This work describes the identification of a <i>Schizosaccharomyces pombe</i> homologue of the <i>Saccharomyces cerevisiae CDC37 </i>gene. Homologues have been previously identified in a variety of organisms, including Drosophila, chicken, mouse, rat and human. The <i>S. pombe </i>gene has been named <i>cdc37<sup>+</sup></i> and encodes a protein with a predicted molecular weight of 52 kDa. The protein shows extremely high sequence similarity with Cdc37p from <i>S. cerevisiae</i> for the first 40 amino acids at the N-terminus but, as with the homologues from other species, this similarity is considerably less over the remainder of the protein. Deletion of <i>cdc37<sup>+</sup></i> results in a lethal phenotype, establishing that the gene is essential for viability. The null mutant could survive when <i>cdc37<sup>+</sup></i> was expressed from the thiamine-regulatable low strength expression vector pREP81; when expression was repressed, the cells showed a variety of phenotypes, becoming swollen, misshapen and/or elongated. The diversity of phenotypes observed suggests that Cdc37 has several biological roles. Elongation of <i>S. pombe</i> cells indicates a cell cycle defect and measurements of cell length during Cdc37 depletion confirmed significant elongation in <i>Dcdc37</i> cells when <i>cdc37<sup>+</sup></i> expression from the plasmid pREP81 was repressed. FACS and cytological analysis indicated that cell cycle defects occurred in both the G2 and mitotic phase of the cell cycle. Genetic analysis carried out to investigate the mechanism of cell cycle arrest suggests that Cdc37 may be involved with the Cdc2/Cdc13 complex, which controls entry into mitosis from the G2 phase, <i>cdc37<sup>+</sup></i> was strongly overexpressed in wild-type cells from the high strength expression vector pREP1 with no effect. However, in strains carrying either of two <i>cdc13<sup>ts</sup></i> alleles, strong <i>cdc37<sup>+</sup></i> overexpression severely reduced in restrictive temperature. A similar but more modest effect was seen in strains carrying various <i>cdc2<sup>ts</sup></i> alleles. Preliminary biochemical studies were also carried out, which showed that the Cdc2 protein level was lower than normal in cells depleted for Cdc37.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:663674 |
| Date | January 2001 |
| Creators | Westwood, Paul |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/14655 |
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