Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-07-31
2001-05-08
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S477000, C435S483000
Reexamination Certificate
active
06228583
ABSTRACT:
BACKGROUND OF THE INVENTION
One of the great challenges of modern biology is an explanation of aging at the organismal level. The fundamental property of aging is manifest in organisms as complex as humans and as simple as the single-celled yeast,
Saccharomyces cerevisiae
. The ability to extend life span and forestall senescence has long been the subject of debate and exploration.
Recent studies of the premature aging disease, Werner's syndrome, provide possible clues about the aging process. Beginning soon after puberty, Werner's syndrome patients display many symptoms of old age, including graying and loss of hair, osteoporosis, cataracts, atherosclerosis, loss of skin elasticity and a propensity for certain cancers. Because cells isolated from Werner's patients divide approximately half as many times in culture as those from normal individuals (Salk et al.,
Cytogenet. Cell. Genet
. 30:108 (1981); G. M. Martin,
Adv. Exp. Med. Biol
. 190:161 (1985)), it is possible that both organismal aging and cellular aging are manifestations of the same process (Lombard and Guarente,
Trends Genet
. 12:283 (1996)).
SUMMARY OF THE INVENTION
The premature aging disease, Werner's syndrome, results from mutations in a single recessive gene, WRN. As described herein, mutation of the yeast WRN homolog, SGS1, causes premature aging in yeast mother cells. The aging-induced phenotype of sterility and redistribution of the Sir3 silencing protein from telomeres to the nucleolus occurs prematurely in sgs1 yeast cells. Further, the nucleolus is enlarged and fragmented in these cells, a change that also occurs in old wild type cells. These findings suggest a remarkably conserved mechanism of cellular aging which may be related to nucleolar structure. The observation that mutation of the yeast Werner's homolog, SGS1, results in premature aging suggests that a common mechanism underlies aging in eukaryotes as diverse as yeast and humans. Thus, insight into the aging process in model systems can provide insight into aging in humans.
The subject invention pertains to methods of identifying agents or compounds which inhibit the replication and/or accumulation of DNA circles, e.g., ribosomal DNA (rDNA) circles, comprising an autonomously replicating sequence (ARS), e.g., rDNA ARS, in cells. The invention also encompasses methods of assessing the ability of a compound to extend life span of a cell, comprising contacting a cell or cells (referred to as a test cell or cells) with a compound which inhibits the replication and/or accumulation of DNA circles comprising an ARS in cells, and assessing the life span of the test cell relative to a comparable cell or cells which was not contacted with the compound (a control cell or cells). If the life span of the test cell is longer than the life span of the control cell, then the compound extends life span. The invention also relates to methods of extending life span, comprising administering to a cell a compound which inhibits replication and/or accumulation of rDNA circles in the cell, with the result that the life span of the cell is extended (is longer than it would have been in the absence of the compound). These compounds can be compounds identified by the methods described herein, derivatives (modified forms) of compounds identified by the present methods, or compounds identified by another method (e.g., computer modeling of compounds designed with reference to structures and/or characteristics of compounds identified by the present method) and synthesized by known methods (e.g., chemical synthesis, peptide chemistry).
As described herein, a mouse gene referred to as mWRN, which is a homolog of Sgs1p and WRN, has been discovered. This invention also pertains to isolated mWRN, or an active derivative or fragment thereof; in a particular embodiment, the isolated mWRN protein has the amino acid sequence of SEQ ID NO: 6 (FIG.
5
). In one embodiment, the polypeptide is a fragment having mWRN activity, e.g., binding or enzymatic activity. In another embodiment, the polypeptide is a derivative possessing substantial sequence identity with endogenous mWRN. In particular embodiments, the mWRN protein is purified to homogeneity or is substantially free of other proteins.
The invention also pertains to an isolated nucleic acid molecule which encodes mWRN, or an active derivative or fragment thereof. In a particular embodiment, the nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 5 (FIG.
4
). In one embodiment, the nucleic acid molecule encodes a polypeptide fragment having mWRN activity. In another embodiment, the nucleic acid molecule encodes a derivative of mWRN possessing substantial sequence identity with endogenous mWRN. In particular embodiments, the isolated nucleic acid molecule encodes mWRN with the same amino acid sequence as endogenous mWRN. In another embodiment, the isolated nucleic acid molecule has the same nucleotide sequence as the endogenous gene encoding mWRN.
The invention also relates to DNA constructs comprising the nucleic acid molecules described above operatively linked to a regulatory sequence, and to recombinant host cells, such as bacterial cells, fungal cells, plant cells, insect cells and mammalian cells, comprising the nucleic acid molecules described above operatively linked to a regulatory sequence. The invention also pertains to an antibody, or an antigen-binding fragment thereof, which selectively binds to mWRN, or an active derivative or fragment thereof; in a particular embodiment, the antibody is a monoclonal antibody.
The invention also pertains to a mouse and its progeny having a suppressed level of expression of the mWRN gene. The invention further relates to a mouse and its progeny in which the mWRN gene is suppressed, either physically or functionally. The invention also relates to embryonic stem cell lines containing a mWRN knockout construct.
In another embodiment, the invention relates to a method of screening a compound for the ability to alter life span comprising administering the compound to a mouse with a suppressed level of mWRN expression and assaying the mouse for altered life span. The invention also relates to compounds identified by assays described herein.
REFERENCES:
patent: 6043053 (2000-03-01), Barnes et al.
patent: WO 91/00920 (1991-01-01), None
patent: WO 97/24435 (1997-07-01), None
Gangloff, Serge, et al., “The Yeast Type I Topoisomerase Top3 Interacts with Sgs1, a DNA Helicase Homolog: a Potential Eukaryotic Reverse Gyrase”,Mol. Cell. Biol., 14(12):8391-8398 (1994).
Christman, Michael F., et al., “Mitotic Recombination in the rDNA ofS. cerevisiaeis Suppressed by the Combined Action of DNA Topoisomerases I and II”,Cell, 55:413-425 (1988).
Watt, Paul M., et al., “SGS1, a Homologue of the Bloom's and Werner's Syndrome Genes, Is Required for Maintenance of Genome Stability inSaccharomyces cerevisiae”, Genetics, 144:935-945 (1996).
Watt, Paul M., et al., “Sgs1: A Eukaryotic Homolog ofE. coliRecQ that Interacts with Topoisomerase II In Vivo and Is Required for Faithful Chromosome Segregation”,Cell, 81:253-260 (1995).
Lu, Jian, et al., “Human Homologues of Yeast Helicase”,Nature, 383:678-679 (1996).
Gottlieb, Shoshanna and Esposito, Rochelle E., “A New Role for a Yeast Transcriptional Silencer Gene,SIR2, in Regulation of Recombination in Ribosomal DNA”,Cell, 56:771-776 (1989).
Kennedy, Brian K., et al., “Redistribution of Silencing Proteins from Telomeres to the Nucleolus is Associated with Extension of Life Span inS. cerevisiae”, Cell, 89:381-391 (1997).
de Beus, Elizabeth, et al., “Yeast NOP2 Encodes an Essential Nucleolar Protein with Homology to a Human Proliferation Marker”,J. Cell Biol., 127:1799-1813 (1994).
Kadowaki, Tatsuhiko, et al., “Nuclear mRNA Accumulation Causes Nucleolar Fragmentation in Yeast mtr2 Mutant”,Mol. Biol. Cell, 5:1253-1263 (1994).
Tani, Tokio, et al., “Nucleolar Accumulation of Poly (A)+RNA in Heat-shocked Yeast Cells: Implication of Nucleolar Involvement in mRNA Transport”,Mol. Biol. Cell, 6:1515-1534 (1995).
Oates, Melani
Guarente Leonard P.
Sinclair David A.
Hamilton Brook Smith & Reynolds P.C.
Ketter James
Massachusetts Institute of Technology
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