The Schizosaccharomyces pombe cdc21<sup>+</sup> gene product is related to the Mcm2-3-5 family of replication proteins. By phylogeny analysis of their protein sequences and screening for cdc21<sup>+</sup>-related sequences using molecular probes I have suggested that at least six types of cdc21<sup>+</sup>-related genes may be present in the yeast genome. The isolation of interaction suppressors of the cdc21<sup>ts</sup> mutant was attempted by overexpression of an S. pombe cDNA library. Two cDNAs were isolated, ts11<sup>+</sup> and dom1<sup>+</sup>, whose overexpression specifically affected the viability of cdc21<sup>ts</sup> cells under certain conditions. The predicted dom1 protein is 60% identical to the budding yeast HMG-like Nhp2 protein. I have studied the phenotype of S. pombe cells overexpressing the cdc21<sup>+</sup> gene and amino-terminal truncations of it. Overexpression of the cdc21<sup>+</sup> gene caused cell elongation but cells were not significantly affected in growth rate. Cells overexpressing the carboxyl-terminal part of cdc21<sup>+</sup> arrested in S phase and also entered mitosis in the absence of nuclear division. The possibility that chromosomes in cdc21<sup>ts</sup> arrested cells may be damaged was investigated by pulsed field gel electrophoresis. No differences could be found compared to wild-type chromosomes. I have also studied the arrest phenotype of cdc21 rad1 and cdc21 cdc2.3w double mutants. Both strains entered mitosis at the restrictive temperature indicating that cdc21<sup>ts</sup> cells arrest in S phase and may contain DNA damage. I have generated two new mutant alleles of cdc21<sup>+</sup>. The first allele was made by deleting most of the cdc21<sup>+</sup> open reading frame (ORF). The second allele was constructed by placing the cdc21<sup>+</sup> ORF under control a regulatable promoter. The resulting construct was used to complement the cdc21 deletion. Both mutants were inviable under appropriate conditions arresting in S phase as elongated cells, although a proportion of them (15-20%) entered mitosis in the absence of nuclear division.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:260763 |
| Date | January 1995 |
| Creators | Maiorano, Domenico |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:e4813692-f9c5-4f81-9fb8-2a13413c04bb |
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