This is a proposal to continue research on the genetic control of morphogenesis in yeast. A model has been developed for a morphogenetic hierarchy. The cytoskeletal systems carry out the morphogenetic events by working in complex and interacting modes. The cytoskeletal systems respond to signals from polarity establishment functions which operate under the influence of bud site selection functions. These have been grouped into axial specific and bipolar specific functions. The long terms goal of the project is to define each level of function int he hierarch and to identify the genes that participate. There are two major areas being pursued in the current proposal. One is to investigate the development of cell polarity. Issues of interest here concern the nature of the signals that direct bud site selection, how persistent signals at a previous division sitecan detemined the bipolar pattern of bud site selection, the mechanism by which axial or bipolar spatial information is trnasduced to proteins responsible for directing polarity establishment, and aspects of GTPase cycles by which information for cell polarization gets transmitted to the cytoskeletal elements for cell surface growth. The second major area of focus is on septins which comprise a family of proteins involved in cytokenesis and a variety of other roles in organization of the cell surface. Septins are coiled-coil proteins with predicted nucleotide binding loops. Mutations well be engineered to address structure/function relationships and the properties of the altered proteins will be assessed by two hybrid and phenotype analysis. Other issues of interest concern aspects of septins in localizaing the synthesis of chitin, role of septins in synthesis of the spore wall, and identification of additonal interacting proteins. The course of the proposal proceeds from a genetic approach to the molecular level but the emphasis is on a thorough, detailed anlaysis from the point of view of a cell biologist. The proposal rest heavily on cytological imaging techniques, primarily immunofluorescence. Biochemical procedures are utilized primarily with the end of producing proteins for immunization, although some qualitative methods are proposed for determining cofactor binding and protein-protein interaction. This second major area of investigation utilizes a fairly standard array of molecular genetic procedures including site directed mutagenesis, immunoprecipitation, as well as two hybrid and suppressor analysis to identify additional interacting proteins and genes.
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