No description available.

No description available.

No description available.

The Saccharomyces cerevisiae SKN7 gene has been identified through a search for genes which, at a high copy number, could restore the growth of the $kre9 Delta$ disrupted strain showing a cell wall $ beta$-glucan defect. SKN7 was mapped to the right arm of chromosome VIII, and is predicted to encode a 70 kDa protein, Skn7p, with a region of homology to the DNA binding domain of the yeast heat shock transcription factor, Hsf1p. Skn7p also has a domain which shows similarity to the prokaryotic receiver modules found on an extensive family of two-component response regulators, including the product of the rcsC gene. While restoring the growth rate to near wild type levels, SKN7 does not appear to restore the $ beta$-glucan levels of the $kre9 Delta$ mutant. However, SKN7-suppressed cells show a partially restored cellular morphology, and a restored cell wall resistance to mechanical stress. SKN7 does not suppress other mutations in the ($1 rightarrow6$)-$ beta$-glucan biosynthetic pathway, suggesting that it does not act as a general bypass suppressor of this glucan.

The Saccharomyces cerevisiae KEX1 gene product, Kex1p, has been identified and partially characterised to assess its role in processing secreted protein precursors and to define its intracellular location. Kex1p antiserum identified a 113 kDa protein that was absent in kex1-$ Delta$ cells and more abundant in cells overexpressing KEX1. Kex1p was found to be a type I membrane associated glycoprotein with N-linked carbohydrate. The N-linked oligosaccharide was modified in a progressive manner after synthesis, causing the glycoprotein to slowly increase in mass to 115 kDa. / After a Kex2p-mediated cleavage event at specific pairs of basic amino acids, $ alpha$-factor and K1 killer toxin precursors have COOH-terminal dibasic residue extensions and require a carboxypeptidase B-like activity to process the precursors to maturity. A carboxypeptidase activity, with apparent specificity for basic amino acids, was detected in KEX1 cells. Disruption of the KEX1 gene abolished this activity, while overexpression of KEX1 increased it. These results provide biochemical evidence, consistent with earlier genetic work, that KEX1 encodes a serine carboxypeptidase involved in the processing of precursors to secreted mature proteins. / Immunological and activity studies indicate that most Kex1p is intracellular and suggests that the enzyme is retained within the secretory pathway. COOH-terminal truncations of the protein indicate that the cytoplasmically exposed domain of Kex1p is responsible for correct localisation of the protein, probably in the late Golgi. / When KEX1 was expressed in Schizosaccharomyces pombe, Kex1p was localised in structures consistent with components of the Golgi. Mammalian cells expressing KEX1 produce a membrane associated activity that is not detected in the medium. In immunofluorescence studies on mammalian cells, Kex1p was localised to the ER and Golgi but not to the plasma membrane. Kex1p in such cells was responsible for completing the processing of the neuropeptide, $ gamma$-lipotropin. This in vivo processing of $ gamma$-lipotropin by Kex1p demonstrates a significant functional homology of the basic prohormone processing machinery in yeast and neuroendocrine cells.

This thesis reports on some sequencing and functional studies on chromosome I of Saccharomyces cerevisiae. / Eight open reading frames (ORFs) have been identified in S. cerevisiae which have similarity to the canine 24kD glycoprotein, gp25L (Wada et al., 1991). In chapter 2 of this thesis, I report the gene disruption and functional characterization of three of these ORFs: YAR002Ac, YAL007c and YGL002w. Disruption of YAR002Ac resulted in calcofluor white resistance, disruption of YGL002w increased sensitivity to this compound, while yal007c$ Delta$ mutants had no calcofluor white phenotype. The expression of Kre9p was partially increased in the ygl002w$ Delta$ mutant. All single, double and triple mutants grew, mated and sporulated normally. / The sequence of each S. cerevisiae chromosome, released in GenBank database, lacked most of the telomere sequence. This was due to the fact that a telomeric fragment with one clonable end cannot be integrated into a vector by the classical cloning method. Recently, Louis and Borts cloned all telomeres of S. cerevisiae successfully using an alternative cloning method (Louis and Borts, 1995). In chapter 3 of this thesis, I report the DNA sequence of the right telomeric region of chromosome I. The sequence indicates that this region represents a typical yeast telomeric region and contains 98bp of TG$ sb{1-3}$ repeats and an X subtelomeric element. Another subtelomeric element, the Y$ sp prime$ element, is absent from the right telomeric region of chromosome I. (Abstract shortened by UMI.)

A mutational analysis of genes which confer resistance to the yeast K1 killer toxin has identified a number of components involved in the synthesis of a $ beta(1 to 6)$-linked glucan polymer found in the Saccharomyces cerevisiae extracellular matrix. The KRE9 gene is predicted to encode a 30 kDa serine/threonine rich cell wall protein, which is modified by O-glycosylation before being secreted at the cell surface where it likely functions in $ beta(1 to 6)$-glucan assembly. Null mutations in KRE9 lead to killer resistance, slow vegetative growth, and reduced cell wall $ beta(1 to 6)$-glucan levels which are 10 to 20% of wild type. A study of the KRE11 gene has revealed that its product, Kre11p, is a 63 kDa cytoplasmic protein which appears to be involved in the regulation of glucan assembly. Through genetic interactions and epistasis assignments with these and other KRE genes, the basis of a biosynthetic pathway for the synthesis of this extracellular matrix polymer has emerged. / A search for genes involved in the regulation of cell surface assembly has led to the identification of SKN7. Sequence analysis of Skn7p revealed a region of homology to the DNA-binding domains found on heat-shock transcription factors, and a distinct region of similarity to a large family of bacterial "two-component" signal-transduction proteins. Two-component systems have historically been confined to prokaryotic organisms, and the identification of SKN7 has raised the possibility that two-component signaling pathways involving phospho-histidine and phospho-aspartate transfer reactions may exist in higher eukaryotes. Skn7p appears to function in yeast as a nuclear localized two-component response regulator, whose transcriptional B activity is regulated through aspartic acid phosphorylation. The Skn7p signal transduction pathway may act in concert with the yeast PKC1-mediated MAP-kinase cascade, to regulate cellular growth events at the cell surface.

The kre2-1 mutation is one of a number of K1 killer resistant complementation groups in Saccharomyces cerevisiae. kre2-1 cells bind less K1 killer toxin than wild type cells due to a reduced affinity for the toxin molecule and yet contain wild type levels of (1$ to$6)-$ beta$-D-glucan, a component of the toxin receptor molecule. The KRE2 gene was cloned by complementation of the kre2-1 mutation and is predicted to encode a 433 amino acid protein directed into the secretory pathway. Haploid strains which carried a disruption at the KRE2 locus, grew more slowly than wild type cells, and showed a defect in the N-linked glycosylation of secreted proteins analysed. Genetic and protein analyses showed the defect to be in the assembly of the core oligosaccharide, most likely in the attachment of one or perhaps two mannose residues to the core structure. The mutant core in kre2 cells appeared to be a less efficient substrate for outer chain elaboration. / A plausible interpretation of these results is that those mannose residues added to the core oligosaccharide in a KRE2 dependent manner are involved in the cross-linking of (1$ to$6)-$ beta$-D-glucan to mannoprotein in the cell wall to complete the K1 killer toxin receptor and failure to make such attachments is the basis of resistance to toxin in kre2 cells.

The PMT (Protein O-mannosyl transferase) family members are involved in the initial step of protein-O-mannosylation. A large scale procedure (Systematic Genetic Array) was performed using pmt1, pmt2, pmt3, pmt5, and pmt6 as query deletions and a set of 4700 non-essential array gene deletions, to screen for query/array double deletion mutant combinations affecting growth of Saccharomyces cerevisiae. This procedure revealed a genetic interaction network consisting of 53 interacting genes. Functional grouping of these 53 genes revealed 9 functional categories that were analyzed according to gene function to elucidate how they might buffer defects in protein-O-mannosylation. Synthetic genetic interactions were also identified between PMT family members demonstrating redundancies among them. Protein-O-mannosylation is a protein modification conserved from yeast to human. POMT1 and POMT2 (Human PMTs counterparts) catalyze mannosyl residue transfer in mammals, with mutations identified to be involved in Walker-Warburg Syndrome (WWS). ∼72 % of the genes in the yeast PMT genetic network have human homologs, and ∼55 % of these are associated with human disease. Using the yeast genetic interaction network as a model for human genetic interactions may help in the understanding of complex inherited human disease.

The Mpt5 protein of Saccharomyces cerevisiae has been implicated in a variety of cellular processes including cell cycle regulation, aging, cell wall maintenance and regulation of gene transcription by glucose. Loss of Mpt5p results in temperature-sensitive cell lysis which is osmotically remedial. In this thesis, synthetic lethalities of the mpt5Delta msn5Delta double mutant and the cln1Delta cln2Deltampt5Delta triple mutant are reported. A multicopy suppressor screen identified amino-terminal truncations of Mpt5p and the HMLalpha silent mating type locus as dosage-dependent suppressors of the mpt5Delta is lethality. Differential gene expression analysis in mpt5Delta revealed that several genes which code for cell wall structural proteins and genes which are under control of the Tup1p-repressor are less expressed in the absence of Mpt5p. Finally, Green Fluorescent Protein-tagged Mpt5p has allowed for investigation of Mpt5p cellular localization.