Hartley, Caroline Anne (1995) Cloning and characterisation of SSV genes from Saccharomyces Cerevisiae. Masters thesis, Durham University.
In order to investigate the pleiotropic effects exhibited by mutations of S. cerevisiae defective in genes involved in vacuole biogenesis, a number of mutants were chosen for further study. Three mutants which had been isolated for salt sensitivity (ssv mutants) also exhibited defects in a number of unrelated functions including protein targeting and vacuole inheritance. Attempts were made to clone the corresponding wild type genes defined by these mutations. Plasmid pMLY38 was thought to confer an advantageous growth effect on the ssv7-l mutant strain, complementing the salt sensitivity and enhancing its growth on YPDA media supplemented with 1.5M salt. Further analysis of growth curves indicated that pMLY38 did not enhance the growth of ssv7-l. Subsequent sequencing indicated that pMLY38 contained the SSV7 gene. However, an open reading frame (ORFl) was detected downstream of SSV7 and the sequence was analysed. A gene disruption of ORFl indicated that it was not an essential gene. Computer analysis of the DNA sequence showed that it encoded a 310 amino acid protein which has a highly acidic C-terminal end. Searches of the data bases showed limited homology to SCYKL202W1 and Nucleolar Transcription Factor 1, the significance of which is unknown. Characterisation of ssv 16 strains indicated cells with class B vacuoles. Immunoprecipitation results showed the mutants also secrete large quantities of CPY. They also display a semi-dominant phenotype for CPY mis-sorting. In the mutant/wild type diploid CPY continues to be secreted indicating that the mutant phenotype is not completely suppressed by the wild type gene. Cloning of SSV16 isolated the previously sequenced gene, SISl. A study of the growth curves, secretion of CPY and chromosome mapping of SSV16 failed to ascertain whether SISl is identical to SSV16.Immunofluorescence of the ssv 17-101 strains indicated that it belongs to a small group of mutants defective in vacuole segregation. Four transformation methods were employed in order to clone SSVl 7 via complementation from a gene bank. Difficulties encountered with these methods resulted in a chromosome mapping technique being employed to locate the gene. This placed the SSV17 gene on chromosome XIV. Attempts to narrow down the position on the chromosome by meiotic mapping were unsuccessful. Information that a VACl homologue was present on chromosome XIV led to its cloning by PGR in order to determine if it complemented the ssvl7-101 mutation.
|Item Type:||Thesis (Masters)|
|Award:||Master of Science|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||24 Oct 2012 15:11|