WO1998017823A1 - STRUCTURAL AND FUNCTIONAL CONSERVATION OF THE C. ELEGANS CLOCK GENE $i(clk-1) - Google Patents

STRUCTURAL AND FUNCTIONAL CONSERVATION OF THE C. ELEGANS CLOCK GENE $i(clk-1) Download PDF

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WO1998017823A1
WO1998017823A1 PCT/CA1997/000768 CA9700768W WO9817823A1 WO 1998017823 A1 WO1998017823 A1 WO 1998017823A1 CA 9700768 W CA9700768 W CA 9700768W WO 9817823 A1 WO9817823 A1 WO 9817823A1
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clk
leu
ala
ser
val
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PCT/CA1997/000768
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French (fr)
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Siegfried Hekimi
Jonathan Ewbank
Thomas Barnes
Bernard Lakowski
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Mcgill University
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Priority to EP97944665A priority Critical patent/EP0932701A1/en
Priority to CA002268749A priority patent/CA2268749A1/en
Priority to BR9712368-4A priority patent/BR9712368A/en
Priority to JP10518750A priority patent/JP2001502181A/en
Priority to NZ335335A priority patent/NZ335335A/en
Priority to AU46128/97A priority patent/AU743527B2/en
Publication of WO1998017823A1 publication Critical patent/WO1998017823A1/en

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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
    • C07K14/4354—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
    • C07K14/43545—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes from Caenorhabditis
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K67/0335—Genetically modified worms
    • A01K67/0336—Genetically modified Nematodes, e.g. Caenorhabditis elegans
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention relates to the identification of clk-1 and to show that the clk-1 gene complements the clk-1 phenotype and restores normal longevity.
  • clk-1 The activity of the gene clk-1 in the nematode Caenorhabditis elegans controls how fast the worms live and how soon they die.
  • the timing of a wide range of physiological processes is deregulated. This leads to an average lengthening of such diverse processes as the worms 1 early cell cycles, their embryonic and post-embryonic development, and the period of rhythmic adult behaviors, such as swimming, pharyngeal pumping, and defecation (A. Wong et al., Genetics 139, 1247 (1995)).
  • clk-1 mutants also have an extended life-span.
  • clk-1 mutations exhibit a maternal effect: homozygous mutant ( clk- 1/ clk-1 ) progeny from a heterozygous hermaphrodite ( clk-1/ + ) are phenotypically wild type; only homozygous mutants from a homozygous mother exhibit a Clk phenotype.
  • the maternal rescue not only influences early events, such as embryonic development, but extends to adult phenotypes, such as defecation and longevity (A. Wong et al., Genetics 139, 1247 (1995)). This, and other evidence (A. Wong et al .
  • One aim of the present invention is to provide clk-1 and to show that the clk-1 gene complements the clk-1 phenotype and restores normal longevity.
  • the clk-1 gene which complements the clk-1 phenotype and restores normal longevity.
  • clk-1 The cloned clk-1 gene should allow us to better understand its biological function and its potential experimental or pharmaceutical uses. It should also allow us to identify other genes related to clk-1 . If human clk-1 gene is altered in transformed cells or cell lines, it would indicate that it is involved in cancer and could be used for cancer diagnosis.
  • a clk-1 gene which has a function at the level of cellular physiology involved in developmental rate and longevity, wherein clk-1 mutants have a longer life and a altered cellular metabolism relative to the wild-type.
  • a method for the diagnosis and/or prognosis of cancer in a patient which comprises the steps of: a) obtaining a tissue sample from said patient; b) analyzing DNA of the obtained tissue sample of step a) to determine if the human clk-1 gene is altered, wherein alteration of the human clk-1 gene is indicative of cancer.
  • a mouse model of longevity which comprises a gene knock-out of murine clk-1 gene.
  • a method to increase life span of an animal or a patient which comprises the steps of down- regulating the expression of the clk-1 gene and/or homologues thereof.
  • a method of treatment of pathological conditions causing slow down of physiological rate of tissue and/or organ in a patient, which comprises administering an agent to said patient to promote tissue and/or organ specific overexpression of clk-1 gene to increase the physiological rate.
  • Fig. 1 illustrates the cloning of clk-1
  • Fig. 2 illustrates the extension of life-span exhibited by clk ⁇ l ( e2519 ) is rescued by the presence of extrachromosomal arrays containing the wild-type clk-1 gene ;
  • Fig. 3A illustrates the alignment of the nema- tode CLK-1 (SEQ ID NO: 13), rat C0Q7 (SEQ ID NO: 14) and yeast Cat5p/Coq7p sequences (SEQ ID NO: 17), together with the partial sequence of murine (SEQ ID NO: 15) and human CLK-1 (SEQ ID NO: 16) homologues ;
  • Fig. 3B illustrates the duplication within the CLK-1 sequence (SEQ ID NO: 13) and its homologues;
  • Fig. 4 illustrates that the nematode gene clk-1 restores the ability of a yeast Acat5/coq7 null mutant to utilize glycerol as a carbon source;
  • Fig. 5 illustrates the comparison of the TOC-1 C. elegans sequence (SEQ ID NO: 18) with homologous sequences in humans (SEQ ID NO: 19) and mice (SEQ ID NO: 20) ;
  • Fig. 6 illustrates the schematic of the expression construct pPD9577.2op
  • Fig. 7 illustrates the defecation cycle lengths of individual animals of three genotypes.
  • Fig. 8 illustrates the survival over time of the wild type (N2) and animals expressing CLK-1: :GFP and carrying two wild type chromosomal copies of clk-1.
  • clk-1 complements the Acat5/coq7 mutant pheno- type, demonstrating that clk-1 and CAT5/COQ7 share biochemical function and that clk-1 acts at the level of cellular physiology. Furthermore, this supports the idea that animal aging has a basis in cellular metabo- lism.
  • clk-1 lies on linkage group III, between the genes dpy-1 7 and lon-1 (A. Wong et al . , Genetics 139, 1247 (1995); S. Hekimi et al., Genetics 141, 1351 (1995); Fig. 1).
  • Top line shows the genetic map in the clk-1 region. clk-1 had been previously mapped to this region (A.
  • the insert in pRA41 contains three predicted genes, ZC395.10, ZC395.3 and ZC395.2 (ACeDB; F. H. Eeckman et al., Meth . Cell Biol . 48, 583 (1995)).
  • a deletion in ZC395.2 eliminates clk-1 rescuing activity, while pRA40 rescues clk-1 , indicating that ZC395.2 is the clk-1 gene.
  • S Sac I
  • X Xba I
  • E Eco RI.
  • the nematode EST CEESX93F (ACeDB; F. H. Eeckman et al., Meth . Cell Biol . 48, 583 (1995)) matches the 3' end of toc-1 .
  • clk-1 gene is part of an operon of two genes, toc-1 and clk-1.
  • TOC-1 protein encoded by toc-1
  • TOC-1 protein encoded by toc-1
  • a homologous mouse sequence (mtoc-1) was also established from database entries (Genbank accessions: AA03268A, AA033329, AA097624) and is also provided in Fig. 5.
  • Fig. 1 were assayed for their ability to rescue the mutant phenotype on micro-injection into clk-1 mutants.
  • the cosmid ZC400 was found to rescue the mutant phenotype of both strong ( qm30) and weaker ( e2519) clk-1 alleles, and fully recapitulated the maternal effect.
  • ZC400-containing extrachromosomal arrays lost their ability to rescue fully the slow growth phenotype of clk-1 mutants, but the rescue of slow defecation persisted. This loss of rescue of the developmental phenotype probably reflects a ubiquitous phenomenon in C.
  • transgenic arrays undergo transcriptional silencing of arrays in the germline and in early embryos, possibly because of their complex repeated structure (C. Mello and A. Fire, Meth . Cell Biol . 48, 451 (1995)). If so, it implies that later zygotic expression of clk-l ( + ) is sufficient to rescue adult behavioral defects, but maternal or early zygotic expression is needed to rescue slow development.
  • Injection of ZC400 subclones localized clk-1 to a 1.9 kb Eco RI fragment. Extrachromosomal arrays containing this fragment restored developmental and behavioral rates to wild-type speed for at least one generation.
  • Graphs show the percentage of worms alive on a given day after being laid as eggs during a 2.5 hour period on day 0; N2 (D), clk-H e2519 ) (•), e2519; qmExl 09 ( ⁇ ) (Fig. 2).
  • N2 (D) clk-H e2519 )
  • e2519 qmEx96 ( ⁇ )
  • the mean life-spans, with standard errors, are 20.4 ⁇ 0.8, 28.1 ⁇ 1.4, 20.2 ⁇ 0.9 and 20.4 ⁇ 0.7 days, respectively.
  • the worms were maintained at 18°C throughout and their longevity scored as previously described (B. Lakowski et al . , Science 272, 1010 (1996)).
  • Sample size is 50 worms of each genotype, except for e2519; qmExl 09 which is 48.
  • the clk-1 gene is predicted to encode a 187- residue protein, CLK-1, that is similar to the product of the Saccharomyces cerevisiae gene CAT5/COQ7 (Cat5p/Coq7p; M. Proft et al . , EMBO J. 14, 6116 (1995); B. N. Marbois et al., J. Biol . Chem. 271, 2995 (1996)).
  • Cat5p/Coq7p M. Proft et al . , EMBO J. 14, 6116 (1995); B. N. Marbois et al., J. Biol . Chem. 271, 2995 (1996).
  • a rat homolog of Cat5p/Coq7p has also been described (T. Jonassen et al., Arch . Biochem. Bi ophys . 330, 285 (1996)).
  • the three proteins are 33% identical over 177 residues, although their N-termini show no similarity, either in terms of length or composition (Fig. 3A) .
  • CLK-1 is highly conserved between ne atodes, yeast and rodents. Over the length of the rat protein, the identity between CLK-1 and its yeast and rat homologues is 42% and 53%, respectively. Introduced gaps are marked by dashes. Reduction-of-function alleles of both clk-1 and CAT5/COQ7 are known, and both occur in absolutely conserved residues (indicated by arrows here, boxed in B, below); G ⁇ D in coq7-l (B. N. Marbois et al., J. Biol . Chem.
  • the rat sequence (GenBank accession no. U46149) appears to contain sequencing errors in the vicinity of residues 82-84 and 151-154 (marked by dots). Using the sequence of the rat gene, we were able to identify and partially sequence murine and human homologues of clk- 1.
  • the protein sequences can each be split and aligned to reveal the presence of an 82-residue tandemly-repeated core domain, which we call the TRC domain, for tandemly-repeated in CLK-1 (or Cat5p/Coq7p or rat C0Q7 ; Fig. 3B).
  • TRC domain tandemly-repeated in CLK-1 (or Cat5p/Coq7p or rat C0Q7 ; Fig. 3B).
  • Each of the sequences shown in (Fig. 3A) can be split and aligned to reveal the presence of a tandemly repeated TRC domain.
  • Those residues identical in four or more of the six domains are shown in black lettering, those that are similar in four or more of the domains are shown in dark gray. Positions where there is absolute conservation of a hydrophobic residue are marked by a
  • the two-domain structure seen in the proteins ' primary structure is expected to be reflected by two equivalent domains at the level of the proteins' tertiary structure; it is likely that these proteins evolved from a precursor that contained just one domain, but that was able to homodimerize .
  • the two strongest clk-1 alleles are qm30, a 590 bp deletion that eliminates 35 codons, including the entire last exon, together with all of the 3' untranslated region of the gene, and qm ⁇ l , a point mutation that eliminates the splice acceptor site of the second intron (Fig. 1).
  • the less severe allele e2519 is a point mutation that results in a glutamic acid to lysine change (E148K; Figs. 1 & 3).
  • CAT5/COQ7 appears to be involved in the control of expression of other enzymes of glu- coneogenesis, and those of respiration and the glyoxy- late cycle (M. Proft et al . , EMBO J. 14, 6116 (1995)). But its role in all these processes appears to be indirect and likely part of a complex regulatory mechanism. For example, CAT5/COQ7 is subject to partial glucose repression, and its expression under derepressing con- ditions requires the activities of CATl/SNFl and CAT8 as well as CAT5/COQ7 itself (M. Proft et al., EMBO J. 14, 6116 (1995)).
  • CAT5/COQ7 has also been characterized as being involved in ubiquinone (coenzyme Q) biosynthesis (B. N. Marbois et al., J. Biol . Chem. 271, 2995 (1996)). cat5/coq7 mutants do not synthesize this lipid-soluble two-electron carrier, which is essential for non-fermentative growth.
  • Chemical characterization of the ubiquinone biosynthetic intermediates in a yeast strain with a point mutation in CAT5/COQ7 (B. N. Marbois et al., J. Biol . Chem. 271, 2995 (1996); Fig.
  • CAT5/COQ7 The pleiotropic effects of mutation of CAT5/COQ7 have led to the proposal that there is a co-regulation of respiratory chain components, the biogenesis of mitochondria, and gluconeogenesis, with CAT5/COQ7 being a likely link connecting glucose derepression with respiration (M. Proft et al . , EMBO J. 14, 6116 (1995)).
  • CAT5/COQ7 appears to be important in the regulation of multiple parallel processes of metabolism. This is consistent with our view of clk-1 regulating many disparate physiological and metabolic processes in C. elegans .
  • yeast manipulations were in the SEY6210 background (MATa , leu2-3, ura3-52, his3-A200, lys2-801 , trpl -A901 , suc2-A9 ) .
  • Yeast cells were grown under standard conditions (YNB, YEPD and YEPG). Strains were transformed using the lithium acetate procedure with sheared, denatured carrier DNA.
  • the CAT5/COQ7 locus was disrupted using a PCR-mediated approach (the primers used were ML134 and ML135).
  • the CAT5/COQ7 gene was entirely replaced with a DNA fragment containing a disruption module encoding the Green Fluorescent Protein and the HIS3 gene (R. K.
  • the primer used were ML134 , TTTTCATATACGGGATTTTCAGGAAAAAAAACAATAGAAATCTAT- AAAACATGAGTAAAGGAGAAGAAC (SEQ ID N0:1); ML135, CCGTT- TTCCTTTCAATTCTCCTTTTCTGGCATAACGCGACTGATGTATGCCACGCGCGCC TCGTTCAGAATG (SEQ ID NO: 2); ML136, CGTACTCTGTCTATAT- TTCCC (SEQ ID NO:3); ML137, GCGTTAAAATGCGTAAGGATG (SEQ ID NO:4); ML138, CCACTTGCCACCTATCACC (SEQ ID NO : 5 ) .
  • elegans clk-1 coding sequence within an expression cassette which includes the constitutive promoter and 3' sequence of the ADH1 gene, conferred the ability to grow on glycerol to the Acat5/coq7 strain (Fig. 4).
  • a wild-type strain (A. Wong et al . , Genetics 139, 1247 (1995)) or yeast cells harboring a deletion of the CAT5/COQ7 gene and containing plasmid pVT102-U (multicopy and constitutive ADH1 promoter) with a CAT5/COQ7 insert (S. Hekimi et al . , Geneti cs 141, 1351 (1995)), clk-1 insert (B. Lakowski et al .
  • the CAT5/COQ7 locus was directly amplified from yeast genomic DNA by PCR using Pfu polymerase (Stratagene) and primers SHP69 and SHP70.
  • a cDNA corresponding to the entire clk-1 coding sequence was ob- tained by PCR amplification, also with Pfu polymerase, and nested primer pairs SHP57/SHP59 and SHP57/SHP58 on single-stranded cDNA that had been synthesized by priming with SHP59.
  • the respective yeast and nematode PCR products were digested with Hind III and ligated to Hind Ill-cut and dephosphorylated pVT102-U (T. Vernet et al., Gene 52, 225 (1987)).
  • both the CAT5/COQ7 and clk-1 containing plasmids restored the ability of the ⁇ cat5/coq7 strain to grow on ethanol (YEPE3 medium).
  • the primers used were SHP57, CAGAAGCTTCCCAGTTACTCAA- GATGTTCCG (SEQ ID NO : 6 ) ; SHP58, CAGAAGCTTTGTTCAAAT- TTTCTCAGC (SEQ ID NO : 7 ) ; SHP59, ATGGAAGAAAAGGGACAC (SEQ ID NO: 8); SHP69, CAGAAGCTTCTATAAAACATGTTTCC (SEQ ID N0:9); SHP70, CAGAAGCTTAAATTCTTTCGGCACTCC (SEQ ID N0:10) .
  • clk-1 affects the longevity of C. elegans .
  • the functional complementation of ⁇ cat5/coq7 by clk-1 in yeast cells highlights one other aspect of the character of this gene: it acts at the level of a single cell (a phenotype previously noted in single-celled C. elegans eggs; A. Wong et al., Genetics 139, 1247 (1995)).
  • a number of observations suggest that the cellular equivalent of organismal aging is a limit to proliferative capacity (known as cellular senescence; reviewed in J. Campisi, Cell 84, 497 (1996); L. Guarente, Cell 86, 9 (1996)). Consequently it will be of interest to determine whether ⁇ cat5/coq7 mutants live longer than wild-type yeast cells .
  • Reporter gene expressing a CLK-1: :GFP fusion protein We have also constructed a reporter gene expressing a fusion protein containing the entire CLK-1 amino acid sequence fused at the C-terminal end to green fluorescent protein (GFP). The reporter gene expresses the protein in the context of the clk-1 operon (Fig. 6).
  • This construct utilized the vector pPD95.77 (gift from Dr. Andrew Fire) which was designed to allow a protein of interest to be transcriptionally fused to Green Fluorescent Protein (GFP).
  • GFP Green Fluorescent Protein
  • the clk-1 operon (including a 5* upstream region presumably containing the full promoter as it extends to the next predicted gene, toc-1, clk-1 and the toc-l/cl ⁇ -1 inter- genie region) was amplified by the polymerase chain reaction (PCR) and cloned into pPD95.77.
  • the primers used were SHP121 (AAAAGGGTCGACCGAAAAGAGAACTAGACGG (SEQ ID N0:1D) and SHP122 (AAGGGGATCCAATTTTCTCAGCAATCGC (SEQ ID N0:12)). These primers had restriction sites, Sail and BamHI respectively, built into their 5' ends to allow the directed cloning of the amplified product.
  • exonic DNA is represented by boxes; shading indicates coding sequence.
  • the clk-1 gene is included in its entirety, with the exception of the termination codon which was excluded to allow translation through gfp.
  • the hashed region in the figure between clk-1 and gfp is part of the vector's multiple cloning site (102 base pairs) and codes for 34 amino acids which provide a linker between CLK-1 and GFP.
  • the reporter construct fully rescues the phenotype of a clA;-l(qm30) mutant upon injection and extrachromosomal array formation, indicating that the fusion to the GFP moiety does not significantly inhibit the function of CLK-1.
  • clk-1 is expressed in all cells at all stages.
  • fluorescence microscopy showed that the CLK-1: :GFP fusion protein is localized to mitochondria.
  • the localization was confirmed by co-localization with a mitochondrion-specific vital dye, G6-rhodamine.
  • the reporter gene was also injected into wild type worms and individuals carrying extrachromosomal arrays were examined for phenotypic alterations .
  • the defecation cycle was examined at two times (day 1 and day 4 of adulthood) in three genotypes (wild type, cJ7-l(qm30) and animals expressing CLK-1: :GFP in a wild type background (Fig. 7)).
  • tissue- or organ-specific overexpression of clk-1 could allow one to increase the physiological rate of tissues or organs whose functional rates are slowed because of a pathological condition.
  • MOLECULE TYPE cDNA
  • Ala Asp Arg lie Tyr Ala Gly Gin Leu Ala Val Leu Gin Gly Ser Ser
  • Glu Leu lie Gly Gin His Tyr Asn Asp Gin Leu Lys Glu Leu Leu Ala
  • MOLECULE TYPE protein

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Abstract

The present invention relates to a clk-1 gene which has a function at the level of cellular physiology involved in developmental rate and longevity, wherein clk-1 mutants have a longer life and a altered cellular metabolism relative to the wild-type. There is also provided a method for the diagnosis and/or prognosis of cancer in a patient, which comprises the steps of: a) obtaining a tissue sample from the patient; b) analyzing DNA of the obtained tissue sample of step a) to determine if the human clk-1 gene is altered, wherein alteration of the human clk-1 gene is indicative of cancer. There is also provided a method of treatment of pathological conditions causing slowdown of physiological rate of tissue and/or organ in a patient.

Description

STRUCTURAL AND FUNCTIONAL CONSERVATION OF THE C. ELEGANS CLOCK GENE clk-1
BACKGROUND OF THE INVENTION
(a) Field of the Invention The invention relates to the identification of clk-1 and to show that the clk-1 gene complements the clk-1 phenotype and restores normal longevity.
(b) Description of Prior Art
The activity of the gene clk-1 in the nematode Caenorhabditis elegans controls how fast the worms live and how soon they die. In clk-1 mutants, the timing of a wide range of physiological processes is deregulated. This leads to an average lengthening of such diverse processes as the worms1 early cell cycles, their embryonic and post-embryonic development, and the period of rhythmic adult behaviors, such as swimming, pharyngeal pumping, and defecation (A. Wong et al., Genetics 139, 1247 (1995)). clk-1 mutants also have an extended life-span. This pleiotropic alteration of developmental and behavioral timing defines the Clock (Clk) phenotype, also exhibited by worms carrying mutations in any one of the genes clk-2, clk-3 and gro-1 (A. Wong et al., Genetics 139, 1247 (1995); S. Hekimi et al., Genetics 141, 1351 (1995)). Mutations in these four genes interact genetically to affect developmental rate and longevity (B. Lakowski et al., Science 272, 1010 (1996)).
Many of the phenotypes of clk-1 mutant worms result from changes in processes believed to be con- trolled by discrete biological clocks (A. Wong et al., Genetics 139, 1247 (1995)). These procedures have widely different periods, from the sub-second time scale to hours, and were not previously thought to be connected in any mechanistic way (A. Wong et al., Genetics 139, 1247 (1995)). We have speculated that clk-1 mutations affect the function of a central bio- logical clock that would ensure control, and temporal coordination, of these different processes, and govern the response of the organism to changes in temperature (A. Wong et al . , Genetics 139, 1247 (1995)). One key feature of the clk-1 phenotype is that clk-1 mutations exhibit a maternal effect: homozygous mutant ( clk- 1/ clk-1 ) progeny from a heterozygous hermaphrodite ( clk-1/ + ) are phenotypically wild type; only homozygous mutants from a homozygous mother exhibit a Clk phenotype. The maternal rescue not only influences early events, such as embryonic development, but extends to adult phenotypes, such as defecation and longevity (A. Wong et al., Genetics 139, 1247 (1995)). This, and other evidence (A. Wong et al . , Genetics 139, 1247 (1995); S. M. Jazwinski, Science 273, 54 (1996)) suggests the existence of a pervasive timing mechanism, a "clock", whose intrinsic rate is determined, or "set", early in development and that subsequently influences diverse timed processes throughout the worm's life.
It would be highly desirable to be provided with clk-1 and to show that clk-1 gene complements the clk-1 phenotype and restores normal longevity.
SUMMARY OF THE INVENTION
One aim of the present invention is to provide clk-1 and to show that the clk-1 gene complements the clk-1 phenotype and restores normal longevity.
In accordance with the present invention there is provided the clk-1 gene which complements the clk-1 phenotype and restores normal longevity.
The cloned clk-1 gene should allow us to better understand its biological function and its potential experimental or pharmaceutical uses. It should also allow us to identify other genes related to clk-1 . If human clk-1 gene is altered in transformed cells or cell lines, it would indicate that it is involved in cancer and could be used for cancer diagnosis.
In accordance with the present invention there is provided a clk-1 gene which has a function at the level of cellular physiology involved in developmental rate and longevity, wherein clk-1 mutants have a longer life and a altered cellular metabolism relative to the wild-type.
In accordance with the present invention there is provided a method for the diagnosis and/or prognosis of cancer in a patient, which comprises the steps of: a) obtaining a tissue sample from said patient; b) analyzing DNA of the obtained tissue sample of step a) to determine if the human clk-1 gene is altered, wherein alteration of the human clk-1 gene is indicative of cancer.
In accordance with the present invention there is provided a mouse model of longevity, which comprises a gene knock-out of murine clk-1 gene. In accordance with the present invention there is provided a method to increase life span of an animal or a patient, which comprises the steps of down- regulating the expression of the clk-1 gene and/or homologues thereof. In accordance with the present invention there is provided a method of treatment of pathological conditions causing slow down of physiological rate of tissue and/or organ in a patient, which comprises administering an agent to said patient to promote tissue and/or organ specific overexpression of clk-1 gene to increase the physiological rate.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 illustrates the cloning of clk-1 ; Fig. 2 illustrates the extension of life-span exhibited by clk~l ( e2519 ) is rescued by the presence of extrachromosomal arrays containing the wild-type clk-1 gene ;
Fig. 3A illustrates the alignment of the nema- tode CLK-1 (SEQ ID NO: 13), rat C0Q7 (SEQ ID NO: 14) and yeast Cat5p/Coq7p sequences (SEQ ID NO: 17), together with the partial sequence of murine (SEQ ID NO: 15) and human CLK-1 (SEQ ID NO: 16) homologues ;
Fig. 3B illustrates the duplication within the CLK-1 sequence (SEQ ID NO: 13) and its homologues; Fig. 4 illustrates that the nematode gene clk-1 restores the ability of a yeast Acat5/coq7 null mutant to utilize glycerol as a carbon source;
Fig. 5 illustrates the comparison of the TOC-1 C. elegans sequence (SEQ ID NO: 18) with homologous sequences in humans (SEQ ID NO: 19) and mice (SEQ ID NO: 20) ;
Fig. 6 illustrates the schematic of the expression construct pPD9577.2op;
Fig. 7 illustrates the defecation cycle lengths of individual animals of three genotypes; and
Fig. 8 illustrates the survival over time of the wild type (N2) and animals expressing CLK-1: :GFP and carrying two wild type chromosomal copies of clk-1.
DETAILED DESCRIPTION OF THE INVENTION
Mutations in the Caenorhabdi tis elegans gene clk-1 affect biological timing and extend longevity. In accordance with the present invention, we report the identification of clk-1 and show that the cloned gene complements the clk-1 phenotype and restores normal longevity. CLK-1 is conserved among eukaryotes, including humans, and is structurally similar to the yeast metabolic regulator Cat5p/Coq7p. These proteins contain a tandem duplication of a core 82-residue (TRC) domain. clk-1 complements the Acat5/coq7 mutant pheno- type, demonstrating that clk-1 and CAT5/COQ7 share biochemical function and that clk-1 acts at the level of cellular physiology. Furthermore, this supports the idea that animal aging has a basis in cellular metabo- lism. clk-1 lies on linkage group III, between the genes dpy-1 7 and lon-1 (A. Wong et al . , Genetics 139, 1247 (1995); S. Hekimi et al., Genetics 141, 1351 (1995); Fig. 1). Top line shows the genetic map in the clk-1 region. clk-1 had been previously mapped to this region (A. Wong et al., Genetics 139, 1247 (1995); S. Hekimi et al . , Genetics 141, 1351 (1995)) and we refined its location to give the map position shown in Fig. 1. Some cosmids tested for rescuing activity are shown here (C. Mello and A. Fire, Meth . Cell Biol . 48, 451 (1995)). Stable rescue that persisted for many generations was obtained only with ZC400. + indicates rescue of all phenotypes; ± indicates rescue of just the defecation phenotype; - indicates no rescue. pRA41 is a derivative of ZC400 with an internal Sac I deletion. The insert in pRA41 contains three predicted genes, ZC395.10, ZC395.3 and ZC395.2 (ACeDB; F. H. Eeckman et al., Meth . Cell Biol . 48, 583 (1995)). A deletion in ZC395.2 eliminates clk-1 rescuing activity, while pRA40 rescues clk-1 , indicating that ZC395.2 is the clk-1 gene. S = Sac I X = Xba I, E = Eco RI. The nematode EST CEESX93F (ACeDB; F. H. Eeckman et al., Meth . Cell Biol . 48, 583 (1995)) matches the 3' end of toc-1 .
We have first described that the clk-1 gene is part of an operon of two genes, toc-1 and clk-1. However, we had few details about the protein (TOC-1) encoded by toc-1, except that it is somewhat similar to members of a family of divalent cation transporting transmembrane transporters, the CDF family (Paulsen I.T. and Saier M.H., J. Membr . Biol., 156:99-103 (1997)). We have now identified by database searches a true human homologue of toc-1 which we call htoc-1. We have used several database entries (Genbank accessions: AA214573, AA134438, AA134439, AA213498, AA505430) to obtain a consensus sequence which predicts a protein with high homology to TOC-1 (Fig. 5). Identical residues are shown in bold and are indicated by a dot above the residue. For humans the middle part of the sequence is not yet available and for mice only the C- terminal part of the sequence is available at this time.
A homologous mouse sequence (mtoc-1) was also established from database entries (Genbank accessions: AA03268A, AA033329, AA097624) and is also provided in Fig. 5.
We resequenced clk-1 from e2519 , qm30 and qm.51 . e2519, a G→A transition introduces a new Hind III site, and causes an E→K missense alteration in an absolutely conserved residue (see Fig. 3). qm30 results in a 590- bp deletion, starting 12 nucleotides 3' of the lesion in e2519, and encompassing the entire last exon. qmδl alters the absolutely conserved terminal G in the intron 2 splice acceptor. Sequencing of allele qmll , which has a phenotype essentially identical to e2519, revealed an identical lesion. The low probability of independently obtaining the same mutation twice suggests that the original allele was lost. Overlapping cosmids from the candidate region
(Fig. 1) were assayed for their ability to rescue the mutant phenotype on micro-injection into clk-1 mutants. The cosmid ZC400 was found to rescue the mutant phenotype of both strong ( qm30) and weaker ( e2519) clk-1 alleles, and fully recapitulated the maternal effect. Over a number of generations (10-20), ZC400-containing extrachromosomal arrays lost their ability to rescue fully the slow growth phenotype of clk-1 mutants, but the rescue of slow defecation persisted. This loss of rescue of the developmental phenotype probably reflects a ubiquitous phenomenon in C. elegans, where transgenic arrays undergo transcriptional silencing of arrays in the germline and in early embryos, possibly because of their complex repeated structure (C. Mello and A. Fire, Meth . Cell Biol . 48, 451 (1995)). If so, it implies that later zygotic expression of clk-l ( + ) is sufficient to rescue adult behavioral defects, but maternal or early zygotic expression is needed to rescue slow development. Injection of ZC400 subclones localized clk-1 to a 1.9 kb Eco RI fragment. Extrachromosomal arrays containing this fragment restored developmental and behavioral rates to wild-type speed for at least one generation. Stably transformed lines, however, showed no amelioration of the slow-growth phenotype, but we observed that as adults these worms defecated at a wild-type rate. This fragment is predicted to contain a single gene, ZC395.2, which is altered in three clk-1 alleles, thereby confirming the identity of this gene as clk-1 (Fig. 1). The weaker clk-1 allele is a missense mutation; the stronger ones involve the disruption of entire exons .
We previously demonstrated that all phenotypes of clk-1 mutants can be fully maternally rescued, that all alleles exhibit the same pattern of phenotypes and that all alleles fail to complement each other for all phenotypes. Given this, the molecular evidence presented here unequivocally established that mutations in the clk-1 gene are responsible for all of the pheno- types seen in clk-1 mutant worms. The clk-1 gene lies just downstream of the predicted gene, ZC395.3 (Fig. 1). Using reverse transcription PCR, we established the 5* and 3' ends of both genes and their splicing patterns. We found that clk-1 is exclusively trans-spliced (D.A. Zorio et al., Nature 372, 270 (1994); J. Spieth et al . , Cell 73, 521 (1993)) to the splice leader SL2 at its 5' end, while the upstream gene is trans-spliced to SL1, in both cases immediately upstream of the initiator AUG codon. The 3' end of the upstream genes lies 105 bp from the 5' end of clk-1 . The pattern of trans-splicing and the intergenic distance are typical of genes organized into operons in C. elegans (T. Blumenthal, Trends Genet . 11, 132 (1995)), suggesting that the two genes share a pro- moter 5' of the upstream gene. The introns in clk-1 were correctly predicted, but the real product of ZC395.3 (Fig. IC) lacks the first predicted exon. This gene potentially encodes a protein that has similarity to a family of divalent metal ion transporters, so we have named it toc-1 , for transporter-like protein in an operon with £lk-l . Although there are cases of functionally related genes occurring together in operons in C. elegans and being coordinately expressed (T. Blumenthal, Trends Genet . 11, 132 (1995)), there is no obvious functional relationship between clk-1 (see below) and toc-1 .
One phenotype of clk-1 mutants is their extended life-span. To demonstrate that the lesion in clk- l ( e2519 ) was responsible for the mutant worms' extended longevity, rather than it resulting from a difference in genetic background, we assayed the longevity of fully-rescued e2519 mutant worms carrying the ZC400- containing extrachromosomal array qmExl 09, as well as those carrying qmEx96 extrachromosomal array contain- ing the clk-1 1.9 kb Eco RI fragment. As adults, e2519; qmEx96 worms show full rescue of their defecation cycle even though their development is slow. We found that the presence of either qmEx96 or qmExl 09 restores a wild-type life-span to e2519 mutant worms. The life-span of e2519; qmExl 09 worms was indistinguishable from that of N2 worms (Fig. 2). The extension of life-span exhibited by clk-l ( e2519 ) is rescued by the presence of an extrachromosomal array containing the wild-type clk-1 gene. Graphs show the percentage of worms alive on a given day after being laid as eggs during a 2.5 hour period on day 0; N2 (D), clk-H e2519 ) (•), e2519; qmExl 09 (Δ) (Fig. 2). For e2519 ; qmEx96 (Δ) a 6-hour limited hatching was used. The mean life-spans, with standard errors, are 20.4±0.8, 28.1±1.4, 20.2±0.9 and 20.4±0.7 days, respectively. The worms were maintained at 18°C throughout and their longevity scored as previously described (B. Lakowski et al . , Science 272, 1010 (1996)). Sample size is 50 worms of each genotype, except for e2519; qmExl 09 which is 48.
The clk-1 gene is predicted to encode a 187- residue protein, CLK-1, that is similar to the product of the Saccharomyces cerevisiae gene CAT5/COQ7 (Cat5p/Coq7p; M. Proft et al . , EMBO J. 14, 6116 (1995); B. N. Marbois et al., J. Biol . Chem. 271, 2995 (1996)). A rat homolog of Cat5p/Coq7p has also been described (T. Jonassen et al., Arch . Biochem. Bi ophys . 330, 285 (1996)). The three proteins are 33% identical over 177 residues, although their N-termini show no similarity, either in terms of length or composition (Fig. 3A) . CLK-1 is highly conserved between ne atodes, yeast and rodents. Over the length of the rat protein, the identity between CLK-1 and its yeast and rat homologues is 42% and 53%, respectively. Introduced gaps are marked by dashes. Reduction-of-function alleles of both clk-1 and CAT5/COQ7 are known, and both occur in absolutely conserved residues (indicated by arrows here, boxed in B, below); G→D in coq7-l (B. N. Marbois et al., J. Biol . Chem. 271, 2995 (1996)) and E-»K in e2519. The rat sequence (GenBank accession no. U46149) appears to contain sequencing errors in the vicinity of residues 82-84 and 151-154 (marked by dots). Using the sequence of the rat gene, we were able to identify and partially sequence murine and human homologues of clk- 1.
Over the available predicted sequences of 43 and 126 amino acids, the human and mouse proteins are 93% and 97% identical to the rat protein, respectively (Fig. 3). These five proteins are unrelated to any other known sequence, and there are few indications as to their biochemical function.
The protein sequences can each be split and aligned to reveal the presence of an 82-residue tandemly-repeated core domain, which we call the TRC domain, for tandemly-repeated in CLK-1 (or Cat5p/Coq7p or rat C0Q7 ; Fig. 3B). Each of the sequences shown in (Fig. 3A) can be split and aligned to reveal the presence of a tandemly repeated TRC domain. There is a single site of insertions for both N- and C-terminal domains; these have been removed for this alignment, as marked by the small black dots. Those residues identical in four or more of the six domains are shown in black lettering, those that are similar in four or more of the domains are shown in dark gray. Positions where there is absolute conservation of a hydrophobic residue are marked by a φ.
Overall, for all repeats, residues are absolutely conserved at 8 positions, and at an additional 12 positions all residues are similar. For each pro- tein, its two TRC domains are juxtaposed without any linking sequence. For each domain, there appears to be only a single point at which insertions are tolerated, flanked by regions predicted to be helical (Fig. 3B). Within these helical regions, (residues 34-56 and 116- 144 for CLK-1) the spacing of conserved hydrophobic residues is suggestive of an interface for protein- protein interaction, such as a surface for dimerization (Fig. 3B). The two-domain structure seen in the proteins ' primary structure is expected to be reflected by two equivalent domains at the level of the proteins' tertiary structure; it is likely that these proteins evolved from a precursor that contained just one domain, but that was able to homodimerize .
The two strongest clk-1 alleles (A. Wong et al . , Genetics 139, 1247 (1995)) are qm30, a 590 bp deletion that eliminates 35 codons, including the entire last exon, together with all of the 3' untranslated region of the gene, and qmδl , a point mutation that eliminates the splice acceptor site of the second intron (Fig. 1). The less severe allele e2519 is a point mutation that results in a glutamic acid to lysine change (E148K; Figs. 1 & 3). As the phenotypes measured in e2519 worms are quantitatively intermediate between wild type and qm30, there would appear to be a correlation between the severity of the Clk phenotype and the amount of residual clk-1 gene activity, relative to that in wild-type worms.
What is the function of the yeast homolog CAT5/COQ7 ? Wild type yeast grow most rapidly with glucose as a carbon source. When glucose is present, the expression of many genes, including those involved in glu- coneogenesis, is strongly repressed. When yeast are transferred to a non-fermentable carbon source, such as glycerol or ethanol, the derepression of PCK1 , which encodes the gluconeogenic enzyme phospho-enolpyruvate carboxykinase, requires the activity of CAT5/COQ7 (M. Proft et al., EMBO J. 14, 6116 (1995)). This derepres- sion of PCK1 is mediated by a carbon source-responsive element (CSRE) in its promoter (M. Proft et al . , EMBO J. 14, 6116 (1995)). As well as being necessary for the formation of a specific CSRE transcriptional activation complex, CAT5/COQ7 appears to be involved in the control of expression of other enzymes of glu- coneogenesis, and those of respiration and the glyoxy- late cycle (M. Proft et al . , EMBO J. 14, 6116 (1995)). But its role in all these processes appears to be indirect and likely part of a complex regulatory mechanism. For example, CAT5/COQ7 is subject to partial glucose repression, and its expression under derepressing con- ditions requires the activities of CATl/SNFl and CAT8 as well as CAT5/COQ7 itself (M. Proft et al., EMBO J. 14, 6116 (1995)).
CAT5/COQ7 has also been characterized as being involved in ubiquinone (coenzyme Q) biosynthesis (B. N. Marbois et al., J. Biol . Chem. 271, 2995 (1996)). cat5/coq7 mutants do not synthesize this lipid-soluble two-electron carrier, which is essential for non-fermentative growth. Chemical characterization of the ubiquinone biosynthetic intermediates in a yeast strain with a point mutation in CAT5/COQ7 (B. N. Marbois et al., J. Biol . Chem. 271, 2995 (1996); Fig. 3) revealed the accumulation of 5-demethoxyubiquinone, a late intermediate in the biosynthetic pathway. Strains in which CAT5/C0Q7 was deleted, however, are reported to accumulate 3-hexaprenyl-4-hydroxybenzoic acid, an earlier intermediate (B. N. Marbois et al., J. Biol . Chem. 271, 2995 (1996)). Thus CAT5/C0Q7 appears to control ubiquinone synthesis at two or more steps, although its mode of action is obscure. The pleiotropic effects of mutation of CAT5/COQ7 have led to the proposal that there is a co-regulation of respiratory chain components, the biogenesis of mitochondria, and gluconeogenesis, with CAT5/COQ7 being a likely link connecting glucose derepression with respiration (M. Proft et al . , EMBO J. 14, 6116 (1995)). Thus CAT5/COQ7 appears to be important in the regulation of multiple parallel processes of metabolism. This is consistent with our view of clk-1 regulating many disparate physiological and metabolic processes in C. elegans . To test whether the structural similarity extended to functional equivalence, we constructed a Δ cat5/coq7 yeast strain, which as expected (M. Proft et al., EMBO J. 14, 6116 (1995); B. N. Marbois et al . , J. Biol . Chem. 271, 2995 (1996)) failed to grow on glyc- erol.
All yeast manipulations were in the SEY6210 background (MATa , leu2-3, ura3-52, his3-A200, lys2-801 , trpl -A901 , suc2-A9 ) . Yeast cells were grown under standard conditions (YNB, YEPD and YEPG). Strains were transformed using the lithium acetate procedure with sheared, denatured carrier DNA. The CAT5/COQ7 locus was disrupted using a PCR-mediated approach (the primers used were ML134 and ML135). The CAT5/COQ7 gene was entirely replaced with a DNA fragment containing a disruption module encoding the Green Fluorescent Protein and the HIS3 gene (R. K. Niedenthal et al., Yeast 12, 773 (1996)). Haploid cells were transformed with the PCR product and HIS3 integrants were selected on minimal medium lacking histidine. Gene disruptions were confirmed by PCR analysis using primers ML136, ML137 and ML138. The Acat5/coq7 strain failed to grow on YEPG, or YEPE3 which contains ethanol (M. Proft et al., EMBO J. 14, 6116 (1995)). The primer used were ML134 , TTTTCATATACGGGATTTTCAGGAAAAAAAACAATAGAAATCTAT- AAAACATGAGTAAAGGAGAAGAAC (SEQ ID N0:1); ML135, CCGTT- TTCCTTTCAATTCTCCTTTTCTGGCATAACGCGACTGATGTATGCCACGCGCGCC TCGTTCAGAATG (SEQ ID NO: 2); ML136, CGTACTCTGTCTATAT- TTCCC (SEQ ID NO:3); ML137, GCGTTAAAATGCGTAAGGATG (SEQ ID NO:4); ML138, CCACTTGCCACCTATCACC (SEQ ID NO : 5 ) . Introduction of a multicopy plasmid containing the C. elegans clk-1 coding sequence, within an expression cassette which includes the constitutive promoter and 3' sequence of the ADH1 gene, conferred the ability to grow on glycerol to the Acat5/coq7 strain (Fig. 4). A wild-type strain (A. Wong et al . , Genetics 139, 1247 (1995)) or yeast cells harboring a deletion of the CAT5/COQ7 gene and containing plasmid pVT102-U (multicopy and constitutive ADH1 promoter) with a CAT5/COQ7 insert (S. Hekimi et al . , Geneti cs 141, 1351 (1995)), clk-1 insert (B. Lakowski et al . , Science 272, 1010 (1996)) or alone (S. M. Jazwinski, Science 273, 54 (1996)) were tested for growth on YEP plates containing 3% glycerol, and incubated at 30°C for seven days. Transformants were grown on selective medium containing 2% glucose prior to streaking.
The CAT5/COQ7 locus was directly amplified from yeast genomic DNA by PCR using Pfu polymerase (Stratagene) and primers SHP69 and SHP70. A cDNA corresponding to the entire clk-1 coding sequence was ob- tained by PCR amplification, also with Pfu polymerase, and nested primer pairs SHP57/SHP59 and SHP57/SHP58 on single-stranded cDNA that had been synthesized by priming with SHP59. The respective yeast and nematode PCR products were digested with Hind III and ligated to Hind Ill-cut and dephosphorylated pVT102-U (T. Vernet et al., Gene 52, 225 (1987)). They were separately transformed into competent TGI cells and the desired recombinant plasmids recovered and each re-transformed into the Acat5/coq7 strain. As well as restoring growth on glycerol (Fig. 4), both the CAT5/COQ7 and clk-1 containing plasmids restored the ability of the Δ cat5/coq7 strain to grow on ethanol (YEPE3 medium). The primers used were SHP57, CAGAAGCTTCCCAGTTACTCAA- GATGTTCCG (SEQ ID NO : 6 ) ; SHP58, CAGAAGCTTTGTTCAAAT- TTTCTCAGC (SEQ ID NO : 7 ) ; SHP59, ATGGAAGAAAAGGGACAC (SEQ ID NO: 8); SHP69, CAGAAGCTTCTATAAAACATGTTTCC (SEQ ID N0:9); SHP70, CAGAAGCTTAAATTCTTTCGGCACTCC (SEQ ID N0:10) .
This functional complementation of the Acat5/ coq7 null by clk-1 is consistent with the reported functional complementation of the yeast mutant by the rat homolog (T. Jonassen et al., Arch . Biochem. Biophys . 330, 285 (1996)) and is indicative of a common biochemical function for these three genes. In spite of their common biochemical function and roles in regulatory mechanisms, one must be cautious in attempting to understand the physiological defects seen in clk-1 worms in terms of the phenotypic defects of cat5/coq7 mutant strains since yeast have some highly specialized systems of metabolic regulation not seen in most other eukaryotes . We are currently investigating, however, whether glucose metabolism and ubiquinone biosynthesis are affected in clk-1 mutants. Nevertheless, the interspecific functional complemen- tation raises the possibility that a central mechanism of metabolic coordination, which regulates distinct downstream regulators, is conserved in all eukaryotes, including humans.
In accordance with the present invention, we have shown that clk-1 affects the longevity of C. elegans . The functional complementation of Δ cat5/coq7 by clk-1 in yeast cells, however, highlights one other aspect of the character of this gene: it acts at the level of a single cell (a phenotype previously noted in single-celled C. elegans eggs; A. Wong et al., Genetics 139, 1247 (1995)). A number of observations suggest that the cellular equivalent of organismal aging is a limit to proliferative capacity (known as cellular senescence; reviewed in J. Campisi, Cell 84, 497 (1996); L. Guarente, Cell 86, 9 (1996)). Consequently it will be of interest to determine whether Δ cat5/coq7 mutants live longer than wild-type yeast cells .
In conclusion, we have identified the C. elegans gene clk-1 and shown that it is structurally and functionally homologous to a yeast central metabolic regulator. This supports our previous speculation that the long life of clk-1 mutants might be a consequence of slower cellular metabolism, with an attendant reduction in the rate of production of detrimental byproducts (B. Lakowski et al . , Science 272, 1010 (1996)). Our findings also lend support to the idea that multicellular organisms age because their cells age (J. Campisi, Cell 84, 497 (1996); L. Guarente, Cell 86, 9 (1996)).
The present invention will be more readily understood by referring to the following example which is given to illustrate the invention rather than to limit its scope. EXAMPLE I
Reporter gene expressing a CLK-1: :GFP fusion protein We have also constructed a reporter gene expressing a fusion protein containing the entire CLK-1 amino acid sequence fused at the C-terminal end to green fluorescent protein (GFP). The reporter gene expresses the protein in the context of the clk-1 operon (Fig. 6).
This construct utilized the vector pPD95.77 (gift from Dr. Andrew Fire) which was designed to allow a protein of interest to be transcriptionally fused to Green Fluorescent Protein (GFP). In this case, the clk-1 operon (including a 5* upstream region presumably containing the full promoter as it extends to the next predicted gene, toc-1, clk-1 and the toc-l/clλ-1 inter- genie region) was amplified by the polymerase chain reaction (PCR) and cloned into pPD95.77. The primers used were SHP121 (AAAAGGGTCGACCGAAAAGAGAACTAGACGG (SEQ ID N0:1D) and SHP122 (AAGGGGATCCAATTTTCTCAGCAATCGC (SEQ ID N0:12)). These primers had restriction sites, Sail and BamHI respectively, built into their 5' ends to allow the directed cloning of the amplified product. In the figure, exonic DNA is represented by boxes; shading indicates coding sequence. The clk-1 gene is included in its entirety, with the exception of the termination codon which was excluded to allow translation through gfp. The hashed region in the figure between clk-1 and gfp is part of the vector's multiple cloning site (102 base pairs) and codes for 34 amino acids which provide a linker between CLK-1 and GFP. The reporter construct fully rescues the phenotype of a clA;-l(qm30) mutant upon injection and extrachromosomal array formation, indicating that the fusion to the GFP moiety does not significantly inhibit the function of CLK-1. We found that clk-1 is expressed in all cells at all stages. Furthermore, fluorescence microscopy showed that the CLK-1: :GFP fusion protein is localized to mitochondria. The localization was confirmed by co-localization with a mitochondrion-specific vital dye, G6-rhodamine. The reporter gene was also injected into wild type worms and individuals carrying extrachromosomal arrays were examined for phenotypic alterations . Presumably, the expression of CLK-1: :GFP from the extrachromosomal array together with the expression of normal CLK-1 from the endogenous wild type gene corre- sponds to an overexpression of clk-1 activity. As a measure of physiological rates, we examined the length of the defecation cycle in aging worms. The defecation cycle was examined at two times (day 1 and day 4 of adulthood) in three genotypes (wild type, cJ7-l(qm30) and animals expressing CLK-1: :GFP in a wild type background (Fig. 7)).
Five inter-defecation periods were scored for each animal and the means calculated and plotted. Ani- mals were maintained at 20 °C throughout the experiment. The cycle lengths are plotted in 2 seconds intervals.
We found that, as seen previously, the rate of defecation of clk-1 mutants at day 1 is much slower on average than that of wild type animals. However, on day 4 there was no significant difference between these two genotypes (Fig. 7 top and middle panels). This suggests that the normal slowing down of defecation between day 1 and day 4 in the wild type is due to down-regulation of clk-1 . Furthermore, we found that a significant proportion of the animals overexpressing the clk-1 activity do not have slow defecation on day 4 (Fig. 7 bottom panel). It should be remembered that the animals which carry a free extrachromosomal array are mosaic, which could explain the variability of the effect observed. Together these observations suggest that a downward regulation of the activity of clk-1 underlies a slowing down of physiological rates during aging which can be prevented by an artificial increase of clk-1 activity. We have previously argued that clJ-1 mutant animals live longer because their physiological rates are reduced. We have now observed that the defecation rate of animals overexpressing clk-1 appears to be increased over the wild type in aging animals (4 days of adult- hood) . The defecation represents only one measure of the physiology of the worms but it is likely that the observed increase reflects a general increase in physiological rates. We have therefore examined the life span of animals overexpressing clk-1 (animals expressing CLK-1: :GFP in a wild type background). We found that these animals indeed live a significantly shorter life than the wild type (Fig. 8).
Animals were maintained at 20 °C throughout the experiment. The sample size was 100. These new results suggest that the level of clk-
1 activity controls physiological rates as well as life span. A reduced level of clk-1 activity as found in mutants leads to slower physiological rates and an increased life span, while an increased activity leads to faster physiological rates (as seen on day 4 of adulthood) and a shortened life span. It suggests that artificially down-regulating the expression of the clk- 1 gene or its homologues in others organisms (for example by anti-sense therapy or pharmacological means) would lead to an increased life span. If artificial down-regulation could be targeted to a particular tissue or organ, it could lead to a specific physiological slowing-down of this tissue or organ and a concomitant slower rate of degradation by the aging process. Alternatively, the tissue- or organ-specific overexpression of clk-1 could allow one to increase the physiological rate of tissues or organs whose functional rates are slowed because of a pathological condition. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims .
SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: McGILL UNIVERSITY et al .
(ii) TITLE OF THE INVENTION: STRUCTURAL AND FUNCTIONAL
CONSERVATION OF THE C. ELEGANS CLOCK GENE clk-1
(iii) NUMBER OF SEQUENCES: 20
(iv) CORRESPONDENCE ADDRESS:
(A ADDRESSEE: SWABEY OGILVY RENAULT (B STREET: 1981 McGill College Ave . - Suite 1600 (C CITY: Montreal (D STATE: QC (E COUNTRY: Canada (F ZIP: H3A 2Y3
(V) COMPUTER READABLE FORM:
(A MEDIUM TYPE: Diskette (B COMPUTER: IBM Compatible (C OPERATING SYSTEM: DOS (D SOFTWARE: FastSEQ for Windows Version 2.0
( i) CURRENT APPLICATION DATA: (A APPLICATION NUMBER: (B FILING DATE: (C CLASSIFICATION:
(vii PRIOR APPLICATION DATA: (A APPLICATION NUMBER: 60/028,977 (B FILING DATE: 21-OCT-1996
(A APPLICATION NUMBER: 60/033,196 (B FILING DATE: 18-DEC-1996
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Cδte, France
(B) REGISTRATION NUMBER: 4166
(C) REFERENCE/DOCKET NUMBER: 1770-160PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 514 845-7126
(B) TELEFAX: 514-288-8389
(C) TELEX:
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 69 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: TTTTCATATA CGGGATTTTC AGGAAAAAAA ACAATAGAAA TCTATAAAAC ATGAGTAAAG 60
GAGAAGAAC 69
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 72 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
CCGTTTTCCT TTCAATTCTC CTTTTCTGGC ATAACGCGAC TGATGTATGC CACGCGCGCC 60 TCGTTCAGAA TG 72
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: CGTACTCTGT CTATATTTCC C 21
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GCGTTAAAAT GCGTAAGGAT G 21
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: CCACTTGCCA CCTATCACC 19
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: CAGAAGCTTC CCAGTTACTC AAGATGTTCC G 31
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: CAGAAGCTTT GTTCAAATTT TCTCAGC 27
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: ATGGAAGAAA AGGGACAC 18
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
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(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: CAGAAGCTTC TATAAAACAT GTTTCC 26
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: CAGAAGCTTA AATTCTTTCG GCACTCC 27
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
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(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: AAAAGGGTCG ACCGAAAAGA GAACTAGACG G 31
(2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: AAGGGGATCC AATTTTCTCA GCAATCGC 28
(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 187 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
Met Phe Arg Val lie Thr Arg Gly Ala His Thr Ala Ala Ser Arg Gin
1 5 10 15
Ala Leu lie Glu Lys lie lie Arg Val Asp His Ala Gly Glu Leu Gly
20 25 30
Ala Asp Arg lie Tyr Ala Gly Gin Leu Ala Val Leu Gin Gly Ser Ser
35 40 45
Val Gly Ser Val lie Lys Lys Met Trp Asp Glu Glu Lys Glu His Leu
50 55 60
Asp Thr Met Glu Arg Leu Ala Ala Lys His Asn Val Pro His Thr Val 65 70 75 80
Phe Ser Pro Val Phe Ser Val Ala Ala Tyr Ala Leu Gly Val Gly Ser
85 90 95
Ala Leu Leu Gly Lys Glu Gly Ala Met Ala Cys Thr lie Ala Val Glu
100 105 110
Glu Leu lie Gly Gin His Tyr Asn Asp Gin Leu Lys Glu Leu Leu Ala
115 120 125
Asp Asp Pro Glu Thr His Lys Glu Leu Leu Lys lie Leu Thr Arg Leu
130 135 140
Arg Asp Glu Glu Leu His His His Asp Thr Gly Val Glu His Asp Gly 145 150 155 160
Met Lys Ala Pro Ala Tyr Ser Ala Leu Lys Trp lie lie Gin Thr Gly
165 170 175
Cys Lys Gly Ala lie Ala lie Ala Glu Lys lie 180 185
(2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 179 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Met Thr Leu Asp Asn lie Asn Arg Ala Ala Val Asp Arg lie lie Arg
1 5 10 15
Val Asp His Ala Gly Glu Tyr Gly Ala Asn Arg He Tyr Ala Gly Gin
20 25 30
Met Ala Val Leu Gly Arg Thr Ser Val Gly Pro Val He Gin Lys Met
35 40 45
Trp Asp Gin Glu Lys Asn His Leu Lys Lys Phe Asn Glu Leu Met Val
50 55 60
Ala Phe Arg Val Arg Pro Thr Val Leu Met Pro Leu Trp Asn Val Ala 65 70 75 80
Gly Phe Ala Leu Gly Ala Gly Thr Ala Leu Leu Gly Lys Glu Gly Gly
85 90 95
Met Ala Cys Thr Val Ala Val Glu Glu Ser He Ala His His Tyr Asn 100 105 110 Asn Gin He Arg Met Leu Met Glu Glu Asp Ala Glu Lys Tyr Glu Glu
115 120 125
Leu Leu Gin Val He Lys Gin Phe Arg Asp Glu Glu Leu Glu His His
130 135 140
Asp Thr Gly Leu Glu His Asp Ala Glu Leu Ala Pro Ala Tyr Thr Leu 145 150 155 160
Leu Lys Arg Leu He Gin Ala Gly Cys Ser Ala Ala He Tyr Leu Ser
165 170 175
Glu Arg Phe
(2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 133 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
Lys Met Trp Asp Gin Glu Lys Asn His Leu Lys Lys Phe Asn Glu Leu
1 5 10 15
Met He Ala Phe Arg Val Arg Pro Thr Val Leu Met Pro Leu Trp Asn
20 25 30
Val Ala Gly Phe Ala Leu Gly Ala Gly Thr Ala Leu Leu Gly Lys Glu
35 40 45
Gly Ala Met Ala Cys Thr Val Ala Val Glu Glu Ser He Ala Asn His
50 55 60
Tyr Asn Asn Gin He Arg Met Leu Met Glu Glu Asp Pro Glu Lys Tyr 65 70 75 80
Glu Glu Leu Leu Gin Val He Lys Gin Phe Arg Asp Glu Glu Leu Glu
85 90 95
His His Asp Thr Gly Leu Asp His Asp Ala Glu Leu Ala Pro Ala Tyr
100 105 110
Ala Leu Leu Lys Arg He He Gin Ala Gly Cys Ser Ala Ala He Tyr
115 120 125
Leu Ser Glu Arg Phe 130
(2) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 46 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Lys Met Trp Asp Gin Glu Lys Asp His Leu Lys Lys Phe Asn Glu Leu
1 5 10 15
Met Val Met Phe Arg Val Arg Pro Thr Val Leu Met Pro Leu Trp Asn 20 25 30 Val Leu Gly Phe Ala Leu Gly Ala Gly Thr Ala Leu Leu Gly 35 40 45
(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 272 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
Met Phe Pro Tyr Phe Tyr Arg Arg Glu Phe Tyr Ser Cys Glu Asn Val
1 5 10 15
Val He Phe Ser Ser Lys Pro He Gin Gly He Lys He Ser Arg He
20 25 30
Arg Glu Arg Tyr He Glu He Met Leu Ser Arg Val Ser Val Phe Lys
35 40 45
Pro Ala Ser Arg Gly Phe Ser Val Leu Ser Ser Leu Lys He Thr Glu
50 55 60
His Thr Ser Ala Lys His Thr Glu Lys Pro Glu His Ala Pro Lys Cys 65 70 75 80
Gin Asn Leu Ser Asp Ala Gin Ala Ala Phe Leu Asp Arg Val He Arg
85 90 95
Val Asp Gin Ala Gly Glu Leu Gly Ala Asp Tyr He Tyr Ala Gly Gin
100 105 110
Tyr Phe Val Leu Ala His Arg Tyr Pro His Leu Lys Pro Val Leu Lys
115 120 125
His He Trp Asp Gin Glu He His His His Asn Thr Phe Asn Asn Leu
130 135 140
Gin Leu Lys Arg Arg Val Arg Pro Ser Leu Leu Thr Pro Leu Trp Lys 145 150 155 160
Ala Gly Ala Phe Ala Met Gly Ala Gly Thr Ala Leu He Ser Pro Glu
165 170 175
Ala Ala Met Ala Cys Thr Glu Ala Val Glu Thr Val He Gly Gly His
180 185 190
Tyr Asn Gly Gin Leu Arg Asn Leu Ala Asn Gin Phe Asn Leu Glu Arg
195 200 205
Thr Asp Gly Thr Lys Gly Pro Ser Glu Glu He Lys Ser Leu Thr Ser
210 215 220
Thr He Gin Gin Phe Arg Asp Asp Glu Leu Glu His Leu Asp Thr Ala 225 230 235 240
He Lys His Asp Ser Tyr Met Ala Val Pro Tyr Thr Val He Thr Glu
245 250 255
Gly He Lys Thr He Cys Arg Val Ala He Trp Ser Ala Glu Arg He 260 265 270
(2) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 428 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
Met Val Ser Phe Ser Trp Val Ser Arg Ser Leu His His Pro Gin Gly
1 5 10 15
Lys Arg Gly Leu He Ser Thr Leu He Cys Leu Ala Cys Val Gly He
20 25 30
Leu Ala Tyr Cys Val Ser Thr Ser His Ser He Val Leu Met Ser Thr
35 40 45
Leu Trp He Thr He Phe Ser Phe Cys Ser Gin Phe Ala Ser Leu Tyr
50 55 60
Ser Met Ser He Thr Glu Lys Pro Thr His Lys Phe Ser Tyr Gly Leu 65 70 75 80
Ala Arg Val Pro Val Leu Ala Val Phe Ser Thr Thr Val Leu Ala Gin
85 90 95
Leu Phe Ser He Phe Leu Ser Lys Glu Ser Phe Glu His Leu Leu Ser
100 105 110
Pro Asp His His Gly Ser His Asp Ala Ser Ala Ala His Glu His Glu
115 120 125
Val Glu Glu He Gly Gly Trp Pro Tyr Phe Val Gly Ser Ala Ala Ser
130 135 140
Ser Val Ala Leu Leu Leu Ser Ala Tyr Ala Leu Lys Asn Gin Pro Phe 145 150 155 160
Gin His Val Leu Gin Ser Ala Thr Ala Ser Ser Leu Gin Glu His Ala
165 170 175
Ala Asp Leu Ser His Ala Val Cys Trp Val He Pro Gly Leu Ser Arg
180 185 190
Leu Leu Leu Pro Arg He Asn Ser Met Val Leu Leu Ala Leu Thr Thr
195 200 205
Thr Gly Leu Asn Leu Leu Cys Glu His Phe Lys His Asp Phe Ala Trp
210 215 220
Ala Asp Pro Val Cys Cys Leu Leu Leu Ser Val Ala Val Phe Ser Thr 225 230 235 240
Met Tyr Pro Leu Ser Thr Tyr Thr Gly Met He Leu Leu Gin Thr Thr
245 250 255
Pro Pro His Leu He Asn Gin He Asp Arg Cys He Ser Glu Ala Ser
260 265 270
His He Asp Gly Val Leu Glu Leu Lys Ser Gly Arg Phe Trp Gin Leu
275 280 285
Asp Phe Asn Ser Leu Val Gly Thr Val Asp Val Arg Val Arg Arg Asp
290 295 300
Ala Asp Glu Gin Asn Val Leu Ala His Val Thr Glu Lys Phe Ser Ser 305 310 315 320
Val He Thr Val Leu Thr Val Gin Val Val Lys Asp Ala Ala Trp Ser
325 330 335
Ala Gly Glu Gin Val Pro Tyr Ser Asn Gly His He His Lys Ser Glu
340 345 350
Gly Asn His Ser His Asp Asn Gly His Gly His Ser His Asp His Asn
355 360 365
Asp His Gly His Ser His Gly His Asp Asp His Gly His Asp Ser His
370 375 380
Gly His Ser His Asp His Asn Glu His Asp His Gly His Ser His Gly 385 390 395 400
Gly Asn Asn Asp Asn His Gly His Ser His Ser Ala Gly Ser Asp Asn
405 410 415
His His Gly His Ser His Asp Gly Val Phe Tyr His 420 425 (2) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 234 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:
Met Gly Thr He His Leu Phe Arg Lys Pro Gin Arg Ser Phe Phe Gly
1 5 10 15
Lys Leu Leu Arg Glu Phe Arg Leu Val Ala Ala Asp Arg Ser Met Gly
20 25 30
Arg Tyr Met Leu Phe Gly Val He Asn Leu He Cys Thr Gly Phe Leu
35 40 45
Leu Met Trp Cys Ser Ser Thr Asn Ser He Ala Leu Xaa Ala Tyr Thr
50 55 60
Tyr Leu Thr He Phe Asp Leu Phe Ser Leu Met Thr Cys Leu He Ser 65 70 75 80
Tyr Trp Val Thr Leu Arg Lys Pro Ser Pro Val Tyr Ser Phe Gly Phe
85 90 95
Glu Arg Leu Glu Val Leu Ala Val Phe Ala Ser Thr Val Leu Ala Gin
100 105 110
Leu Gly Ala Leu Phe He Leu Lys Glu Ser Ala Glu Arg Xaa Leu Glu
115 120 125
Gin Ser Xaa Leu Xaa Leu Cys He Pro Xaa Ser Val Tyr Ser Gly Lys
130 135 140
Val Xaa Leu Gin Thr Thr Pro Pro His Val He Gly Gin Leu Asp Lys 145 150 155 160
Leu He Arg Glu Val Ser Thr Leu Asp Gly Val Leu Glu Val Arg Asn
165 170 175
Glu His Phe Trp Thr Leu Gly Phe Gly Ser Leu Ala Gly Ser Val His
180 185 190
Val Arg He Arg Arg Asp Ala Asn Glu Gin Met Val Leu Ala His Val
195 200 205
Thr Asn Arg Leu Tyr Thr Leu Val Ser Thr Leu Thr Val Gin He Phe
210 215 220
Lys Asp Asp Trp He Arg Pro Gly Phe Thr 225 230
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 183 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Tyr Pro Met Ser Val Tyr Ser Gly Lys Val Leu Leu Gin Thr Thr Pro
1 5 10 15
Pro His Val He Gly Gin Leu Asp Lys Leu He Arg Glu Val Ser Thr
20 25 30
Leu Asp Gly Val Leu Glu Val Arg Asn Glu His Phe Trp Thr Leu Gly
35 40 45
Phe Gly Ser Leu Ala Gly Ser Val His Val Arg He Arg Arg Asp Ala
50 55 60
Asn Glu Gin Met Val Leu Ala His Val Ser Asn Arg Leu Cys Thr Leu 65 70 75 80
Val Ser Thr Leu Thr Val Gin He Phe Lys Asp Asp Trp He Arg Pro
85 90 95
Ala Leu Ser Ser Gly Pro Val Ala Pro Asn Val Leu Asn Phe Ser Asp
100 105 110
His His Val He Pro Met Pro Leu Leu Lys Asn Val Asp Glu Arg Thr
115 120 125
Pro Val Thr Ser Thr Pro Ala Lys Pro Ser Ser Pro Ser Pro Glu Phe
130 135 140
Ser Phe Asn Thr Pro Gly Lys Asn Val Ser Pro Val He Leu Leu Asn 145 150 155 160
Thr Gin Thr Arg Pro Tyr Ser Leu Gly Leu Asn Arg Gly His Thr Pro
165 170 175
Tyr Ser Ser Val Phe Ser Gin 180

Claims

I CLAIM:
1. A clk-1 gene which has a function at the level of cellular physiology involved in developmental rate and longevity, wherein clk-1 mutants have a longer life and a altered cellular metabolism relative to the wild- type.
2. A method for the diagnosis and/or prognosis of cancer in a patient, which comprises the steps of: a) obtaining a tissue sample from said patient; b) analyzing DNA of the obtained tissue sample of step a) to determine if the human clk-1 gene is altered, wherein alteration of the human clk-1 gene is indicative of cancer.
3. A mouse model of longevity, which comprises a gene knock-out of murine clk-1 according to claim 1.
4. A method to increase life span of an animal or a patient, which comprises the steps of downregulating the expression of the clk-1 gene of claim 1 and/or homologues thereof.
5. A method of treatment of pathological conditions causing slow down of physiological rate of tissue and/or organ in a patient, which comprises administering an agent to said patient to promote tissue and/or organ specific overexpression of clk-1 in to increase the physiological rate.
PCT/CA1997/000768 1996-10-21 1997-10-17 STRUCTURAL AND FUNCTIONAL CONSERVATION OF THE C. ELEGANS CLOCK GENE $i(clk-1) WO1998017823A1 (en)

Priority Applications (6)

Application Number Priority Date Filing Date Title
EP97944665A EP0932701A1 (en) 1996-10-21 1997-10-17 STRUCTURAL AND FUNCTIONAL CONSERVATION OF THE C. ELEGANS CLOCK GENE clk-1
CA002268749A CA2268749A1 (en) 1996-10-21 1997-10-17 Structural and functional conservation of the c. elegans clock gene clk-1
BR9712368-4A BR9712368A (en) 1996-10-21 1997-10-17 Gene clk-1, mouse model of longevity and processes for diagnosis and / or prognosis of cancer in a patient, to increase the life span of an animal or a patient, and to treat pathological conditions that cause the reduction the rhythm of the physiological rate of tissue and / or organ in a patient.
JP10518750A JP2001502181A (en) 1996-10-21 1997-10-17 C. Preservation of structure and function of elegance clock gene clk-1
NZ335335A NZ335335A (en) 1996-10-21 1997-10-17 Identification of clk-1 and shows that clk-1 gene complements the clk-1 phenotype and restores normal longevity
AU46128/97A AU743527B2 (en) 1996-10-21 1997-10-17 Structural and functional conservation of the C. elegans clock gene clk-1

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US2897796P 1996-10-21 1996-10-21
US60/028,977 1996-10-21
US3319696P 1996-12-18 1996-12-18
US60/033,196 1996-12-18

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US7572575B2 (en) 2000-12-13 2009-08-11 Massachusetts Institute Of Technology SIR2 activity
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999058671A2 (en) * 1998-05-08 1999-11-18 Research Corporation Technologies, Inc. Human and nematode homologues of the yeast longevity assurance gene lag1
WO1999058671A3 (en) * 1998-05-08 2000-11-09 Res Corp Technologies Inc Human and nematode homologues of the yeast longevity assurance gene lag1
US8642284B1 (en) 1999-12-15 2014-02-04 Massachusetts Institute Of Technology Methods for identifying agents that alter NAD-dependent deacetylation activity of a SIR2 protein
US8652797B2 (en) 1999-12-15 2014-02-18 Massachusetts Institute Of Technology Methods of NAD-dependent deacetylation of a lysine residue in a protein
WO2001098478A2 (en) * 2000-06-22 2001-12-27 Mcgill University Clk-2, cex-7 and coq-4 genes, and uses thereof
WO2001098478A3 (en) * 2000-06-22 2003-05-01 Univ Mcgill Clk-2, cex-7 and coq-4 genes, and uses thereof
US7572575B2 (en) 2000-12-13 2009-08-11 Massachusetts Institute Of Technology SIR2 activity
US8546074B2 (en) 2000-12-13 2013-10-01 Massachusetts Institute Of Technology Sir2 activity
US8546090B2 (en) 2005-04-21 2013-10-01 Massachusetts Instittue Of Technology SIRT4 activities
WO2015067727A1 (en) 2013-11-07 2015-05-14 Centre National De La Recherche Scientifique Snorna, compositions and uses

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