Studies of cellular reproduction and differentiation are central to basic biology and should also improve understanding of important diseases such as cancer. Successful analyses of these complex processes will be most likely if they are approached in relatively simple systems, such as the unicellular eukaryote yeast, in which the full power of both classical and molecular genetic methods can be brought to bear. The proposed research will use yeast to address three distinct but interconnected problesm. First, it will attempt to resolve the """"""""gene-number paradox"""""""" (i.e., the general discrepancy between classical genetic and molecular estimates of numbers of genes) and thus enhance the utility of genetic method for the study of cellular and developmental processes. The principal approach to this problem will be the molecular analysis of a chromosome on which few genes have been identified by classical mutational analyses. Additional genes will be identified as transcribed regions on cloned segmants of the chromosome, and the reason for their """"""""invisibility"""""""" to classical analyses will be explored using Southern-blotting and DNA-mediated gene disruption. Second, the genetic control of the cell cycle will be studied; the results should also help elucidate the reasons for the gene-number paradox. New cell-cycle genes will be sought by analysis of cold-sensitive-lethal mutants that arrest at specific cell-cycle stages. Analysis of extragenic suppressors of such mutants and of similar high-temperature-sensitive mutants should reveal additional genes as well as evidence about interactions among cell-cycle genes and their products. The mutant phenotypes will be characterized to derive clues to the molecular functions of the gene products and to the functional organization of cell-cycle events. New genes and evidence about intergenic interactions will also be sought by screening recombinant-DNA libraries for genes whose overexpression can suppress mutations in other genes of interest. Third, the molecular basis of cellular morphogenesis will be explored. The genetic studies just described will focus on genes that control the morphogenetic processes of the cell cycle. In addition, such genes will be cloned. Clues to the molecular functions of the gene products will then be sought by sequencing the genes and by raising antibodies that can be used to localize the gene products in the cells. The results should illuminate the roles of cytoskeletal elements and of other machanisms both in cellular reproduction and in cellular morphogenesis.
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