Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-10-06
2001-10-09
Martin, Jill D. (Department: 1632)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C530S350000, C536S023100, C435S320100, C435S325000, C435S477000, C514S04400A, C424S093210, C424S277100
Reexamination Certificate
active
06300084
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates generally to the identification and characterization of fungal genes and proteins, and in particular to mitotic spindle proteins useful as development candidates for anti-mitotic agents and processes using such agents.
The mitotic spindle undergoes a remarkable series of transitions in response to cell cycle control signals. At each mitotic cell division, the spindle assembles, it forms attachments to each chromosome, it orients itself properly within the cell, and then, with extraordinarily high fidelity, it carries out chromosome segregation. Then it disassembles.
Proper spindle assembly and function involves coordination of many events and processes including modulation of microtubule dynamics and creation of at least three distinct microtubule populations (kinetochore, polar, and astral microtubules). In addition, connections must be established between different spindle microtubule subpopulations, between spindle microtubules and chromosomes, between spindle microtubules and microtubule-associated proteins and motor proteins, and between spindle microtubules and the cell cortex (reviewed by Waters and Salmon, 1997). Proper spindle assembly is monitored by a cellular surveillance system which activates a mitotic checkpoint if the spindle is not assembled correctly (reviewed by Hardwick, 1998; Rudner and Murray, 1996). Once the spindle is assembled, a carefully orchestrated set of molecular events results in chromosome to pole movement (anaphase A) and separation of spindle poles (anaphase B).
Genetic approaches to the study of spindle mechanics and regulation in
S. cerevisiae, S. pombe, A. nidulans,
and in a variety of other organisms have complemented studies in Xenopus extracts and mammalian and plant cells (reviewed by Nicklas, 1997; Sobel, 1997). Each different approach has provided an extremely powerful and unique avenue toward identification of mitotic spindle components and elucidation of their functions. Budding yeast contains five kinesin-related motor proteins and one dynein (reviewed by Winsor and Schiebel, 1997). Elegant genetic studies in yeast have revealed how the forces generated by these proteins work both synergistically and antagonistically to assemble and orient spindles, and to separate chromosomes (Cottingham and Hoyt, 1997; Gambino et al., 1984; Oakley and Morris, 1980; Oakley and Rinehart, 1985; Saunders and Hoyt, 1992).
It is believed that a large number of proteins in the spindle function in concert with tubulin, the major spindle protein. Genetic studies have identified and provided functional tests of &ggr;-tubulin and many other proteins associated with spindle pole bodies (Marschall et al., 1996; Oakley, 1994; Rout and Kilmartin, 1990; Sobel and Snyder, 1995; Spang et al., 1995). Also, a number of spindle accessory proteins have been found and studied functionally (Berlin et al., 1990; Machin et al., 1995; Pasqualone and Huffaker, 1994; Pellman et al., 1995; Wang and Huffaker, 1997). These genetic studies have been particularly valuable both because non-tubulin spindle components are typically low in abundance, making their discovery difficult by other means, and because genetic analysis facilitates tests of function in vivo.
As indicated above, the mitotic spindle has been the subject of considerable research. The study of mitotic spindle proteins has yielded anti-mitotic compounds with important applications in cancer chemotherapy, and therapeutic agents targeted against fungal pathogens. For example, several plant and fungal secondary metabolites such as colchicine, vinblastine and taxol have been shown to interfere with mitotic spindle function in a wide variety of eukaryotes.
The demonstrated effectiveness of these anti-mitotic compounds in important medical and agricultural applications demonstrates the desirability of identifying and characterizing anti-mitotic compound development candidates.
SUMMARY OF THE INVENTION
To achieve the foregoing, the present invention provides a genetic identification and characterization of a gene which encodes an essential yeast mitotic spindle protein. The protein functions in proper spindle assembly and anaphase spindle elongation. The invention also provides an identification of a protein which interacts with this mitotic spindle protein. The proteins identified and characterized by the present invention are useful as development candidates for cancer chemotherapeutic agents, anti-fungal compounds, and other anti-mitotic agents.
In one aspect, the present invention provides a nucleic acid vector including a gene which encodes an essential mitotic spindle protein, and a plasmid capable of incorporating that gene. In preferred embodiments, the gene is the yeast gene YGL061c or the yeast gene YGR113w, and the plasmid is Bluescrpit SK
+
vector (pDD478).
In another aspect, the invention provides a substantially pure protein essential to mitotic spindle formation. In preferred embodiments, the protein includes an amino acid sequence coded for by the yeast gene YGL061c or the yeast gene YGR113w.
In still another aspect, the present invention provides a binding protein for a protein essential to mitotic spindle formation. The binding protein includes an amino acid sequence coded for by the yeast gene YGR113w.
In yet another aspect, the present invention provides a composition useful as a development candidate for an anti-mitotic agent. The development candidate includes an amino acid sequence selected from at least one of an amino acid sequence coded for by the yeast gene YGL061c, and an amino acid sequence coded for by the yeast gene YGR113w.
In an additional aspect, the invention also provides an anti-mitotic agent identified by a screening method using one or more proteins essential to mitotic spindle formation. In preferred embodiments, the one or more proteins include an amino acid sequence selected from at least one of an amino acid sequence coded for by the yeast gene YGL061c, and an amino acid sequence coded for by the yeast gene YGR113w.
In a further aspect, the invention provides a method of disrupting mitotic spindle formation in a cell. The method involves administering to the cell an anti-mitotic agent that disrupts the activity of one or more proteins essential to mitotic spindle formation. The one or more proteins include an amino acid sequence selected from at least one of the amino acid sequence coded for by the yeast gene YGL061c, and the amino acid sequence coded for by the yeast gene YGR113w.
REFERENCES:
J. M. Nigro et al., Molecular and Cellular Biology, “Human p53 and CDC2Hs Genes Combine To Inhibit the Proliferation ofSaccharomyces cerevisiae,” Mar. 1992, vol. 12, No. 3, pp. 1357-1365.*
W. French Anderson. Human gene therapy. Nature, vol. 392, pp. 25-30, 1998.*
T. Gura. Systems for identifying new drugs are often faulty. Science, vol. 278, pp. 1041-1042, 1997.*
Hartwell et al. Integrating genetic approaches into the discovery of anti-cancer drugs. Science, vol. 278, pp. 1064-1067, 1997.*
Hofmann, Christian, et al, “Saccharomyces cerevisiaeDuo 1p and Dam 1p, Novel Proteins Involved in Mitotic Spindle Function”, Nov. 16, 1998, Department of Molecular and Cell Biology, University of California, Berkeley, The Journal of Cell Biology, vol. 143, No. 4, pp. 1029-1040.
Jones, Michele H., et al., “Yeast Dam1p Is Required to Maintain Spindle Integrity during Mitosis and Interacts with the Mps1p Kinase”, Jul. 1999, Department of Molecular, Cellular, and Developmental Biology, University of Colorado, vol. 10, pp. 2377-2391.
Search SGD, http://genome-www.standford.edu/saccharomyces/.
Cheesman, Iain M., “DUO1p and DAM1p, Two Novel Proteins Important for Mitotic Spindle Function inSaccharaomyces Cerevisiae” Abstracts, Nov. 1998, Supplement to Molecular Biology of the Cell, vol. 9, p. 248.
Pasqualone, Danielle, et al., “STU1, a Suppressor of a B-Tubulin Mutation, Encodes a Novel and Essential Component of the Yeast Mitotic Spindle”, Dec. 1994, Section of Biochemistry, Molecular and Cell Biology, Cornell University, vol. 127, No. 6, Part 2, p
Drubin David G.
Hofmann Christian J.
Beyer Weaver & Thomas LLP
Martin Jill D.
Paras, Jr. Peter
The Regents of the University of California
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