frequency of Caucasians, frequency of Japanese) were as follows: 114 (0.006 0), 122 (0, 0.009); 124 (0.011, 0); 128 (0253, 0.079); 130 (0.020), 132 (0.006, 0.009); 134 (0.046, 0.096); 136 (0.086, 0.009); 138 (0.011, 0); 140 (0.034, 0); 142 (0.052, 0.035); 144 (0.023, 0.061); 146 (0.023, 0.053); 148 (0.034, 0.132); 150 (0.098, 0.070); 160 (0.046, 0.018); 162 (0.029, 0.009); 166 (0, 0.009); 168 (0, 0.009). The 5 mutations identified showed that SW mutations are not restricted to the 3 'end of the gene, but are also found in other regions of WRN. In addition, mutations 5 and 7-9 interrupt each any part or all of the helicase region. Therefore, subjects with SW homozygous for this mutation will completely lack the domains of the WRN heiicase as well as the 3 'end of the protein. Although there is a possibility that the truncated 368 amino acid protein has some partial remaining function, mutation 5 probably results in the complete loss of all WRN protein activity. However, the SW phenotype in these subjects is not appreciably different from the SW phenotype generated by other mutations described herein. Therefore, all mutations in the SW gene may be the complete loss of function mutations. EXAMPLE 8 Identification of the Mouse WRN Gene This WRN cDNA from mice was isolated by screening a cDNA library of splenocytes from mice with low restriction with human WRN cDNA as a probe. The DNA sequence of mice is presented in Figure 9. The homology between the WRN cDNA sequence of humans and mice is about 80%. At the amino acid level, the product of the WRN gene of humans and mice show an identity of approximately 90%. Notably, the repeated exon in human WRN cDNA (exons 10 and 11) is only present once in WRN cDNA from mice. The WRN clone of genomic mice was isolated using WRN-specific primers from mice to screen genomic BAC from mice. The genomic DNA sequence was presented in Figure 6. The genomic DNA sequence was presented in Figure 7 and the SEQ ID Nos .: 207-209. The DNA sequence was presented in Figure 6 and SEQ ID NOS: 205 and 206. EXAMPLE 9 Location of the WRN Gene Product A rabbit polyclonal antiserum raised for a peptide of the WRN gene product was used as an analysis of Indirect immunofluorescence to determine the intracellular localization of the WRN protein. A rabbit polyclonal antiserum arose for the peptide Phe-Pro-Gly-Ser-Glu-ile-Cys-Ser-Ser-Ser-Lys-Arg (FPGSEEICSSSKR) (SEQ ID NO: 204) by normal methods (see Harlow and Lane, Antibodies, A Laboratory Manual, CSH Press, Cold Spring Harbor, 1989, Current Protocols in Immunology, Greene Publishing, 1995). The peptide corresponds to residues 1375 to 1387 of the WRN polypeptide.

The cells, such as epithelial cells develop on a plastic or glass surface, fixed with 3% of formaidehyde and permeabilized for 2 minutes with a pH buffer containing 0.5% Triton X-100, 10 mM PIPES, pH 6.8, 50 mM NaCl, 300 mM sucrose and 3 mM MgCl2 (see for example, Fey et al., J Biol. Chem, 98: 1973, 1984). The cells are then stained for 20 minutes with a suitable dilution of the antipeptide antibody (1: 1500), washed, stained with a suitable second antibody (e.g., goat anti-rabbit antibody conjugated to FITC), washed and mounted for visualization by fluorescence microscopy. The control stains include bis-benzoimidine (Sigma, St. Louis, MO), which stain DNA and phalloidin (Molecular Probes, OR, BODIPY 558/568 phalloidin) that stain filamentous actin. As seen in Figure 9, the product of the WRN gene is almost completely localized in the nucleus. Nuclear staining is easily observed in the epithelial cells on the lower left in panel A. These cells are near the periphery of the expanding clone of human prostate epithelial cells. Cells that do not divide rapidly (eg, cells closer to the center of the clone), such as those seen in the upper right of panel A, stain in the cytoplasm and nucleus. The location and size of the nuclei in these cells are shown by staining DNA with the interspersed dye bis-benzoimidine (Hoeschst 33258), panel B. The overall size of the cells and in some cases the key cyclokeletal characteristics are revealed by F-staining. -actin as shown in panel C. EXAMPLE 10 Isolation of a Protein that binds to the WRN Gene Product An interaction screen of 2 yeast hybrids (Hollenberg et al., Mol Cell Biol. 13: 3813, 1995) it was used to identify and isolate a cellular protein that binds to the 443 amino acids terminated in carboxyl (residues 990 to 1432) of the WRN gene product. A bank of 1.1 x 106 independent cDNA clones generated from RNA isolated from stimulated human peripheral blood mononuclear cells were generated in pACT-2 (Clontech, Palo Alto, CA) that creates fusions of cDNA / GAL4 activation domain are cotransfected in yeast containing pLEXA with the fragment of the WRN gene to generate fusion of WRN / LEXA DNA. The host yeast cells, L40, develop in the medium lacking leucine, tryptophan and histidine and containing 4 mM of 3AT, a catabolite toxic to histidine. 67 colonies developed in this medium. Of these, 60 were cured of the pLEXA plasmid by development in the medium containing cycloheximide and matched with the yeast strain expressing a "sticky" laminin fusion and the GAL4 activation domain. 19 clones did not activate the sticky protein and underwent DNA sequence analysis. Of these, 6 sequences that are not equal to any sequence in GenBank were followed by BLAST research. Two different clones encoded carnitine palmitoyl transferase I and prolyl 4-hydroxylase B subunit. Six independent clones encoded a 70K component of the Ul snRNP complex (GenBank, Access No. M22636). In addition, all six were derived from the RNA recognition motif region of the 70K protein. From the foregoing, it will be appreciated that, although the specific embodiments of this invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except by the appended claims.

Claims (2)

1. CLAIMS 1. An isolated nucleic acid molecule that encodes a product of the WRN gene.
2. 2. A nucleic acid molecule isolated from the group consisting of: (a) an isolated nucleic acid molecule as shown in the Figures or complementary sequence thereof; (b) an isolated nucleic acid molecule that specifically hybridizes to the nucleic acid molecule of (a) under conditions of high restriction; and (c) an isolated nucleic acid encoding a product of the WRN gene. 4. The expression vector according to claim 3, wherein said promoter is selected from the group consisting of the CMV IE promoter, SV40 early promoter and MuLV LTR 5. The expression vector according to claim 3, wherein The promoter is a specific promoter for tissue 6. A viral vector capable of directing the expression of a nucleic acid molecule according to claims 1 or 2. 7. The viral vector according to claim 6, wherein the vector is select from! group consisting of viral vectors of herpes simplex, adenoviral vectors, vectors associated with adenovirus and retroviral vectors 8 A host cell carrying a vector according to any of claims 3 to 7 9. The host cell according to claim 8, wherein said cell is selected from the group consisting of human cell, dog cell, monkey cell, rat cell and mouse cell. An isolated protein comprising a product of the WRN gene 11 An antibody that specifically binds to the protein according to claim 10 The antibody according to claim 11, wherein said antibody is a monoclonal antibody. according to claim 11, wherein said antibody is selected from the group consisting of a Fab fragment, an Fv fragment and a single chain antibody. A hibpdoma capable of producing an antibody according to claim 12. of nucleic acid that is capable of specifically hybridizing to a WRN gene under conditions of high restriction 16 A pair of primers capable of specifically amplifying all or a portion of a nucleic acid molecule according to any of claims 1 or 2 17. A transgenic animal whose germ cells and somatic cells contain a WRN gene that is operably linked to an effective promoter for the expression of said gene, said gene being introduced into said mouse, or an ancestor of said mouse, in an embryonic stage. 18. The transgenic animal according to claim 17, wherein the animal is selected from the group consisting of a mouse, a rat and a dog. 19. The transgenic animal according to claim 17, wherein WRN is expressed from a vector according to any of claims 3 to 7. 20. An agonist of a product of the WRN gene. 21. An antagonist of a product of the WRN gene.