Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1997-11-28
2001-11-20
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C536S023200, C435S320100, C435S252300, C435S192000, C435S348000, C435S419000, C435S254200, C424S094400, C514S04400A
Reexamination Certificate
active
06319708
ABSTRACT:
Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
BACKGROUND OF THE INVENTION
C. elegans
and other nematodes can halt development under appropriate conditions (overcrowding, reduction in food supply) by becoming a nonfeeding stage (an alternative third larval stage) called the dauer larva. This stage is similar to the dispersal stage of many parasitic nematodes (Roberts and Janovy, Jr., 1996).
C. elegans
dauers can exist for extended periods (at least two months) without effects on subsequent post-dauer life-span or progeny production (Klass and Hirsh, 1976).
Several genes affect the transition into and out of the dauer state (Riddle et al., 1981). These dauer formation (daf) genes can be mutated to cause animals to always (the Daf-C, dauer constitutive, phenotype) or never (the Daf-D, dauer defective, phenotype) form dauer larvae. Most researchers agree that two interlinked genetic pathways control dauer formation (Vowels and Thomas, 1992; Thomas et al., 1993, Gottlieb and Ruvkun, 1994; Larsen et al., 1995).
Recent work has implicated one of these pathways in the control of aging. Specifically, Kenyon et al. (1993) showed that a daf-c mutation in the daf-2 gene caused a doubling of life-span in
C. elegans
. This increase was suppressed by a daf-d mutation in the daf-16 gene. Larsen et al. (1995) extended these observations, finding that daf-c mutations in daf-2 and daf-23 extended life-span. This extension of life-span was suppressed completely by mutations in daf-16 and partially by mutations in daf-18. These workers also found that certain daf-2; daf-12 double mutants had greatly extended life-spans, even though daf-12 mutations on their own did not affect aging.
These results place the most famous
C. elegans
aging gene, age-1, in a broader context. An age-1 mutation was identified by Klass (1983) and studied by Friedman and Johnson (1987), who showed that it extended both average and maximum life-span in
C. elegans
. The age-1 mutation is now known to be an allele of daf-23 (Malone et al., 1996). Moreover, Morris et al. (1996) have found that daf-23 encodes a subunit of a PI 3-kinase, indicating a role of signaling in determinating life-span.
The dauer pathway, when expressed in adults, allows animals to survive for relatively long periods of time (at least a four-fold extension in mean life-span). The targets of the dauer pathway genes that allow life-span extension, however, have not been identified, previously. Some of the ultimate targets for the daf genes may be genes encoding antioxidant enzymes, since catalase and superoxide dismutase (SOD) activities are approximately five times higher in the dauer larvae than in L3 worms (Anderson, 1982; Larsen, 1993). Moreover, unlike the activities in wild-type animals, total SOD and catalase activities increase with age in age-1 mutants (Vanfleteren, 1993; Larsen, 1993).
Several investigators have hypothesized that oxidative damage to cells is a major cause of cellular and organismal senescence (Gershmann et al., 1954; Harman, 1956; Sohal and Allen, 1990). Most relevant to our studies are experiments in
Drosophila melanogaster
where overexpression of Cu/Zn SOD and catalase, but not either alone, increased mean adult life-span by 33% (Seta et al., 1990; Stavely et al., 1990; Orr and Sohal, 1992, 1993, 1994). These results not only suggest that control of reactive oxygen species is an important determinant of longevity, but also underline the need to balance SOD and catalase activities for the control of oxidative stress (see also Phillips et al., 1989 and Amstad et al., 1991).
Here we show that
C. elegans
contains two catalase genes. One gene, ctl-1, appears to be needed for normal life-span and for the extension of life-span seen in daf-c adults. One striking feature of the ctl-1 catalase is its localization in the cytosol, not in peroxisomes. This localization is unusual, since cytosolic catalases have rarely, if ever, been seen in animals. The second
C. elegans
catalase gene, ctl-2, appears to encode the peroxisomal catalase. We suggest that the ctl-1 catalase is needed during periods of starvation, such as the dauer larva, and that its expression in daf-c adults enables them to live longer. As such ctl-1 would represent a true life-span extension gene.
In nematodes an alternative third larval stage, often called the dauer stage in free-living animals, allows animals to weather periods of low food availability (if free living) or to disperse (if parasitic). Mutations in several genes that control entry into and exit from the dauer stage of the nematode
Caenorhabditis elegans
profoundly affect the life-span of adults. The ctl-1 gene, which encodes an unusual, cytosolic catalase, is required in
C. elegans
for the life-span extension exhibited by animals with these dauer mutations. Cytosolic catalase may have evolved in nematodes to allow prolonged periods of dormancy before reproductive maturity.
SUMMARY OF THE INVENTION
This invention provides a composition comprising an amount of a polypeptide effective to increase the life-span of cells wherein the polypeptide has the amino acid sequence of a cytosolic catalase and a suitable carrier.
This invention also provides a composition comprising an amount of a nucleic acid molecule comprising a nucleotide sequence encoding a cytosolic catalase effective when introduced into cells to produce a sufficient amount of cytosolic catalase to increase the life-span of cells and a suitable carrier.
This invention also provides an isolated nucleic acid molecule encoding a cytosolic catalase.
This invention also provides a host vector system for the production of a polypeptide having the biological activity of catalase which comprises the above-described vectors in a suitable host.
This invention also provides a method for prolonging cell life, comprising: (a) linking the above-described nucleic acids to a regulatory element such that the expression of the above-described nucleic acids is under the control of the regulatory element; and (b) introducing the linked nucleic acid into cells for expression of the nucleic acid, thereby prolonging cell life.
REFERENCES:
R. Weiser et al. “Heat Shock Factor-Independent Heat Control of Transcription of the CTT1 Gene Encoding the Cytosolic Catalase T ofSaccharomyces cerevisiae”, J. Biol. Chem. 266(19): 12406-12411, Jul. 1991.*
S.M. Brown et al. “Cloning and Characterization of the katB Gene of Pseudomonas aeruginosaEncoding a Hydrogen Peroxide-Inducible Catalase”, J. Bacteriol. 177(22): 6536-6544, Nov. 1995.*
P.E. Perdue et al. “Targeting of Human Catalse to Peroxisomes is Dependent Upon a Novel COOH-Terminal Targeting Sequence”, J. Cell Biol. 134(4): 849-862, Jul. 1991.*
Riddle, D. L.,A Genetic Pathway For Dauer Larva Formation In C. elegans, Stadler Genetics Symposium(1977) 9:101-120 (Exhibit B).
Amstad, P., Peskin, A., Shah, G., Mirault, M.E., Moret R., Zbinden I., and Cerutti, P., “The balance between Cu, Zn-superoxide dismutase and catalase affects thesensitivity of mouse epidermal cells to oxidative stress.”Biochem. (1991) vol. 30, 9305-9313, (Exhibit 1).
Anderson, G. L., “Superoxide dismutase activity in dauerlarvae ofCaenorhabditis elegans” (Nematoda: Rhabditidae)Can. J. Zool. (1982) vol. 60, 288-291, (Exhibit 2).
Friedman, D. B., and Johnson, T. E., “A mutation in theage-1gene inCaenorhabditis eleganslengthens life and reduces hermaphrodite fertility.” (1987)Geneticsvol. 188, 75-86 (Exhibit 3).
Gerschman, R., Gilbert, D. L., Nye, S., Dwyer, P., and Fenn, W. O., “Oxygen poisoning and X-irradiation: A mechanism in common.” (1954)Sciencevol. 119, 623-626, (Exhibit 4).
Gottlieb, S., and Ruvkun, G., “daf-2, daf-16, and daf-23: Genetically interacting genes controlling dauer formation inCaenorhabditis elegans.” (1994)Geneticsvol. 137, 107-120, (Exhibit 5).
Harman, D., “Aging: A theory based on free radical and
Chalfie Martin
Hahn Jang-Hee
Ma Charles
Rothblatt Jonathan
Taub James J.
Cooper & Dunham LLP
Prouty Rebecca E.
The Trustees of Columbia University in the City of New York
White John P.
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