The primary objective of this work was the isolation and characterisation of gene products which interacted with Mts2p. This was achieved by the use of the yeast '2-hybrid' screen, and enabled isolation of a previously identified <I>S. pombe</I> gene <I>let1</I><SUP>+</SUP>, which is homologous to a gene from the budding yeast <I>S. cerevisiae</I>. The product of this homologue, <I>SUG1,</I> also a member of the AAA family, was thought to be in transcription or transcriptional regulation. Characterisation of the phenotype resulting from a disruption of <I>let1</I><SUP>+<I> </I></SUP>suggested that like <I>mts2</I><SUP>+</SUP>, it encoded a subunit of the 26S proteasome. Further evidence is presented in favour of this argument. In addition to <I>let1</I><SUP>+</SUP>, a novel <I>S. pombe </I>gene <I>aps1</I><SUP>+</SUP> was located. <I>Aps1</I><SUP>+</SUP> encodes the <I>S. pombe</I> homologue of the mouse MSS1 gene, which was originally isolated as a suppressor of a mutation in a yeast genes encoding a protein kinase. In this laboratory, however, MSS1 was isolated as a multicopy suppressor of the <I>ts</I> phenotype of <I>mts2-1</I>. The interactions between Mts2p, Let1P and Aps1p, the products of <I>mts2</I><SUP>+</SUP>, <I>let1</I><SUP>+ </SUP>and <I>aps1</I><SUP>+<I> </I></SUP>respectively, were studied using the yeast 2-hybrid<I><SUP> </SUP></I>system. The region of interaction between Let1p and Mts2p were defined, and the results suggest that, as is the case for two other ATPase subunits of the 26S proteasome, the N-terminus of each protein is important in mediating this interaction. In a separate screen to look for genes which were involved in the enhancement of position effect variegation at the centromere, 4 cold sensitive (<I>cs</I>) alleles of <I>mts2</I> were isolated. Mutation analysis was performed on these and on the three <I>ts</I> alleles of <I>mts2, </I>which had been isolated in the original drug resistance screen. All of the mutations lie in a 230 amino acid region which is highly conserved between all members of the AAA protein family. The phenotype of all of these mutants was studied with respect to morphology, DNA content and MBC resistance. The results indicate that the three <I>ts </I>alleles are more drug resistant and have a greater morphological deformation than the <I>cs</I> alleles.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:666244 |
| Date | January 1997 |
| Creators | McGurk, Gordon Benedict |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/11130 |
Page generated in 0.0019 seconds