U529238.pdf (40.06 MB)
Analysis of host/plasmid interactions in yeast.
thesisposted on 2015-11-19, 09:06 authored by Katherine Ann. Duffy
The 2mum plasmid of Saccharomyces cerevisiae is an autonomously replicating, multicopy, nuclear DNA plasmid. The plasmid requires both plasmid and host-encoded functions to ensure its stable maintenance. To date, no positive phenotype has been associated with the presence of the 2mum plasmid in the host cell. It is proposed, therefore, that host proteins involved in 2mum plasmid stability will have general DNA maintenance roles within the nucleus. The role of the host in 2mum plasmid replication has been well documented. It has been demonstrated that the 2mum ARS element interacts with host replication proteins in the same way as chromosomal replicator sequences. Prior to this study, host genes involved in replication have been identified by the study of mutants defective in plasmid and minichromosome maintenance. More recently, the host has been implicated in the 2mum plasmid partitioning mechanism. One mutant defective specifically in 2mum plasmid partitioning has been isolated (plm1; plasmid maintenance). This study describes the isolation of four further 2mum plasmid maintenance mutants. These are temperature-sensitive and were shown to represent two complementation groups, plm2 and plm3. Plasmid stability and copy number analyses suggest that plm2 is not defective in the replication of plasmid molecules. Genetic complementation of the plm2 mutation with a genomic library was successfully carried out. PLM2 is a previously unidentified ORF on chromosome IV, 1.6kb in length encoding for a protein of 521 amino acids. Sequence analysis revealed that PLM2 has a homologue on chromosome XII, also of unknown function. A strain carrying a disrupted allele of plm2 created by integration of a Tn1000::HIS3 cassette is viable. Analysis of the Plm2p sequence demonstrated homology with nuclear proteins involved in DNA binding, either as DNA-directed RNA polymerases or as transcription factors. It is proposed that Plm2p has a general role in DNA maintenance within the yeast nucleus, perhaps as a protein of the nuclear scaffold.
Date of award1996-01-01
Author affiliationModern Languages
Awarding institutionUniversity of Leicester