This proposal is focused on the dissection of the process of conjugation in the simple eukaryote S. cerevisiae. There are four specific aims of this proposal: 1) investigation of the function of three genes identified in previous studies as involved in nuclear congression during conjugation; 2) investigation of the function in mating and vegetatively growing cells of three genes implicated in nuclear fusion during mating; 3) characterization of several new genes involved in cell fusion; and 4) identification of new mutants affected in genes which are required in both parental cells for proper conjugation. The first specific aim encompasses studies of the class I genes KAR3, KAR9, and KIP2. The products encoded by this set of genes are involved in microtubule-dependent orientation and movement of the two nuclei towards each other.
The aim of these studies is to localize the proteins, determine their possible interactions with other proteins, and establish their physical and functional relationship to microtubules. The second specific aim targets the class II karyogamy genes KAR5, KAR7, and KAR8 whose produts affect fusion of the nuclear envelope. The objective here is to understand more precisely the events that are catalyzed by this set of proteins. The possible requirement of class II genes for fission of nuclei and nuclear envelope proliferation during vegetative growth will also be assessed. The last two sections of the proposal touch on the FUS genes that affect fusion of the two parental cells. The applicant has isolated mating defective mutants (distinct from ste mutants) identifying new FUS genes that act either in a mating type-specific or independent manner. This third specific aim will strive to clarify if and how the proteins encoded by this group of genes may influence early steps of zygote formation such as fusion of the cytoplamsic membrane and establishement of cell polarity. During the previous period some of the mutants were obtained from bilateral screens in which karyogamy of cell fusion defects are observed only when both partners carry mutations. Since the number of mutants isolated in the earlier screens suggested that not all the genes have been saturated, the last specific aim of the project is to complete the analysis of mutants generated from a more recent large scale screen.
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