Ochotorena, Iciar Lourdes; (2000) A novel mechanism of polyploidisation in fission yeast. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The pop3 mutant was isolated from a genetic screen performed in fission yeast in order to obtain mutants that are defective in the maintenance of genome ploidy. The screen was designed to isolate mutants that were sterile, and became polyploid due to a failure to arrest in G1 under nitrogen starvation conditions. The pop3 mutant showed diploidising phenotypes even in vegetative cycles. The mutant strain was also found to show retarded growth in most media and showed reduced viability at 26°C. The mutant was also cold and temperature sensitive for growth. The pop3 + gene was cloned through complementation of the temperature-sensitive phenotype with a wild type fission yeast genomic library. The sequence obtained encodes a highly conserved 314 amino acid protein. The Pop3 protein consists solely of six (maybe seven) WD repeats, a known protein-protein interacting domain, but no other known domains are found. Pop3 has homologues in several organism including budding yeast and humans, with budding yeast showing the greatest identity at 67% Due to the lack of information provided by the sequence and the homologues, potential roles for pop3 in proteolysis, cell cycle, cell morphology or transcription were studied. The pop3 mutant was found to have low levels of α-tubulin and Cig2 (S-phase cyclin) proteins. This led me to study the state of microtubules within the mutant cells. In the pop3 mutant, microtubule structures are partially disrupted, either faint or shorter microtubule arrays were observed. Consistent with this finding, pop3 mutants are super-sensitive to the microtubule destabilising drug, thiobendazole. Furthermore, occasional unequal sister chromatid segregation was observed. The lack of microtubule integrity and chromosome mis-segregation in the pop3 mutant could result in the polyploidisation phenotype. Further studies also showed that the pop3 mutant is defective in the maintenance of mRNA levels. All mRNA levels tested were found to be lower than wild type, however this only resulted in a decrease in protein level for a subset of genes. Hence, the inefficient transcription of the mutant and the resulting low levels of some proteins such as Cig2 and α-tubulin could lead to genome instability, temperature sensitivity and growth retardation. Further studies are required to establish whether Pop3 plays a role in the synthesis of transcription or in the maintenance of transcript stability. To date I have shown that the pop3 mutant is defective in the maintenance of the mRNA levels.

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