We have used in vivo genomic footprinting to investigate the protein-DNA interactions within the conserved DNA elements (CDE I, CDE II, and CDE III) in the centromere from chromosome III of the yeast Saccharomyces cerevisiae. The in vivo footprint pattern obtained from wildtype cells shows that some guanines within the centromere DNA are protected from methylation by dimethylsulfate (DMS). These results are consistent with studies demonstrating that yeast contain sequence-specific centromere DNA binding proteins. Our in vivo experiments on chromosomes with mutant centromeres show that some mutations which affect chromosome segregation also alter the footprint pattern caused by proteins bound to the centromere DNA. The results of this study provide the first fine-structure map of proteins bound to centromere DNA in living yeast cells and suggest a direct correlation between these protein-DNA interactions and centromere function. We have identified a putative Saccharomyces cerevisiae homologue of a phosphoinositide-specific phospholipase C (PI-PLC) gene, PLC1, which encodes a protein most similar to the $\delta$-class of PI-PLC enzymes. The PLC1 gene was isolated during a study of yeast strains that exhibit defects in chromosome segregation. plc1-1 cells show an 8-9 fold increase in aberrant chromosome segregation and arrest primarily as large-budded or unbudded cells. Molecular analysis revealed that PLC1 encodes a predicted protein of 94 kD with approximately 50% and 26% identity to the highly conserved X and Y domains of PI-PLC isozymes from human, bovine, rat, and Drosophila melanogaster. The putative yeast protein also contains a consensus EF-hand domain that is predicted to bind calcium. Interestingly, the temperature sensitive and chromosome segregation plc1-1 phenotypes are suppressed by exogenous calcium, suggesting a possible relationship between the PI-PLC signaling pathway and control of nuclear events in yeast.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-8644 |
| Date | 01 January 1993 |
| Creators | Payne, William Everett |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
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