WO1999002684A1

WO1999002684A1 - Methods of identifying and screening genes associated with increased longevity and slowed aging

Info

Publication number: WO1999002684A1
Application number: PCT/US1998/014223
Authority: WO
Inventors: Shin Murakami; Thomas E. Johnson
Original assignee: University Technology Corporation
Priority date: 1997-07-09
Filing date: 1998-07-09
Publication date: 1999-01-21
Also published as: AU8296198A

Abstract

The present invention provides methods of identifying polynucleotides and polypeptides associated with life extension in a transgenic nematode system. The invention also provides screening methods using the polynucleotides and polypeptides thus identified, including tkr-1, a kinase receptor gene from C. elegans.

Description

METHODS OF IDENTIFYING AND SCREENING GENES ASSOCIATED WITH INCREASED LONGEVITY AND SLOWED AGING

CROSS-REFERENCE TO RELATED APPLICATIONS This applications claims the benefit of U.S. Provisional Application No. 60/051,983, filed July 9, 1997, which is incorporated herein by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH Research relating to this invention was supported in part by grants from the National Institutes of Health (NIH) (AG08322, AG08761 and AA00195). The government may have certain rights in this invention.

FIELD OF THE INVENTION The present invention relates to methods of identifying and using genes involved in the aging process. More particularly, the invention provides methods to identify polynucleotides and polypeptides associated with an increased lifespan and/or the slowing of aging in a nematode transgenic system, as well as screening methods using these polynucleotides and polypeptides.

BACKGROUND OF THE INVENTION As the proportion of the older population dramatically increases, age-related phenomena, including diseases and physical manifestations of aging, will become an ever more pressing issue. In the United States, the percentage of elderly individuals (those over 65) is larger than ever: approximately 12.5% versus 8.1% in 1950 and 4.1% in 1900. By the time the last cohort of "baby-boomers" reaches 65 around 2030, the percentage of elderly will be over 20%. However, relatively little is known about the causes of age- related phenomena.

Apart from the ever-present desire to retain "youth", aging is presenting very real problems in terms of diseases and quality of life. Aging-related diseases include cancer, stroke, heart disease, osteoporosis, and diseases of the central nervous systems (including Alzheimer's and Parkinson's disease).

One promising system in which to study the mechanisms of aging is the nematode C. elegans, a self-fertilizing hermaphroditic nematode species that is used in genetic research. The only mutants directly identified on the basis of extended longevity in any metazoan have been isolated in C. elegans. The self-fertilization minimizes inbreeding depression and allows rapid inbreeding, which facilitates the isolation of mutants and genetic analyses (Russell and Jacobson, 1985 In: Handbook of the Biology of Aging (2nd ed.), Finch, C.E. and Schneider, E.L. (Editors), pp. 128-145, Van Nostrand Reinhold, New York; Johnson, 1990 in: Handbook of the Biology of Aging (3rd ed.), Schneider, E.L. and Rowe, J.W. (Editors), pp. 45-59, Academic Press, New York; Johnson and Lithgow, 1992 J. Am. Gerontol Soc. 40, 936-945; Lithgow, 1996 In: Handbook of the Biology of Aging (4th ed.), Schneider, E.L. and Rowe, J.W. (Editors), pp. 55-73, Academic Press, New York).

In the past, two approaches have been used with C. elegans to isolate genetic variants having longer life spans: (1) the production of recombinant-inbred (RI) strains, and (2) the isolation of mutants having extended life spans.

Recombinant Inbred Strains. Only slight progress in isolating long-lived worms has been made from the analysis of RI strains. Ebert et al., 1993 Genetics 135, 1003-1010, analyzed a large population of worms from crosses between the strains N2 and Bergerac that were segregating a variety of age-related genotypes. They carefully maintained polymoφhisms and then examined multiple markers throughout the genomes of individual worms, comparing marker distribution in the longest-lived worms with the distribution of markers obtained from young worms. They found several regions of the genome that were associated with increases in lifespan.

Two studies (Brooks and Johnson, 1991 Heredity 67, 19-28; Shook et al., 1996 Genetics 142, 801-817) have used a series of RI strains derived from the same two parents to study aging. Intermediate levels of heritability (0.05 to 0.36) for lifespan and for fertility were found confirming earlier studies (Johnson and Wood, 1982 Proc. Natl. Acad. Sci. USA 79, 6603-6607; Johnson, 1987 Proc. Natl. Acad. Sci. USA 84, 3777-3781). Quantitative trait loci (QTL) were found for hermaphrodite self-fertility on linkage groups 2, 3, and 4 and QTLs for life span on groups 2 and 4 and the X chromosome. Little evidence was found to suggest that reduced fertility was associated with increased life span, except for a locus on chromosome 2 where a major QTL affected both fertility and lifespan, both in the same direction.

Mutant strains. A second approach to studying the aging processes in C. elegans has been the isolation of long-lived mutants (Klass, 1983 Mech. Aging Dev. 22, 279-286). The first mutant identified (age-1) causes a 70% increase in life expectancy (Johnson, 1990, Science 249, 908-912), maps to the middle of chromosome 2, and has little affect on fertility, length of reproduction, or rate of development (Friedman and Johnson, 1988 Genetics 118, 75-86; Johnson and Lithgow, 1992, supra; Johnson et al.. 1993 Genetica 91, 65-77). The mutant allele slows the exponential rate of increase of the mortality rate (Johnson, 1990, supra). Mutant strains containing age-1 move better at all ages and keep moving until later in life, suggesting that mutations that increase longevity increase overall vitality and "health" (Duhon and Johnson, 1995 J. Gerontol Biol. Sci. 50, B254-B261).

There are other published reports of mutations in C. elegans that result in longer life. (1) Mutations in spe-26 result in life extensions of about 80% for the hermaphrodite and the mated male (Van Voorhies, 1992 Nature 360, 456 458) presumably because of decreased sperm formation. (2) At 16°C and normal oxygen tension, rad-8 mutant worms live almost 50% longer than wild-type worms (Ishii et al., 1994, J. Gerontol. Biol. Sc. 49, Bl 17-B120). Most or all of the increased life span is the result of an extended developmental phase. (3) daf-2 mutants result in a more than twofold extension of mean life span at the permissive temperature (Kenyon et al., 1993 Nature 366, 461-464), and this extension is blocked by the action of the daf-16 mutation, daf-12 mutants interact with daf-2 to cause an almost fourfold increase in mean life span (Larsen et al., 1995 Genetics 139, 1567-1583). These mutations affect the dauer formation pathway. The dauer is an alternative developmental path taken by C. elegans under "hard times" conditions (Riddle, 1988 In: The Nematode Caenorhabditis elegans, Wood, W.B. (Editor), pp. 393-412, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY ). (4) Larsen et al., 1995 Genetics 139, 1567-1583 showed that other dauer mutants also affect length of life: daf-23 doubles the normal adult life span. (5) Mutations in clk-1 and other elk genes alter development, the cell cycle and some alleles prolong life (Wong et al., 1995 Genetics 139, 1247-1259; Lakowski and Hekimi, 1996 Science 272, 1010-1013; Eubank et al., 1997 Science 275, 980-983). These dauer-formation mutants define a signal transduction pathway in which homologs to mammalian genes involved in signal-transduction can be identified, daf-1 codes for a translational product that has homology to a serine/threonine kinase in the raf superfamily and which may be a cell-surface receptor (Georgi et al., 1990 Cell 61, 635- 645). daf-4 is the nematode homolog of human bone moφhogenetic protein (BMP) receptor (Estevez et al, 1993 Nature 365, 644 649). age-lldaf-23 has recently been shown to encode a P13 kinase (Morris et al., 1996, Nature 382, 536-539).

The relationship between age-1 and the other age genes is not completely clear. age-1 daf-16 double mutants have wild-type or shorter life spans similar to daf-16 alone (Murakami and Johnson, 1996 Genetics 143, 1207-1218). The long-life trait of all other mutants described above is suppressed by daf-16, as are the stress resistance effects (see below). These gerontogenes are part of a genetic pathway that shares the daf-16 gene downstream. Three gerontogenes have been cloned, daf-23 phosphatidylinositol-3-OH kinase (Morris et al., 1996, supra.), spe-26/actin associated protein (Varkey et al., 1995 Genes & Develop. 9, 1074-1086) and daf-2. Though controversial, age-1 may be daf-23. It is probable that these gerontogenes negatively regulate stress resistance and life extension. Genes that have an opposite function (positive regulation of stress response and longevity) to these gerontogenes have not been isolated or characterized.

Involvement of Stress Response in Determining Life Span in C. elegans. Larsen (1992) Proc. Natl. Acad. Sci. USA 90, 8905-8909, and Vanfleteren (1993) Biochem. J 292, 605-608 showed that age-1 mutants are resistant to oxidative stress and have elevated levels of superoxide dismutase (SOD) and catalase in late life. Worms with the age-1 mutation survive thermal stress better than do wild type worms (Lithgow et al., 1994 J Gerontol. Biol. Sci. 49 B270-B276; 1995 Proc. Natl Acad. Sci. USA 92, 7540-7544). This phenotype has been called increased thermotolerance (Itt). Itt was mapped to the age-1 interval on chromosome 11 (Lithgow et al., 1995, supra) and was shown to be associated with age-1 in both sterile and nonsterile worms (Lithgow et al., 1994, supra). New age-1 mutants are all Itt, as well as UV -resistant and resistant to reactive oxidants (Duhon et al., 1996 Dev. Genet. 18, 144-153). Other Age strains, Including constitutive dauer mutations, daf-2, daf-28, daf-4, and daf-7 are also Itt (Lithgow et al., 1995, supra). Very recent studies have also shown that all long-lived mutants in C. elegans are UV resistant as well (Murakami and Johnson, 1996, supra). Life-extension mutants must have altered genes specifying the rate-determining process(es) for life span. The observation that repair processes are increased to higher levels in many longevity mutants suggests that these are the rate-determining processes limiting longevity in a variety of species.

A "gerontogene" is a gene that can be altered to slow aging, extend life and enhance late-life health. Genes affecting aging have also been called "longevity assurance genes" (D'mello et al, 1994 J. Biol. Chem. 269, 15451-15459) or "longevity associated genes" by the NIA (McCormick, personal communication) both of which can be conveniently abbreviated using the term "LAG." This term is similar to the term "gerontogene" as used by Rattan (1985) Bioessays 2, 226-228.

These classes of genes are a starting scheme for classifying genes involved in aging and senescence. Null mutants in any of the first three types of genes should result in life extension while a null mutant in the last class should result in life shortening. Notably, all life-extension mutants previously identified in C. elegans result from hypo- or nullo- moφhic mutations.

The longevity genes, or life-extension genes, are of obvious interest and importance, both for their life-extension potential and the possibility of their contributing to the enhancement of the quality of life, particularly later during the lifespan. However, very few of these genes have been identified, and even less is understood about how these genes act to prevent aging and promote life extension.

There is an increasingly pressing need to discover genes whose function is associated with life-extension and/or stress resistance, particularly those genes which positively regulate life extension (i.e., genes in which over-expression confers an increased life span). Such genes and their products could provide the basis for, inter alia, screening for promising anti-aging (including anti-disease) agents.

Throughout this application, various publications, patents, and published patent applications are referred to by an identifying citation. The disclosures of these publications, patents, and published patent specifications referenced in this application are hereby incoφorated by reference in their entirety into the present disclosure. SUMMARY OF THE INVENTION

This invention is based upon the identification of genes (i.e., polynucleotide sequences and the polypeptide sequences encoded therein) which are associated with life extension, particularly those genes which positively modulate life extension. Application of these methods has identified a gene from C. elegans, designated tkr-1, which is associated with life-extension as well as stress resistance.

Accordingly, the present invention provides methods of identifying a polynucleotide associated with longevity comprising measuring lifespan in a transgenic nematode, such as C. elegans, said transgenic nematode comprising the polynucleotide, wherein a polynucleotide is identified as associated with longevity if its expression confers longer lifespan than a suitable control. In some embodiments, the method comprises 1) computer similarity searches for known gerontogenes or genes suspected to be involved in stress resistance or aging, and 2) determining the life-span increasing capability (if any) of the genes in transgenic nematodes. Further provided is a method for identifying genes leading to increased stress resistance in an organism, in particular, heat shock, UV resistance and oxidative resistance. Such genes may increase lifespan in organisms containing the gene or provided with the product encoded thereby.

In another aspect of the invention, the life-extension gene (i.e., polynucleotide) and/or the protein encoded thereby could be used to discover drug candidates that are targeted to the gene, or to the protein encoded thereby, as site-specific interventional agents. Accordingly, the invention provides screening methods in which an agent is identified by its ability to modulate the life-extension polynucleotide and/or polypeptide function(s) (i.e., functional characteristic). Accordingly, the present invention encompasses methods of identifying agents that may have life-extension activity comprising contacting a transfected cell comprising a life-extension polynucleotide identified in the methods described herein or a polypeptide encoded by the life extension polynucleotide with the agent, wherein the agent is identified by its ability to modulate a functional characteristic of a life-extension polynucleotide or polypeptide.

In a further aspect of the invention, a transgenic nematode is provided, wherein the transgene is a chimeric tkr-1 gene. Such chimeric tkr-1 transgenes may comprise, for example, sequences encoding a human FGF receptor kinase domain. Also provided is a transgenic nematode comprising a tkr-1 polynucleotide. A transgenic nematode of the present invention can be used in the screening methods described herein. The present invention also provides a chimeric nematode tkr-1 gene.

Other objects of the present invention will be readily apparent to those of ordinary skill in the appropriate art.

DESCRIPTION OF THE FIGURES

Fig. 1 is a schematic depicting the physical map positions cosmids and YACs initially tested in the transgenic system. Transgenic C. elegans were constructed carrying multiple-copies of the YAC (thick bar) or cosmid DNA (thin bar) in order to over-produce the sequences of interest. The bar indicates DNA clones that confer (black) and do not confer (white) increased resistance to heat. The lower section is an amplified region showing the organization of tkr-1. The abbreviations of the restriction sites are as follows: Bg (Bgl II), Bs (Bstx 7), E (Eco RI), H (Hind III) and Sp (Spe I).

Fig. 2 is a schematic as in Fig. 1, with additional data regarding a frameshift and a deletion tkrl constructs. Parentheses show numbers of independent transgenics per total that display increased resistance to heat, UV and life extension. The frameshift mutant gene, tkr-1 Δkin, lacks the cytoplasmic domain carrying the kinase region of the protein encoded thereby (see Fig. 4 for detail). The truncation mutation, tkr-1 ABB, lacks the whole coding region except for the N-terminal 15 amino acids, as shown schematically in Figure 3.

Fig. 3 is a schematic summarizing all tkr-1 derivative constructs as well as the lifespan and stress (UV and thermotolerance) data. Designations are as in previous figures. tkr-1 FGFR is a construct in which the kinase domain from fibroblast growth factor receptor has been substituted for the kinase domain of tkr-1. The percentage shown represent the increase in mean life span (Life), UV resistance (UV), or thermotolerance (Heat). Abbreviations: TM, transmembrane domain; TK1, kinase domain 1; TK2, kinase domain 2, KI, kinase insert.

Fig. 4 shows the nucleotide (SEQ ID NO: 12) and predicted amino acid sequence (SEQ ID NOS:13-23) of tkr-1. The predicted transmembrane domain is underlined. Four bp of a frameshift mutation was introduced in the Bgl II site resulting in the deletion of a kinase region (tkr-1 Δkin; arrows). The potential autophosphorylation sites are circled. Figs. 5(a) - (c) show the alignment of the amino acid sequences (SEQ ID NOS:24- 81) of the kinase domain of tkr-1 and tkr-2. The tkr-1 nematode gene family members and related mammalian receptor kinases are shown. Identical residues are shown by filled-in boxes with white letters.

Fig. 6 is a sequence comparison of tkr-1 and tkr-2 with human fibroblast growth receptor 1 (FGFR-1) (SEQ ID NOS:82-99). The positions of the predicted signal peptide (amino acids 18-29) and the HRDLALRN motif (amino acids 319-326) specific to the kinase subdomain VIB are shown by a line above and a dotted line below, respectively. A potential cleavage site of the signal peptide is shown by the arrowhead. Predicted autophosphorylation sites are shown by asterisks. Abbreviations: TM, transmembrane domain; TK1, kinase domain 1; TK2, kinase domain 2; KI, kinase insert.

Fig. 7 is a schematic depicting the evolutionary history of kinase domains most closely related to the kinase domain of tkr-1.

Figs. 8 (a) and (b) shows increased survival of tkr 1-1 and tkrl-2 as compared to controls and to tkr-2 (tkr2-l and tkr2-2). Fig. 8(a) shows increased survival after heat shock; Fig. 8(b) shows increased survival after UV exposure.

Figs. 9 (a) and (b) are graphs depicting life extension conferred by the tkr-1 gene (tkr 1-1 and tkrl-2; Fig. 9 (a)) when compared to controls and to tkr-2 (tkr2-l and tkr2- 2(Fig. 9 (b)).

Figs. 10 (a) and (b) are schematic depictions of tandemly clustered tkr-1 family members.

Figs. 11 (a) - (c) are graphs depicting increased thermotolerance, UV resistance and life span of animals expressing the construct encoding the hybrid tkr-1 -FG¥R, containing the kinase domain from human FGF-Rl . Panel a shows that thermotolerance is increased by tkr 1 -FGFR (pO.OOOl). Panel b shows that UV resistance is increased by tkrl-FGFR (p<0.0001). Panel c shows that life is extended by the chimeric construct, tkr- FGFR (pO.OOOl). Each panel also depicts the lack of effect of animals carrying a deletion of the kinase domain (tkr-1 Δkin).

Figure 12 is a graph depicting suppression of the effects of tkr-1 by a reduction of function mutation of da 16. The daf-16(m26) mutation is epistatic to the tkr-1 phenotypes. Fig. 13 is a schematic depicting a construct encoding a tkr-1 -green fluorescent protein (GFP) fusion protein.

Fig. 14 (a) and (b) are graphs depicting the response of transgenic animals carrying the construct encoding a tkr-1 -green fluorescent protein (GFP) fusion protein, shown schematically in Figure 13, after exposure to UV (upper panel) or heat (lower panel). Induction of tkr-1 was measured as the intensity of GFP fluorescence. Starvation also induces expression of the tkr-1 -GVV fusion protein in these animals.

Figure 15 shows an amino acid sequence (SEQ ID NO: 100) of the kinase domain of human FGFR.

DETAILED DESCRIPTION OF THE INVENTION We have discovered an effective method to identify polynucleotides associated with life extension, particularly those polynucleotides which positively modulate life extension (i.e., overexpression positively modulates life extension). This powerful transgenic nematode system can provide polynucleotides which in turn can be used in screening methods to identify agents which may be useful in treating aging-related phenomena and/or delaying the aging process and/or lengthening life span. One such polynucleotide, denoted tkr-1, has been identified. Overexpression of tkr-1, which encodes a putative receptor tyrosine kinase, increases longevity 40-100% (average, 65%) and confers increased resistance to heat and ultraviolet light in transgenic nematodes, and does not alter development or fertility. Unlike previously identified gerontogenes, tkr-1 positively modulates longevity and stress resistance. The tkr-1 polynucleotides described herein share a region of homology with a part of a human gene, FGFR (fibroblast growth factor receptor), and the FGFR kinase domain functionally substitutes for the kinase domain of tkr-1. Polynucleotides and polypeptides identified by these methods may likely function to modulate a variety of other target genes to slow aging. Thus, identification of these polynucleotides and the polypeptides encoded thereby is useful for locating other such similar genes, including those in other organisms such as mammals and including humans.

In another aspect of the invention, the polynucleotides identified as associated with life extension (and/or polypeptides encoded thereby) can be used in screening assays to discover drugs that modify the activity of such polynucleotides, or the polypeptides encoded by these polynucleotides, to produce site-specific interventional agents. In some embodiments, the transgenic nematode may be used for screening, especially the transgenic nematode comprising a tkr-1 polynucleotide or the kinase domain of FGFR..

General Techniques

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook, Fritsch, and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, (F.M. Ausubel et al. eds., 1987); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.); PCR 2: A PRACTICAL APPROACH (M.J. McPherson, B.D. Hames and G.R. Taylor eds., 1995); ANIMAL CELL CULTURE (R.I. Freshney. Ed., 1987) and METHODS IN CELL BIOLOGY, VOL. 48, CAENORHABDITIS ELEGANS: MODERN BIOLOGICAL ANALYSIS OF AN ORGANISM, (H.F. Epstein and D.C. Shakes eds. , 1995, Academic Press, San Diego).

Definitions

A polynucleotide or polypeptide that is "associated with longevity" is one whose function correlates with increased lifespan. "Longevity" and "life-extension", used interchangeably herein, also include delay and/or stabilizing the aging process. Preferably, the longevity is due to an extension of the mature life phase, as opposed to an extension of the immature life phase (i.e., delay in maturity). Such polynucleotides or polypeptides that are found to be correlated with longevity are generically denoted herein as "life-extension" polynucleotides or polypeptides. A "function" of a polynucleotide can be on any level, including DNA binding, transcription, translation, processing and/or secretion of expression product, interaction (such as binding) of expression product with another moiety, and regulation (whether repression or de-repression) of other genes. It is understood that a life-extension polynucleotide or polypeptide includes fragments, or regions, of a polynucleotide or polypeptide, as long as the requisite life-extension phenotype is observed.

The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably to refer to polymeric forms of nucleotides of any length. The polynucleotides may contain deoxyribonucleotides, ribonucleotides, and/or their analogs. Nucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The term "polynucleotide" includes single-, double-stranded and triple helical molecules. "Oligonucleotide" refers to polynucleotides of between about 5 and about 100 nucleotides of single- or double-stranded DNA. Oligonucleotides are also known as oligomers or oligos and may be isolated from genes, or chemically synthesized by methods known in the art.

The following are non-limiting embodiments of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A nucleic acid molecule may also comprise modified nucleic acid molecules, such as methylated nucleic acid molecules and nucleic acid molecule analogs. Analogs of purines and pyrimidines are known in the art, and include, but are not limited to, aziridinycytosine, 4-acetylcytosine, 5- fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl- aminomethyluracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1- methylpseudouracil, 1 -methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, pseudouracil, 5- pentylnyluracil and 2,6-diaminopurine. The use of uracil as a substitute for thymine in a deoxyribonucleic acid is also considered an analogous form of pyrimidine.

If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non- nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications included in this definition are, for example, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g. , acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s).

Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid supports. The 5' and 3' terminal OH groups can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups.

Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, but not limited to, 2'-O-methyl-, 2'-O- allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside.

Although conventional sugars and bases will be used in applying the method of the invention, substitution of analogous forms of sugars, purines and pyrimidines can be advantageous in designing a final product, as can alternative backbone structures like a poly amide backbone.

As used herein, the term "tkr-1 gene(s)" or "tkr-1 " refers to the C. elegans tkr-1 gene. As is understood in the art, the tkr-1 gene includes, not only the coding sequences, but also 5' and 3' flanking sequences, as shown in SEQ ID NO: 1. A "fragment" of tkr-1 is a portion of the tkr-1 gene, and as such may contain coding and/or non-coding sequences. Preferably, a fragment of tkr-1 comprises at least 10 contiguous nucleotides, more preferably at least 15, more preferably at least 25, more preferably at least 30, more preferably at least 50, more preferably at least 100 contiguous nucleotides.

"tkr-1" refers to a protein (polypeptide) product encoded in the C. elegans tkr-1 gene. The sequence of full-length tkr-1 is shown in SEQ ID NO: 2 as well as in Figure 4. A "fragment" of full-length tkr-1 is a portion of the tkr-1 gene product. A "tkr-1 polynucleotide" refers to any of the polynucleotide embodiments described herein based on the tkr-1 gene polynucleotide sequence (SEQ ID NO: 2). A "tkr-1 " polynucletoide also includes fusion or hybrid constructs, such as those described herein. A "tkr-1 polypeptide" refers to a polypeptide product encoded by or within tkr- 1; thus, a"tkr-l polypeptide" refers to any of the polypeptide embodiments described herein based on the coding region of tkr-1, including full-length tkr-1 and fragments of tkr-1.

"Isolated" polynucleotides or polypeptides are substantially free of those substances with which they are associated in nature. "Substantially free" refers to compositions containing less than about 50%, preferably less than about 70%, and more preferably less than about 90% of the materials with which they are usually associated with in nature.

The term "vector" refers to a DNA molecule that can carry inserted DNA and be peφetuated in a host cell. Vectors are also known as cloning vectors, cloning vehicles or vehicles. The term includes vectors that function primarily for insertion of a nucleic acid molecule into a cell, replication vectors that function primarily for the replication of nucleic acid, and expression vectors that function for transcription and/or translation of the DNA or RNA. Also included are vectors that provide more than one of the above functions.

A "host cell" includes an individual cell, cell culture or cell which is part of a whole animal which can be or has been a recipient for vector(s) or for incoφoration of nucleic acid molecules and/or proteins. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in moφhology or in total DNA complement) to the original parent due to natural, accidental, or deliberate mutation. A host cell includes cells transfected with the polynucleotides of the present invention.

The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acids and does not refer to a specified length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. Included within this definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally and non-naturally occurring. "Analogs" of tkr-1 proteins are any naturally or non-naturally occurring peptide or peptide mimicking molecule which exhibit the same biological effects as tkr-1 proteins, for example increasing life span.

A "individual " is a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.

As used herein, "expression" refers to the process by which polynucleotides are transcribed into mRNA and translated into peptides, polypeptides, or proteins. The term includes transcription as well as translation.

A "transgenic" animal refers to a genetically engineered animal or offspring of genetically engineered (i.e., altered) animals. As used herein, a transgenic nematode may or may not be genetically altered by using a polynucleotide from at least one unrelated organism, such as from a virus, plant, or other animal. Thus, a "transgenic nematode" is one that has been stably transfected with heterologous (foreign) or native (self) polynucleotide. A "transgene" is any gene that is inserted to create a transgenic animal. The transgene can be homologous or heterologous. For example, a homologous transgene is shown infra, namely a stress-related gene such as tkr-1. A heterologous transgene can be, for example, a chimeric gene which is created by insertion of a heterologous sequence that does not normally occur in the host animal, e.g., nematode. An example of a heterologous transgene is the tkr-1 FGF-Rl construct described herein.

"Transformation" or "transfection" refers to the insertion of an exogenous polynucleotide into a host cell or animal, irrespective of the method used for the insertion, for example, lipofection, transduction, infection or electroporation. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host cell genome.

As used herein, "expression" includes transcription and/or translation.

As used herein, the term "agent" means a biological or chemical compound such as a simple or complex organic or inorganic molecule, a peptide, a protein, oligonucleotide, polynucleotide, carbohydrate, or lipoprotein. A vast array of compounds can be synthesized, for example oligomers, such as oligopeptides and oligonucleotides, and synthetic organic compounds based on various core structures, and these are also included in the term "agent". In addition, various natural sources can provide compounds for screening, such as plant or animal extracts, and the like. Compounds can be tested singly or in combination with one another.

An agent that "modulates" life-extension is an agent that affects life-extension, or lifespan, whether directly or indirectly, whether negatively or positively.

Methods to identify polynucleotides associated with longevity

The present invention provides methods of identifying a polynucleotide associated with longevity comprising measuring lifespan in a transgenic nematode, said transgenic nematode comprising the polynucleotide, wherein the polynucleotide is identified as associated with longevity if its expression confers longer lifespan than a suitable control. The method used to identify gerontogenes whose over-expression causes increased stress resistance and increased life span could be effective for isolating such genes in mammals and other organisms. Genomic screening of transgenic animals would also be useful to identify sequences known or suspected to be associated with the stress response which are also associated with longevity.

The methods of this invention identify particularly useful sequences in that their over-expression is associated with longevity, i.e., their over-expression positively modulates life extension. Polynucleotides are overexpressed in the transgenic nematode, leading to increased lifespan. Accordingly, transgenic worms carrying cosmid or YAC clones as set forth in the Figures were constructed, and tested whether the transgenic worms showed life extension as well as an increased stress resistance in young adult hermaphrodites.. As detailed in the Examples below, the genomic screening specifically covered a 0.5 Mb region to the left of unc-4 on chromosome II of the nematode Caenorhabditis elegans. A gene, tkr-1, with a newly defined role in stress response and longevity was identified. The nucleotide sequence of tkr-1 and also the amino acid sequence of the polypeptide encoded thereby is shown in Figure 4.

The methods entail measuring the lifespan of a transgenic nematode, preferably C. elegans, that contains the polynucleotide to be tested. It is understood that, for purposes of this invention, a polynucleotide identified by conferring increased lifespan (when compared to a suitable control) means that any polypeptide encoded by that polynucleotide has also been identified. It is also understood that fragments, or regions, of the polynucleotides and/or polypeptides encoded therein may also be identified by testing for lifespan conferring function as described herein. Such polynucleotides and polypeptides identified by these methods are denoted "life-extension polynucleotide(s)" and "life- extension polypeptide(s)" herein.

The polynucleotide(s) to be screened may be from any source, and of any function, either known or unknown. The polynucleotide may or may not be derived from a nematode such as C. elegans and may be, for example, mammalian, preferably human. The significant degree of homology (i.e., shared sequences) across species and even various genuses indicates that this screening method would be suitable for using polynucleotide sequence from sources other than nematodes such as C. elegans. Similarly, C. elegans sequences may indicate other mammalian sequences that could be tested for life-extension properties. We have shown, for example, that the kinase domain from FGFR can substitute for the kinase domain from tkr-1 in terms of life extension and stress resistance.

A polynucleotide to be tested may be prepared by any technique known to those of skill in the art using the polynucleotide sequences provided herein. For example, they can be prepared by isolating the polynucleotides from a natural source, or by chemical synthesis, or by synthesis using recombinant DNA techniques.

Methods of making transgenic nematodes, such as C. elegans, are described in the Examples. These methods are also applicable to other nematodes, for example C. briggsae. Generally, any acceptable method of introducing the polynucleotide of interest so that it is stably inherited in the organism is acceptable. The polynucleotide is preferably introduced by direct injection into the gonads of the nematode. Generally, a selectable marker, such as the rol-6 (sulOOό) gene, which confers the rolling phenotype is co- transfected to detect transfection. Stable transgenics are then selected and propagated for testing.

The polynucleotide is introduced to the nematode in any one of a number suitable vectors, such as cloning and/or expression plasmids, BACs, YACs, cosmids, many of which are commercially available (Examples 1 and 4). Such vectors are known in the art and need not be described in detail herein.

The ability of the polynucleotide to confer increased lifespan is measured and compared to a suitable control. For puφoses of the present invention, a suitable control is one which does not over-express the polynucleotide. "Over-expression" is a term well- understood in the art, and, in this context of transgenic C. elegans, means that there is a copy number of the polynucleotide higher than that generally found in nature. "Overexpression" includes transcription and/or translation. Copy number can be readily measured (if necessary) by techniques such as Southern blotting. Degree of transcription can be measured using, for example, Northern analysis. Amount of expression product may be measured by Western analysis (if an antibody is available) or by a functional assay that detects the amount of protein, such as kinase activity. The polynucleotide' s function(s) which is associated with life extension may be a coding and/or non-coding sequence (such as a transcriptional regulatory element). Generally, one skilled in the art would assume that a transgenic nematode, since it de facto contains an additional copy (or copies) of the polynucleotide, would "over-express" the polynucleotide. Methods of varying the number of copies are known in the art and include, for example, varying concentration during injection. Suitable controls include, but are not limited to, a transgenic nematode that contains a selectable marker only (i.e., has not been transformed with the polynucleotide to be tested) and a nematode that has not been transformed.

In some embodiments, polynucleotides known or suspected to be associated with a stress response are tested. In other embodiments, polynucleotides known or suspected to be associated with stress resistance are tested. Polynucleotides known or suspected to confer stress resistance may be obtained in a variety of ways, such as using those published in the literature or searching databases for sequences homologous to sequences known to be associated with stress resistance. As discussed below, a C. elegans polynucleotide (gene) tkr-1 was identified using this method.

In other embodiments, C. elegans genes similar to the mammalian tyrosine receptor kinases that are involved in cellular stress response are used. Other stress response genes could also be used as a basis for the homolog screening. These include, for example, members of the MAP kinase family or other kinases, for example Jun N-terminal kinase (JNK SAPK) in the cosmid T07A9, p38/HOG-l in the cosmid B0218, ELKl in the cosmid F19F10, CL100 in the cosmid C05B10, and transcription factors, for example, AP-1 (c- Fos/c-Jun) in the cosmid T24H10 and TCF/Elk-1. Several receptor kinases also share in (and perhaps cross-regulate) response to UV or other stimuli. Also included are mammalian homologs of these genes. These kinases include the c-kit protooncogene, PDGFR (platelet derived growth factor receptors), IGF-R (insulin like growth factor I receptors), and EGF-R (epidermal growth factor receptors). For example, binding of c-kit to its ligand, stem cell factor (SCF), confers an increased radiation resistance to bone marrow progenitor cells (Shul et al., 1995 Cancer Res. 55, 3431-3437). Several additional receptor kinases also are activated or phosphorylated after exposure to UV irradiation and/or oxidative stress, including EGF-R. insulin receptor and T cell receptor (Schieven et al., 1994 J. Biol. Chem. 269. 20718-20721 : Coffer et al, 1995 Oncogene 11, 561-569). These polynucleotide and polypeptide sequences are readily available in the literature and publicly accessible databases and need not be described herein.

Accordingly, the invention includes a method of identifying a polynucleotide associated with longevity comprising (a) conducting a database search using a polynucleotide (or polypeptide) sequence known to be (or suspected to be) associated with stress resistance; and (b) testing a polynucleotide identified in step (a) for ability to confer increased lifespan in a transgenic nematode comprising the polynucleotide.

If desired, additional tests may be conducted using the polynucleotide(s) identified above to further characterize the nature of the polynucleotide' s function with respect to longevity. For example, egg laying may also be measured to determine whether the longevity occurs by delaying maturity. As another example, other phenotypes associated with other gerontogenes could be tested to determine whether the identified polynucleotide shares functional pathways with these other genes.

The invention also includes transgenic nematodes comprising (i.e., transfected with) any of the polynucleotide(s) identified herein.

tkr-1, a gene associated with longevity

Using the above methods, a gene denoted tkr-1 (SEQ ID NO: 12) was identified from overlapping cosmids showing life extension and stress resistance when introduced in high copy in C. elegans. This tyrosine kinase receptor gene, with sequence similarity to the mammalian c-kit protooncogene and fibroblast growth factor receptor gene, confers increased resistance to heat and ultraviolet light, and also increases longevity up to 65%. The cytoplasmic kinase domain of tkr-1 must be present to cause these forms of stress resistance. A tkr-1 homolog, tkr-2, promotes no detectable stress resistance and moderate life extension. Unlike the C. elegans gerontogenes previously identified (such as age-1, daf-2, spe-26 and clk-1), tkr-1 has an "anti-aging" function that positively modulates stress resistance and longevity.

The tkr-1 gene was identified using the following two-part strategy: (1) performing a computer homolog search using the C. elegans database and (2) a genomic screening using transgenics, to identify an "anti-aging" gene that confers stress resistance and life extension. Genes conferring stress resistance in adult C. elegans when over-expressed were screened and then tested to see whether these genes extended the life span. The present inventors have also shown that the life extension and stress resistance effects of tkr-1 are suppressed by daf-16 mutants (Figure 12). In particular, the daf-16(m26) mutation is epistatic to the tkr-1 -associated phenotypes. Without being bound by one theory, this suggests that tkr-1 functions in the same genetic pathway utilized by other gerontogenes.

Because of their association with life extension, tkr-1 polynucleotides and polypeptides are useful in screening agents which modulate tkr-1 function, and thus may be involved in the aging process, particularly in life extension. These uses are discussed below. Further, because of the significant discovery that the kinase domain of FGFR, particularly human FGF-Rl, substitutes for the kinase domain of tkr-1, this implies that (a) the chimeric gene construct comprising tkr-1 with the substituted FGFR kinase domain can be used in screening for potentially useful anti-aging agents; (b) the kinase domain of FGFR may have important functional implications in longevity, and thus may be a basis for screening for anti-aging agents; (c) FGFR may have important function in longevity, and thus polynucleotides encoding FGFR can be screened for anti-aging agents. These and other screening embodimennts are discussed below.

Screening methods using polynucleotides and/or polypeptides associated with longevity

The invention also provides methods of in vitro and in vivo screening using the life- extension polynucleotides identified above and/or any polypeptides encoded therein. The life-extension polypeptides and polypeptides to be used in these screening methods may be obtained using standard synthetic methods known in the art, including, but not limited to, isolation from natural sources, recombinant methods, chemical synthetic methods, and enzymatic digestion followed by purification. The screening methods generally employ an expression construct comprising the polynucleotide(s), although in in vitro methods the polynucleotide(s) and/or polypeptide(s) alone may be used.

The correlation of over-expression of tkr-1 with life extension indicates that the up- regulation of a functional characteristic of life-extension polynucleotide (discussed below) confers longevity, and that agents which likewise cause this up-regulation may be useful in treating age-related phenomena. Conversely, an agent which causes down-regulation is indicated as a toxic agent in the sense that it negatively affects longevity. Thus, the screening methods described herein are applicable to a number of contexts, including screening for potentially useful anti-aging substances as well as screening for potentially harmful age-inducing substances.

Further, our finding that the kinase domain of FGFR can substitute for the kinase domain of tkr-1 implies shared functionality between the kinase domain of FGFR, and possibly FGFR, and tkr-1 with respect to longevity and/or stress resistance. The conservation between trk-1 and human receptor kinases indicates that they may share a downstream signaling pathway for increased longevity and/or stress resistance.

Accordingly, the present invention encompasses methods of identifying agents that may have life-extension activity based on their ability to modulate a functional characteristic of a life-extension polynucleotide and/or polypeptide. These methods may be practiced in a variety of embodiments. We have observed that polynucleotide sequences may be identified based on their ability to confer life extension when overexpressed in nematodes, particularly C. elegans. While not being bound to any one theory, this observation suggests that a pathway(s) involving these polynucleotide(s) (such as tkr-1, discussed below) may play a role in life extension. This further suggests that modulation of function of these life-extension polynucleotide(s) (including, but not limited to, modulation of any polypeptide(s) encoded therein) may result in control of the aging and/or survival process. Thus, an agent identified by the methods of the present invention may be useful in the treatment of aging-associated conditions. It is understood that these screening embodiments include, but are not limited to, using the tkr-1 polynucleotides and polypeptides described above.

The methods described herein are in vitro and in vivo cell- and animal (e.g., nematode)-based screening assays. In the in vitro embodiments, an agent is tested for its ability to modulate function of a life-extension polynucleotide and/or life-extension polypeptide using the methods described herein. In the cell-based embodiments, living cells comprising the life-extension polynucleotide(s) and/or life-extension polypeptide(s) are used for testing agents. For puφoses of this invention, an agent may be identified on the basis of modulation of a polynucleotide or polypeptide, although, given that such polynucleotides and/or polypeptides have been identified by their ability to confer life extension due to over-expression, will likely be preferably that the agents confer increased, or enhanced, functionality. These assays can also be conducted on whole animals, using for example, nematodes.

In all of these methods, modulation of function of a life-extension polynucleotide and/or polypeptide may occur at any level. An agent may modulate function by reducing or preventing transcription of a life-extension polynucleotide. An example of such an agent is one that binds to the upstream controlling region, including a polynucleotide sequence or polypeptide. An agent may modulate translation of mRNA. An example of such an agent is one that binds to the mRNA, such as an anti-sense polynucleotide, or an agent which selectively degrades or stabilizes the mRNA. An agent may modulate function by binding to the life-extension polypeptide. An example of such an agent is a polypeptide or a chelator. An agent may modulate function by affecting gene expression of a gene that is regulated by a life-extension gene. An example of such an agent is one that alters expression of a life-extension-regulated gene on any of the levels discussed above.

With respect to these screening methods, any life-extension polynucleotide (and or polypeptide) may be used. Generally, the life-extension polynucleotide (and/or polypeptide) will have been identified using the transgenic methods described above. In some embodiments, a tkr-1 polynucleotide (and/or polypeptide) is used. In other embodiments, a fusion construct in which a polynucleotide encoding the kinase domain of FGFR has replaced the coding region for the kinase domain of tkr-1 (such as that described in Example 4) is used. In other embodiments, a polynucletoide the kinase domain of FGFR is used. In other embodiments, a polynucleotide encoding FGFR is used. In other embodiments, mammalian, preferably human, homologs of the polypeptides identified above can be used. For clearness, these embodiments will generally not be reiterated below, as the various types of assays are discussed. In vitro screening methods

In in vitro screening assays of this invention, an agent is screened in an in vitro system, which may be any of the following: (1) an assay that determines whether an agent is modulating transcription of a life-extension polynucleotide; (2) an assay for an agent which modulates translation of mRNA or a polynucleotide encoding a life-extension polypeptide; (3) an assay for an agent that binds to a life-extension polynucleotide or polypeptide.

For an assay that determines whether an agent modulates transcription of a life- extension polynucleotide, an in vitro transcription or transcription/translation system may be used. These systems are available commercially, and generally contain a coding sequence as a positive, preferably internal, control. A life-extension polynucleotide is introduced and transcription is allowed to occur. Comparison of transcription products between an in vitro expression system that does not contain any agent (negative control) with an in vitro expression system that does contain agent indicates whether an agent is affecting transcription. Comparison of transcription products between control and the life- extension polynucleotide indicates whether the agent, if acting on this level, is selectively affecting transcription of the life-extension polynucleotide (as opposed to affecting transcription in a general, non-selective or specific fashion).

For an assay that determines whether an agent modulates translation of a life- extension mRNA or a polynucleotide encoding a life-extension polypeptide, an in vitro transcription/translation assay as described above may be used, except the translation products are compared. Comparison of translation products between an in vitro expression system that does not contain any agent (negative control) with an in vitro expression system that does contain agent indicates whether an agent is affecting transcription. Comparison of translation products between control and the life-extension polynucleotide indicates whether the agent, if acting on this level, is selectively affecting translation of the life-extension polynucleotide (as opposed to affecting translation in a general, non- selective or specific fashion).

For an assay for an agent that binds to a life-extension polypeptide, a life-extension polynucleotide is first recombinantly expressed in a prokaryotic or eukaryotic expression system as a native or as a fusion protein in which a life-extension polypeptide (or fragment thereof) is conjugated with a well-characterized epitope or protein as are well known in the art. Recombinant life-extension polypeptide is then purified by, for instance, immunoprecipitation using anti-life-extension polypeptide antibodies or anti-epitope antibodies or by binding to immobilized ligand of the conjugate. An affinity column made of life-extension polypeptide or life-extension polypeptide fusion protein is then used to screen a mixture of compounds which have been appropriately labeled. Suitable labels include, but are not limited to flurochromes, radioisotopes, enzymes and chemiluminescent compounds. The unbound and bound compounds can be separated by washes using various conditions (e.g. high salt, detergent ) that are routinely employed by those skilled in the art. Non-specific binding to the affinity column can be minimized by pre-clearing the compound mixture using an affinity column containing merely the conjugate or the epitope. A similar method can be used for screening for agents that competes for binding to a life-extension polypeptide. In addition to affinity chromatography, there are other techniques such as measuring the change of melting temperature or the fluorescence anisotropy of a protein which will change upon binding another molecule. For example, a BIAcore assay using a sensor chip (supplied by Pharmacia Biosensor, Stitt et al. (1995) Cell 80: 661-670) that is covalently coupled to native life-extension polypeptide or life- extension polypeptide fusion proteins, may be performed to determine the life-extension polypeptide binding activity of different agents.

In another embodiment, an in vitro screening assay detects agents that compete with another substance (most likely a polypeptide) that binds a life-extension polypeptide. Competitive binding assays are known in the art and need not be described in detail herein. Briefly, such an assay entails measuring the amount of life-extension polypeptide complex formed in the presence of increasing amounts of the putative competitor. For these assays, one of the reactants is labeled using, for example, ³²P.

It is also understood that the in vitro screening methods of this invention include structural, or rational, drug design, in which the amino acid sequence, three-dimensional atomic structure or other property (or properties) of a life-extension polynucleotide or polypeptide provides a basis for designing an agent which is expected to bind to a life- extension polynucleotide or polypeptide. Generally, the design and/or choice of agents in this context is governed by several parameters, such as the perceived function of the polynucleotide or polypeptide target, its three-dimensional structure (if known or surmised), and other aspects of rational drug design. Techniques of combinatorial chemistry can also be used to generate numerous permutations of candidate agents. For puφoses of this invention, an agent designed and/or obtained by rational drug designed may also be tested in the cell-based assays described below.

Cell-based screening methods

In cell-based screening assays, a living cell containing a functioning life-extension polynucleotide (whether or not an encoding region) is exposed to an agent. The living cell may be in culture or, alternatively, part of a whole, living animal. In contrast (as described above), conventional drug screening assays have typically measured the effect of a test agent on an isolated component, such as an enzyme or other functional protein.

The cell-based screening assays described herein have several advantages over conventional drug screening assays: 1) if an agent must enter a cell to achieve a desired therapeutic effect, a cell-based assay can give an indication as to whether the agent can enter a cell; 2) a cell-based screening assay can identify agents that, in the state in which they are added to the assay system are ineffective to modulate the life-extension polynucleotide and/or polypeptide function, but that are modified by cellular components once inside a cell in such a way that they become effective agents; 3) most importantly, a cell-based assay system allows identification of agents affecting any component of a pathway that ultimately results in characteristics that are associated with life-extension polynucleotide and/or polypeptide function.

In one embodiment, an agent is identified by its ability to modulate, preferably enhance or increase, life-extension polynucleotide and/or polypeptide function in a suitable host cell. A suitable host cell in this context is any host cell which allows such function to be measured. Preferably, the host cell is a nematode cell, either isolated or as part of a whole nematode. Suitable host cells include, but are not limited to, fungi (including yeast), bacterial, insect, mammalian, and amphibian.

In one embodiment, the invention provides methods for identifying an agent that may increase longevity comprising the following steps: (a) contacting at least one agent to be tested with a suitable host cell that has life-extension polynucleotide and/or polypeptide function; and (b) analyzing at least one characteristic which is associated with life-extension polynucleotide and/or polypeptide function in said host cell, wherein an agent is identified by its ability to modulate at least one such characteristic. Characteristics associated with function of a life-extension polynucleotide and/or polypeptide depend upon the polynucleotide or polypeptide. Functional characteristics include, but are not limited to, transcription, translation (including levels of precursor and/or processed polypeptide), location of protein product (such as membrane localization), any enzymatic activities, such as kinase activity, structural and/or functional phenotypes (such as stress resistance or life cycle), and expression (including repression or de-repression) of any other genes known to be controlled (modulated) by the polynucleotide. Any measurable change in any of these and other parameters indicate that the agent may be useful. Measuring these parameters (such as those using reporter genes) involve methods known in the art and need not be discussed herein. Reporter genes include, but are not limited to, alkaline phosphatase, chloramphenicol acetyl transferase, β- galactosidase, luciferase and green fluorescent protein. Identification methods for the products of reporter genes include, but are not limited to, enzymatic assays and fluorimetric assays. Reporter genes and assays to detect their products are well known in the art and are described, for example in Current Protocols in Molecular Biology, eds. Ausubel et al., Greene Publishing and Wiley-Interscience: New York (1987) and periodic updates. Reporter genes, reporter gene assays and reagent kits are also readily available from commercial sources (Stratagene, Invitrogen and etc.).

Introduction of life-extension polynucleotides depend on the particular host cell used and may be by any of the many methods known in the art, such as microinjection, spheroplasting, electroporation, CaCl₂ precipitation, lithium acetate treatment, and lipofectamine treatment.

Polynucleotides introduced into a suitable host cell(s) are polynucleotide constructs comprising a life-extension polynucleotide. These constructs contain elements (i.e., functional sequences) which, upon introduction of the construct, allow expression (i.e., transcription, translation, and post-translational modifications, if any) of life-extension polypeptide amino acid sequence in the host cell. The composition of these elements will depend upon the host cell being used. For introduction into C. elegans, polynucleotide constructs will generally contain a selectable marker such as rol-6 (sulOOό) and the life- extension polynucleotide operatively linked to a suitable promoter (when encoding a life- extension polypeptide), such as tkr-1. Other suitable host cells and/or whole animals include Drosophila, yeast and mammalian cells. Suitable selectable markers for nematode cells are those that enable the identification of cells that have taken up the nucleic acid, such as moφhologic and behavioral markers such as rol-6 or visual markers such as green fluorescent protein. Screening of the transfectants identifies cells or animals that have taken up and express the polynucleotide.

In some embodiments, a life-extension polynucleotide is operatively linked to an inducible promoter. Use of an inducible promoter provides a means to determine whether the agent is acting via a pathway involving the life-extension polynucleotide. If an agent modulates a functional characteristic of a life-extension polynucleotide and/or polypeptide in a cell in which the inducible promoter is activated, an observation that the agent fails to elicit the same result in a cell in which the inducible promoter is not activated indicates that the agent is affecting at least one step or aspect of life-extension polynucleotide function. Conversely, if the functional characteristic is also observed in a cell in which the inducible promoter is not activated, then it can be assumed that the agent is not necessarily acting solely via the life-extension polynucleotide functional pathway.

Cell-based screening assays of the present invention can be designed, e.g., by constructing cell lines or strains of animals in which the expression of a reporter protein, i.e., an easily assayable protein, such as β-galactosidase, chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP) or luciferase, is dependent on life-extension polynucleotide and/or polypeptide function. The cell is exposed to a test agent, and, after a time sufficient to effect β-galactosidase expression and sufficient to allow for depletion of previously expressed β-galactosidase, the cells are assayed for the production of β-galactosidase under standard assaying conditions.

Assay methods generally require comparison to a control sample to which no agent is added. The screening methods described above represent primary screens, designed to detect any agent that may exhibit anti-aging activity. The skilled artisan will recognize that secondary tests will likely be necessary in order to evaluate an agent further. For example, a cytotoxicity assay would be performed as a further corroboration that an agent which tested positive in a primary screen would be suitable for use in living organisms. Any assay for cytotoxicity would be suitable for this puφose, including, for example the MTT assay (Promega).

In some embodiments, a nematode transgenic system (as descibed above) is used for screening. Our discovery that a transgenic nematode comprising tkr-1 exhibits life extension, coupled with our discovery that the kinase domain of FGFR can substitute for the kinase domain of FGFR, indicates that our life-extension transgenic nematode system would be especially useful for screening for agents which may (a) exhibit life-extension activity; or (b) exhibit repression of life-extension.

Accordingly, in one embodiment, the invention provides a method for identifying an agent which may modulate life extension, comprising contacting a transgenic nemotode comprising a tkr-1 polynucleotide with the agent and measuring lifespan. In another embodiment, the transgenic nematode comprises a fusion construct containing a tkr-1 polynucleotide in which the kinase domain of FGFR has been substituted for the kinase domain of tkr-1.

The following examples are provided only for illustrative puφoses, and not to limit the scope of the present invention. In light of the present disclosure numerous embodiments within the scope of the claims will be apparent to those of ordinary skill in the art.

EXAMPLE 1 Screening for polynucleotides associated with life extension using transgenic C. elegans

Computer similarity search using mammalian stress response receptor genes A Blast search (Altschol et al.,1990, J. Mol. Biol. 215, 403-410) for C. elegans genes similar to c-kit identified more than seven genes. Two of the most similar open reading frames were C08H9.5 (Blast score 3.7e-62; same as tkr-l; see below) and ZK938.5 (Blast score 9.6e-59; same as tkr-2; see below). The other identified open reading frames were R151.4 (Blast Score 7.9e-57), F59F3.1 (Blast score 4.5e-53), and F59F3.5 (Blast score 4.1e-51). A homology search for PDGF-R also identified these same C. elegans gene members. Therefore, homology searching identified a group of genes similar to c- fe^'t/PDGF-R receptors.

A homology search for EGF-R and IGF-R identified let-60/EGF-R and two open reading frames, R151.4 and C01G6.8, respectively. Therefore, the computer search of the C. elegans gene database identified genes that were structurally similar to the mammalian stress genes used as a basis for the search. Construction of transgenics

Although the search method described above effectively identified a pool of stress genes, another method was required to identify particular genes that affect the aging process. The genomic screen identified two cosmids, C11D5 and C08H9, that overlap with each other (Figure 2). To test these sequences to determine whether they conferred stress resistance and/or life extension, we constructed transgenic nematodes.

Strains and Media. The C. elegans strains were maintained and handled on nematode growth medium (NGM) agar seeded with E. coli, OP50 as a food source (The Biology of Caenorhabditis elegans, Wood, W.B., Cold Spring Harbor Press (1988)). For the stress resistance and life span assays, we used the NGM plates spread with OP50 over the surface (Wood, W.B., supra).

Plasmids. The p(tkr-l) plasmid is Bluescript Il-based (Stratagene) carrying the 6.5 kb Spe 1 fragment from the cosmid C08H9 which contains the entire tkr-1 ORF plus the entire 5' untranslated region plus some of the upstream ORF (total 5' region, 2240 bp). Proc. Natl. Acad. Sci. USA (1986) 83:7821-7825; Wilson et al (1994) Nature 368: 32-38. The 6.5 kb fragment carrying tkr-1 also contains a 5' truncated chitanase gene, which should not be expressed because it lacks a promoter region. The region around tkr-1 contains a tandem array of chitanase genes, these chitanase genes, including truncated forms, have no effect on thermotolerance and lifespan.

The construction of the mutant plasmid, p(tkr-l Δkin), was as follows: The tkr-1 plasmid, digested with Bgl II, was treated with T4 DNA polymerase and was ligated. The resulting plasmid was recovered by E. coli transformation. The tkr-1 Akin plasmid has a 4 bp additional sequence (5'-GATC-3') resulting in the frameshift mutation (Figures 2 and 3). The predicted termination codon is located just after the Bgl II site. Primary structure of all plasmids was confirmed by sequencing.

Construction of transgenics. We constructed C. elegans transgenics by using a microinjection technique previously described (Mello et al., 1991 EMBOJ. 10, 3959- 3970). A marker plasmid, pRF4 (Mello et al., supra), was used. All transgenic strains were made in the wild-type strain, N2, by microinjecting high copy numbers of each clone together with a plasmid carrying the dominant rol-6 (sulOOό) marker, that causes a "roller" phenotype in the worms when expressed. All the cosmids and yeast artificial chromosomes (YACs) were obtained from Dr. Alan Coulson, Sanger Center (1994 Nature 368, 32-34). Each plasmid (0.02 μg/μl) was co-injected with pRF4 (0.2 μl/μg) into the gonads of young adult hermaphrodites. To minimize the possibility that toxic genes may offset the thermotolerance of other genes when over-expressed, we injected a single YAC or cosmid DNA into the worms instead of injecting a pool of DNAs. Transgenics showing either an Age or Str (stress resistance) phenotype were detected by subsequent comparisons with either N2 or control strains carrying only the rol-6 marker. F2 transgenics carrying the rol- 6 marker were isolated and maintained. More than two transgenics from the independent microinjections were isolated and tested. We did not find any significant effect of the pRF4 on thermotolerance, UV resistance and life span. Integrated tkr-1 strains were derived from tkrl-1 or tkrl-2. Irradiated Rol offspring (F2) were then backcrossed with the N2 strain five times to obtain stably integrated strains free of mutations produced by the irradiation.

Level of tkr-1 expression in transgenic nematodes. We confirmed that the tkr-1 transgenics showed high levels of the tkr-1 RNA and of the tkr-1 protein, suggesting that tkr-1 was over-expressed in the transgenic lines. A typical tkr-1 transgenic line had approximately 20 copies of tkr-1 as assessed by Southern analysis and overexpressed tkr-1 mRNA.. The tkr-2 gene also was over-expressed as RNA in the tkr-2 transgenics.

Transgenic strains containing only tkr-1 together with pRF4 (a plasmid carrying the rol-6(sul006) dominant behavioral marker) were examined for effects on stress resistance and longevity.

Transgenic screening for a gene conferring stress resistance (thermotolerance and UV resistance) and life extension

Stress resistance assays. For the thermotolerance assay, 4-day-old young adult hermaphrodites were incubated at 35°C. Survival was measured every one or two hours after an initial decline in viability was detected. For the UV resistance assay, the young adults (4 days old) were irradiated with 254 nm UV-C light at 20 J/m². Survival was measured every day until all were dead. Each experiment contained two sets of about 20 worms that expressed the marker phenotype. Each experiment was replicated more than twice. The statistical analysis was performed using the Wilcoxon (Gehan) statistic as implemented in the SPSS survival package (SPSS Update, Hull, CH. and Nie, N.H., McGraw Hill, 1979). Independently isolated transgenics were used in both the thermotolerance and UV assay. Lifespan assays. The lifespan assay was performed as described previously. Murakami and Johnson, 1996, Genetics 143, 1207-1218. Survival of the hermaphrodites was measured every day until they stopped laying eggs, and then measured every few days thereafter.

We constructed an extensive array of either YAC or cosmid transgenics that collectively covered the entire 0.5 Mb region. Both overlapping cosmids Cl 1D5 and C08H9 conferred increased thermotolerance by about 30% (range of 15-60%) in transgenic worms (Figure 3). Transgenics carrying all other cosmids or YACs over the 0.5 Mb region (C03H10, F17E3, K07G10, F07G10, F07E3, R09A5, T10B9, T18C7, ZK1320, W04A3, Y53C12 and Y10G6) had normal thermotolerance (transgenic vs. rol-6 control p > 0.1). As shown in Fig. 8a, the tkr-1 transgenics are 35% more heat resistant than control transgenics (control- 1 and control-2) (p<.0001). Survival at 35°C (mean ± SD) was: control-1, 654 ± 14 minutes; control-2, 682 ± 14 minutes; tkr-1, 834 ± 20 minutes; tkrl-2, 793 ± 18 minutes. Three additional tkr-1 transgenics were also thermotolerant. All four tkr-1 transgenics tested were significantly more thermotolerant by 15% to 30%> than wild type (average 20%; p O.0001, typical data are shown in Fig. 8a). No other cosmids or YAC clones conferred such tolerance.

Using subclones, we narrowed the region responsible for the increased thermotolerance to a 6.5 kb fragment containing the common region of the two cosmids and identified a single open reading frame, designated tkr-1 (tyrosine kinase receptor- 1), containing a conceptual receptor tyrosine kinase.

Since a frameshift mutation, tkr-1 Akin, and a truncation mutation, tkr-1 ABB, abolished the thermotolerance phenotype, we concluded that the tkr-1 gene is responsible for the thermotolerance.

The tkr-1 gene consists of 10 exons, encoding 502 amino acid residues (Figure 4). The RACE (rapid amplification of cDNA ends) method (Froman et al., supra) confirmed the 5' and 3' ends of the tkr-1 transcript. The predicted protein sequence has a potential transmembrane domain and shows a strong similarity with the kinase region of the mammalian receptor kinases, c-kit (rat c-kit identity 35%; similarity 5 %), FGF-R (human FGF-Rl ; identity 30%; similarity 56%), and PDGF-R (mouse PDGF-R; identity 35%; similarity 56%). The tkr-1 kinase region has a nucleotide binding site and three potential autophosphorylation sites. The tkr-1 kinase region has a putative ATP binding site and three potential autophosphorylation sites (Figure 4). The predicted extracellular domain is only 40 amino acids. Thus, while tkr-1 may function as a receptor, it may be a receptor with a short extracellular domain or it may form a complex with another membrane protein that contains a longer receptor domain. tkr-1 is identical to C08H9.5, identified in the computer similarity search of Example 1. This suggests that tkr-1 is very similar to the c-Λ/t/PDGF-R gene family in C elegans. However, the similarity is mainly from the cytoplasmic kinase domain (44% identity against rat c-kit) rather than the extracellular ligand binding domain (15% identity against the rat c-kit). tkr-1 lacks the kinase insertion seen in c-kit/FDGF-R family between the kinase subdomains, V and VIA. None of the known genes showed similarity to the tkr- 1 extracellular domain. These data suggest that tkr-1 is a novel tyrosine kinase gene with a similarity to the kinase domain of the c-fe^'t/PDGF-R family and FGF-R.

Kinetics of tkr-1 induction after UV and heat stresses. We constructed a green fluorescent protein reporter system, p(tkr-lGFP) and measured fluorescence after UV irradiation or heat shock (Figure 13). The response of transgenic nematodes carrying p(tkr-l-GFP) to UV light (10 or 20 J/m²) or heat shock (35°C for 2 hours) are shown in Figure 14. The induction of tkr-1 was measured as the intensity of GFP fluorescence. Images of stressed and unstressed transgenic worms were analyzed using the program NIH image. The results show that tkr-1 is induced upon stress.

UV resistance. We tested the UV resistance of four tkr-1 transgenics. Two of the transgenics tested showed 30 to 50% more UV resistance than the control transgenics (average 30%; (Figure 8b). The tkr-1 transgenics were 30 to 50% more resistant to UV irradiation than control transgenics (p<.0001). Observed survivals after UV irradiation at 20 J/m² (mean ± SD) were: control-1, 3.2 ± 0.1 days; control-2, 3.2 ± 0.1 days; tkr-1, 4.8 ± 0.2 days; tkrl-2, 4.6 ± 0.1 days. This suggests that the stress resistance conferred by tkr- 1 is not specific to heat but rather more general and includes other stresses as well. Since multi-stress resistance is a phenotype observed in life-extension mutants in C. elegans, the tkr-1 transgenics share a common phenotype to the life-extension mutants.

Life extension. Four independently isolated tkr-1 transgenics were tested for their life-extension abilities. The results are shown in Figures 9a and 9b. All of them showed an increase in mean life span ranging from 48 % to 65 % (p <.0001). Life expectancies (mean ± SEM) were: (control-T) 24.3 ± 1.1 days, (control-2) 26.9 ± 1.2 days, (tkrl-T) 39.8 ± 1.5 days and (tkrl-2) 40.2 ± 1.3 days, (tkr2-l) 31.1 ± 1.5 days (p<0.05) and (tkr2-2) 34.5 ± 1.2 days (p<0.01). The life expectancy of the four transgenics was 27.6 days (65% extension; wild type 16.9 days) and their maximum life span was 48 days (77% extension; wild type 27 days) tkr-1 and the control transgenics, all of which were hermaphrodites, started to produce eggs at 3 days after hatching, suggesting that tkr-1 does not delay development. Therefore, the tkr-1 life extension is an extension of adult life span. This life extension is similar to that of the previously reported life-extension mutant, age-1 (65%), and larger than spe-26 (30%), and elk-1 (5-40%).

Effect of various deletions in tkr-1 tkr-1 ABB is a truncation mutation of tkr-1 that deletes the whole coding region, except for the portion encoding the first 15 amino acids. This mutation totally abolished thermotolerance (see above), UV resistance and life extension (Table 1). This indicates that the tkr-1 gene is essential for these phenotypes. tkr-1 Akin is a frameshift mutation in tkr-1 that deletes the cytoplasmic portion of the encoded polypeptide, including the kinase domain, with a small C-terminal domain (tkr-1 Akin; see Fig. 2 and Fig. 3). tkr-1 Akin abolished the thermotolerance, the UV resistance, and the life extension observed in the wild type tkr-1 (Table 1). This indicates that the cytoplasmic kinase domain plays a major role in the stress resistance and life extension. In addition, the mutant tkr-1 Akin can still confer a moderate life extension, suggesting that something other than the kinase domain also plays a minor role in the increased longevity, but not in the stress resistance.

The results of a typical experiment are shown in Table 1. More comprehensive data is summarized in Table 2. All experiments were replicated. The tkr-1 transgenics in each experiment were significantly different from the control transgenics (p<.0001), whereas the tkr-1 Akin transgenics were indistinguishable from the controls (p>0.2) except for the life span experiments (p<.0001). The control transgenics carried only a marker plasmid, pRF4, whereas the tkr-1 and tkr-1 Akin transgenics carried the marker plasmid and the tkr-1 plasmid and the tkr-1 Akin plasmid, respectively. Table 1. Effect of deleting the tkr-1 cytoplasmic domain.

Table 2. Effect of the tkr-1, tkr-2, and tkr-1 derivatives

Strain Life Expectancy UV Resistance Thermotolerance

Mean + SEM (N) Mean + SEM (N) Mean + SEM (N)

(days) (days) (minutes)

1. control 25.6+1.3 (123) 3.5 + 0.2 (198) 625 + 3 (152)

2. tkr-1 42.3+2.1 (147) 4.5 + 0.1 (220) 750+1 (88)

3. tkr-2 30.4+3.6 (98) 3.7 + 0.2 (258) 656 + 16 (342)

4. control 16.3 + 5.0 (98) 2.9 + 0.3 (102) 627+10 (99)

5. tkrlAB 17.8 + 2.2 (132) 3.1+0.1 (154) 631+3 (70)

6. control 16.9 + 0.1 (86) 3.2 + 0.1 (123) 623+4 (112)

7. tkrlAkin 16.4+1.2 (74) 3.5 + 0.2 (124) 624 + 2 (158)

8. tkr-FGFRl 28.3 + 0.3 (108) 4.1+0.1 (169) 744+14 (106)

9. control 17.6 + 0.4 (96) 3.2 + 0.1 (113) 664 + 10 (119)

10. tkr- ^' t. 28.3 + 4.1 (102) 4.7 + 0.3 (56) 836 + 32 (52) 11. control 16.0 + 3.3 (80) 2.9 + 0.5 (71) 558 + 2 (67)

12. daf-16 (m26j 15.1 + 1.6 (94) 3.0 + 0.9 (58) 530 + 10 (48)

13. tkr-1 24.4 + 2.9 (94) 4.7 + 0.4 (58) 863 + 3 (53)

14. tkr-1 ; daf- 15.8 + 3.0 (61) 3.5 + 0.9 (47) 644 + 5 (95)

16(m26)

Tkr-1 transgenic strains were further characterized to determine whether they display phenotypic alterations observed in other Age mutants, such as reduced fertility (spe-26), altered developmental timing (Clk mutants), or constitutive dauer formation (age- 1 and daf-2). The tkr-1 transgenic strains have normal fertility (mean fertility +/- SEM 303 +/- 12 for tkr-1 transgenics and 298 +/-14 for the wild type control) and a normal time of fertility (mean +/- SEM 73.0 +/- 3.8 hours after hatching for tkr-1 transgenics and 72.0 +/- 4.3 hours for the wild-type control). Unlike age-1 and daf-2, tkr-1 transgenic strains do not form dauers at 27°C when well fed; instead, they appear wild type with regard to dauer formation. Thus, the effects of tkr-1 are specific to the adult phase and show no other phenotypes previously ascribed to other Age mutants.

EXAMPLE 2 Further characterization of tkr-1 and mapping of clustered tkr-1 family members

Determination of the tkr-1 transcriptional start and end

The RACE method was used as described (Froman et al., 1988 Proc. Natl. Acad. Sci. USA 85, 8998). The first strand was synthesized using a reverse transcriptase system (LifeTechnologies). Five μg of total RNA from a mixed population of the worms was incubated with AP primer for 10 minutes at 70°C in RNase free water. The mixture was incubated at 42°C for 2 minutes in buffer (20 mM Tris-HCl pH 8.4, 50 mM MgCl₂, 2.5 mM MgCl₂, 10 mM DTT, 500 nM AP primer, 2.0 mM dNTP). Reverse transcriptase (Life Technologies) was added to the reaction mixture, incubated at 42°C for 50 minutes, and inactivated at 70°C for 15 minutes. The resulting RNA/DNA hybrids were treated with RNase H. We then amplified the 3' or 5' end of tkr-1 or tkr-2 by using the first primer pair. The resulting PCR product was used to amplify the same region using the second set of the primers (nested PCR). The PCR reaction conditions were 95°C for 10 minutes, 35 cycles of both 95°C for 30 sec and 65°C for 30 sec, and 72°C for 10 minutes using a Taq polymerase (Perkin Elmer). The sequences of the primers used are: AP (5'-GGC CAC GCG TCG ACT AGT AGT ACT-(T)₁₂.₁₅-3') (SEQ ID NO:l), AUAP (5'-GGC CAC GCG TCG ACT AGT AGT ACT-3') (SEQ ID NO:2), tkl-Fl (5'-CCG GAT GTA ATT GGG TAG GAG TTG G-3') (SEQ ID NO:3), tkl-F2 (5'-GTT GGC TCA AGA AGA TAC TCA TCA CC-3') (SEQ ID NO:4), tkl-B2 (5'-CCG CGT TTA GTG AAA GAG GCC TAC-3') (SEQ ID NO:5), tk-B3 (5'-CGT CAG ATT CTT ACA ACA TG-3') (SEQ ID NO:6), tk2- Fl (CCG GAA GTA ATT GGA TAA GGC-3') (SEQ ID NO:7), tk2-F3 (5'-TAA ATC AAC AAG GCC CAA TGA CGG-3') (SEQ ID NO:8), tk2-B4 (5'-CAT TGG GCC TTG ATT TAG T-3') (SEQ ID NO:9), SLl (5'-GGT TTA ATT ACC CAA GTT TGA G-3') (SEQ ID NO: 10), and SL2 (5'-GGT TTT AAC CCA GTT ACT CAA G-3') (SEQ ID NO:l 1). The first and second primer pairs used were: for the 3' end of tkr-1 (AUAP and tkl-B; AUAP and tk-B3), for the 3' end of tkr-2 (AUAP and tk2-B4; AUAP and tk-B3), for the 5' end of tkr-1 (SLl and tkl-FI; SLl and tkl-F2), and for the 5' end of tkr-2 (SLl and tk2-B3; SLl and tk2-Fl). The primer for SL2 was also used instead of the SLl primer as a control. The PCR products were sequenced to determine the ends.

Mapping tkr-1 family members

We mapped the tkr-1 family members. Map positions of all the sequenced cosmids were determined in the C. elegans genome project (Wilson et al., supra). The members formed tandem repeats scattered in three positions (Figure 10). One group, including tkr- 1, tkr-2 and C08H9.8, locates to the left of unc-4 on chromosome II. The direction of the predicted transcription is the same in all three. F59F3.5 and F59F3.1 also form a tandem repeat near daf-12 on chromosome X. Tandem repeats of kin-15 and kin-16 were previously reported and are localized on chromosome II. It has been shown that this kin-15 and kin- 16 pair transcribe in a single transcription unit. Since the intervals between the other members are much larger (from 2 to 13 kb) than the kin- 15 pair (300 bp), it is not clear whether the other members are in the same transcription unit. EXAMPLE 3

The kinase domain of a human receptor substitutes for the kinase domain of tkr-1 in C. elegans

The kinase domain of tkr-1 shows similarity to the mammalian receptor kinases, FGF-R, c-kit and PDGF-R. The tkr-1 kinase domain is similar to that of FGF-R in that they lack regulatory sites seen in the KI domains of c-kit and PDGF-R. Since the kinase domain of the c-kit.PDGF-R family may interact with additional signaling proteins at the KI domain, we chose one of the FGF-R family members, human FGF-Rl, and tested a functional conservation of the cytoplasmic kinase domain between them.

We constructed a chimeric tkr-1 gene, tkrl-FGFR, and substituted its kinase domain with that of the FGF-Rl. The chimeric plasmid, p(tkrl-FGFR), was constructed as follows: p(tkr-l) was digested with Bgl II, treated with T4 DNA polymerase to make it a blunt ended, desalted and concentrated. The plasmid was then digested with^δα 1. The linearized plasmid was ligated with a Fsp 1/Xba 1 fragment from a plasmid carrying the human FGF-Rl kinase domain (Wennstrom et al., 1991 Growth Factors 4, 197-208). This resulted in the tkr-1 and human FGF-R fusion gene substituting the th"-l cytoplasmic kinase domain for the FGF-R kinase domain. The primary DNA structure of all the mutant and the fusion constructs was confirmed by sequencing.

As shown in Figure l ie, the chimeric tkrl-FGFR conferred an increased life expectancy by 68%, resistance to heat by 19% and UV light by 32% (Table 2 and Fig. 1 lc). Survivals (mean ± SD.) at 35°C were: (control-1) 627 ± 91 minutes, (control-2) 619 ± 87 minutes, (tkrl-FGFR-1) 730 ± 86 minutes, (tkr-l-FGFR-2) 758 ± 86 minutes, (tkrl Akin-1) 623 ± 93 minutes (p=0.8), (tkrl Δkin-2) 626 ± 93 minutes (p=0.7). Survival after UV irradiation at 20 J/m² (mean ± SD) were: (control- 1) 3.3 ± 0.9 days, (control-2) 3.1 ± 0.7 days, (tkrl-FGFR-1) 4.3 ± 1.0 days, (tkrl-FGFR-2) 4.2 ± 1.1 days, (tkrl Δkin-l) 3.3 ± 0.8days (p>0.1), (tkrl Δkin-2) 3.0 ± 0.8days (p>0.1). Life expectancies (mean ± SD) were: (control-1) 16.8 ± 6.4 days, (control-2) 16.9 ± 5.7 days, (tkrl-FGFR-1) 28.7 ± 9.6 days, (tkrl-FGFR-2) 28.0 ± 11.1 days, (tkrl Δkin-l) 15.2 ± 7.4 days (p=0.4), (tkrl Δkin-2) 17.5 ± 6.9 days (p=0.6). We also confirmed that the chimeric gene was over-expressed at the RNA level. These data suggest that the kinase domain of tkr-1 can be substituted with that of human FGFR1. This conservation between tkr-1 and the human receptor kinase raises the possibility that they share a downstream signaling pathway for the stress resistance and increased longevity.

EXAMPLE 4 tkr-1 homolog, tkr-2, shows a weaker longevity function

We searched tkr-1 family members in C. elegans using a Blast search (Fig. 5a-c). We identified the best C. elegans tkr-1 homolog, termed tkr-2 (tyrosine kinase receptor-2; >90% identity). Other members that were identified are less similar to tkr-1; kin- 15 (identity 40%) kin-16 (identity 47%), F59F3.5, F59F3.1, MOlB2.e and R09D1.13. tkr-2 encodes a 498 amino acid protein composed of 9 exons and is nearly identical to tkr-1 protein amino acid sequence. The tkr-2 gene is located in cosmid F17E3, near tkr- 1. The primary structures of tkr-1, tkr-2 and FGFR-1 are shown in Figure 6. tkr-1 and tkr- 2 are 91% identical and 93 % similar in the kinase domain.

We tested tkr-2 to see whether it could confer stress resistance and increases in life span like tkr-1. Plasmid ptkr-2 (accession number: z49918;ZK938.5) is a Bluescript-based plasmid that carries a 4.1 kb EcoRI fragment from F17E3, containing the entire tkr-2 ORF plus the entire 5' untranslated region plus some of the upstream ORF (total 5' region, 1245 bp). The primary structure was confirmed by sequencing.

The tkr-2 transgenics showed no detectable difference in resistance to heat and UV light (Figure 8) and a weak but statistically significant (p < 0.05) extension of life span (Figure 9). This suggests that stress resistance and life extension is rather specific to tkr-1 but not a general feature of the receptor kinase genes similar to tkr-1.

While exemplary preferred embodiments of the present invention are described herein with particularity, those having ordinary skill in the art will recognize various changes, modifications, additions, and applications other than those specifically described herein, and may adapt the preferred embodiment and methods without departing from the spirit of the invention.

Claims

CLAIMSWhat is claimed is:

1. A method of identifying a polynucleotide associated with longevity, comprising measuring lifespan of a transgenic nematode comprising the polynucleotide, wherein the polynucleotide is identified by its ability to increase the lifespan of the transgenic nematode compared to a suitable control.

2. The method of claim 1, wherein the nematode is C. elegans.

3. The method of claim 1, wherein the polynucleotide is associated with stress response.

4. The method of claim 3, wherein the polynucleotide encodes a receptor.

5. The method of claim 4, wherein the receptor is a kinase receptor.

6. The method of claim 3, wherein the polynucleotide is selected from the group consisting of Jun N-terminal kinase, p38/HOG-l, ELKl, CL100, C-Jun, c-kit, platelet derived growth factor receptors, insulin-like growth factor receptor, epidermal growth and factor receptor.

7. A method of identifying a polynucleotide associated with longevity, said method comprising a) conducting a computer similarity search for a polynucleotide suspected to be involved in stress resistance and b) measuring lifespan of a transgenic nematode comprising the polynucleotide identified in step (a), wherein the polynucleotide is associated with life extension if expression of the polynucleotide confers longer lifespan than a suitable control.

8. A method of identifying an agent that may modulate life-extension comprising contacting a transfected cell comprising a polynucleotide identified in claim 1 or a polypeptide encoded by the polynucleotide identified in claim 1 with the agent, wherein the agent is identified by its ability to modulate a functional characteristic of a life-extension polynucleotide or polypeptide.

9. The method of claim 8, wherein the polynucleotide comprises a region of at least 15 contiguous nucleotides of SEQ ID NO:2.

10. The method of claim 8, wherein the polypeptide comprises a region of at least 10 contiguous amino acids of SEQ ID NO:3.

11. An in vitro method for screening for an agent that may modulate life extension said method comprising the steps of: a) contacting the agent to be tested with a polynucleotide identified in claim 1 or a polypeptide encoded by the polynucleotide identified in claim 1 ; and b) measuring a functional characteristic of said polynucleotide or polypeptide, wherein the agent is identified by its ability to modulate said functional characteristic.

12. The method of claim 11 , wherein the functional characteristic is transcription.

13. The method of claim 11 , wherein the functional characteristic is translation.

14. The method of claim 11 , wherein the functional characteristic is binding to a chemical moiety.

15. The method of claim 11 , wherein the functional characteristic is enzymatic activity.

16. The method of claim 15, wherein the enzymatic activity is kinase activity.

17. The method of claim 11 , wherein the polynucleotide comprises a region of at least 15 contiguous nucleotides of SEQ ID NO: 12.

18. The method of claim 11 , wherein the polypeptide comprises a region of at least 10 contiguous amino acids of SEQ ID NO: 13.

19. A method for screening for an agent that may modulate life extension; said method comprising the steps of: a) contacting the agent to be tested with a transgenic nematode comprising a tkr-1 polynucleotide; and b) measuring lifespan of the nematode, wherein the agent is identified by its ability to modulate lifespan compared to the absence of the agent.

20. The method of claim 19, wherein the tkr-1 polynucleotide comprises a kinase domain from fibroblast growth factor receptor, said kinase domain replacing a kinase domain of tkr-1.

21. The method of claim 20, wherein the kinase domain is from human FGF- Rl.

22. A method for screening for an agent that may modulate life extension, said method comprising the steps of: a) contacting the agent to be tested with a transgenic nematode comprising a polynucleotide encoding the kinase domain of human fibroblast growth factor receptor; and b) measuring a functional characteristic of said kinase domain, wherein the agent is identified by its ability to modulate said functional characteristic.

23. The method of claim 22, wherein the functional characteristic is kinase activity.

24. A transgenic nematode containing a chimeric tkr-1 gene comprised of a human FGF-Rl receptor kinase domain.

25. A chimeric nematode tkr-1 gene comprised of a human FGF-Rl receptor kinase domain.

26. A transgenic nematode comprising a tkr-1 transgene.