The accurate segregation of chromosomes during mitosis is critical to eucaryotic cell division. Immunological and genetic approaches will be combined to identify chromosome segregation components in the model eucaryote; Saccharomyces cerevisiae. Human autoantibodies from scleroderma patients react with conserved determinants of the mammalian spindle pole (centrosome). These have been used to identify two related antigens in yeast. The subcellular localization of these and other potential antigens within a yeast cell will be determined by indirect immunofluorescence. Genes encoding these proteins will be cloned by immunoscreening a Lambdagt11 yeast genomic DNA library. The genes will be characterized and sequenced and antibodies raised against the gene products. Mutations will be constructed in these genes (in vitro or in E. coli), which will then be substituted for the genomic copies in vivo. This procedure will utilize a novel method for rapidly and crudely mapping antigenic coding regions, and creating insertion mutations using transposition mutagenesis. The phenotypes of these mutations on cell viability and growth, chromosome segregation, zygote formation, and meiosis will be examined. Conditional mutations will be sought that allow the isolation of extragenic second site suppressors. In addition to the immunological approach, a genetic screen will be performed to identify mutations in genes involved in chromosome segregation. This screen uses a colony color assay to identify mutants that exhibit a high frequency of chromosome nondisjunction. A restricted subset of mutants, with phenotypes expected of chromosome segregation mutants will be studied extensively. The combined immunological and genetic approaches will identify components involved in eucaryotic chromosome segregation. Biochemical and genetic analysis of these components in yeast provide a unique opportunity to examine the molecular events that occur during mitosis, including the mechanism of action of individual proteins, the proteins with which they interact, and how the assembly and disassembly of the mitotic apparatus is controlled in the eucaryotic cell cycle. Scleroderma is an autoimmune disease of unknown etiology and unknown pathogenicity. This proposal will identify, for the first time, molecules recognized by these spindle pole scleroderma sera. This information and these approaches will be useful for understanding etiology, pathogenicity and diagnosis of autoimmune disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM036494-03
Application #
3290576
Study Section
Genetics Study Section (GEN)
Project Start
1986-08-01
Project End
1989-07-31
Budget Start
1988-08-01
Budget End
1989-07-31
Support Year
3
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Yale University
Department
Type
Schools of Arts and Sciences
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Hall, David A; Ptacek, Jason; Snyder, Michael (2007) Protein microarray technology. Mech Ageing Dev 128:161-7
Gelperin, Daniel M; White, Michael A; Wilkinson, Martha L et al. (2005) Biochemical and genetic analysis of the yeast proteome with a movable ORF collection. Genes Dev 19:2816-26
Bidlingmaier, Scott; Snyder, Michael (2004) Regulation of polarized growth initiation and termination cycles by the polarisome and Cdc42 regulators. J Cell Biol 164:207-18
Casamayor, Antonio; Snyder, Michael (2003) Molecular dissection of a yeast septin: distinct domains are required for septin interaction, localization, and function. Mol Cell Biol 23:2762-77
Hanrahan, Jessie; Snyder, Michael (2003) Cytoskeletal activation of a checkpoint kinase. Mol Cell 12:663-73
Santos, Beatriz; Snyder, Michael (2003) Specific protein targeting during cell differentiation: polarized localization of Fus1p during mating depends on Chs5p in Saccharomyces cerevisiae. Eukaryot Cell 2:821-5
Bidlingmaier, Scott; Snyder, Michael (2002) Large-scale identification of genes important for apical growth in Saccharomyces cerevisiae by directed allele replacement technology (DART) screening. Funct Integr Genomics 1:345-56
Vallier, Laura G; Segall, Jeffrey E; Snyder, Michael (2002) The alpha-factor receptor C-terminus is important for mating projection formation and orientation in Saccharomyces cerevisiae. Cell Motil Cytoskeleton 53:251-66
Casamayor, Antonio; Snyder, Michael (2002) Bud-site selection and cell polarity in budding yeast. Curr Opin Microbiol 5:179-86
Ni, L; Snyder, M (2001) A genomic study of the bipolar bud site selection pattern in Saccharomyces cerevisiae. Mol Biol Cell 12:2147-70

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