Expression of cDNA clones of the M1 double-stranded RNA killer preprotoxin coding region in Saccharomyces cerevisiae successfully directed the synthesis of secreted active toxin. Transformants harbouring these expression plasmids also displayed a K1 specific immunity phenotype. Immunoprecipitation of intracellular proteins with antitoxin antiserum showed that these transformants synthesize a 42kd glycosylated preprotoxin precursor. Two smaller unglycosylated immunoreactive species could also be resolved. These toxin precursor species were characterized by using secretory-defective hosts, by comparative electrophoretic mobilities, and by tunicamycin susceptibility. Such studies indicate that these protein species represent intermediates generated by signal cleavage of the preprotoxin and its subsequent glycosylation and provide evidence that these events occur post-translationally. Mutational analysis of the 44 amino acid preprotoxin N-terminal leader indicated that it is functionally bipartite, consisting of an N-terminal signal sequence and a C-terminal pro-sequence. Deletion of the leader perturbed but did not eliminate secretion of toxin.

No description available.

Reversible phosphorylation is a major mechanism for regulating the activity, localization and stability of proteins required for vital cellular processes such as glucose metabolism, gene expression, establishment of polarity, mitosis and cytokinesis. Phospho-regulation is driven by the activities of kinases and phosphatases. Together, these enzymes account for ∼3% of eukaryotic genomes and it is estimated that 30% of the eukaryotic proteome is composed of phospho-proteins. Protein kinases (PKs) have been studied extensively, however relatively little is known regarding the signaling networks of protein phosphatases (PPases). The identification of PPase functional networks has been slow due to the redundant nature of the majority of PPases, the complexity of their substrate recognition in vivo, and the lack of large-scale analyses that would facilitate network analysis. We hypothesized that large-scale analysis of genetic interactions using the Synthetic Genetic Array (SGA) and proteomic analyses using 2D-PAGE Difference Gel Electrophoresis (DiGE) could reveal PPase functional networks. Here, we apply this approach to the essential and conserved PP1 PPase Glc7 as it regulates numerous cellular processes in budding yeast. For this study, we created a glc7 hypomorphic mutant (glc7-E101Q) suited for both SGA and DiGE analyses. SGA analysis of glc7-E101Q revealed a broad network of 147 synthetic sick/lethal (SSL) and 178 synthetic rescue (SR) interactions. DiGE comparison of the glc7-E101Q proteome relative to wild-type at medium-resolution (∼1000 proteins) revealed alterations in 39 proteins that changed as a consequence of both the mutation and growth conditions. One of the proteins identified in this analysis was Eno1, a non-essential enolase that is mis-regulated in the presence of glucose and identified a SR mutation in the glc7-E101Q SGA. Subsequent phenotypic analysis suggests a novel, non-metabolic role for Eno1 in the Glc7 interaction network. Our results reveal that parallel analysis, using SGA and DIGE, can reveal novel functions and networks that a single analysis may not detect.

The mannoprotein of Saccharomyces cerevisiae, baker's yeast, is an emulsifying agent which could be used in foods and cosmetics. This glycoprotein emulsifier can be extracted simply with very good yield by autoclaving yeast cells in neutral buffer. The spent yeast from the beer and wine industries is a suitable raw material for its production. Protein detected by binding of Coomassie blue dye was essential for emulsifying activity. Components of the heat extracted material with greatest emulsifying activity included a high molecular weight fraction ($>$200 kDa) which provided viscous and durable emulsions, and a low molecular weight fraction ($<$14 kDA) which was very surface active and readily generated nonviscous emulsions. Mannoprotein and the small molecule surfactants lecithin and cetyltrimethylammonium bromide interacted synergistically to increase emulsifying activity at a weight ratio of 100:1 mannoprotein to surfactant. A correction was made to the formula for the Emulsifying Activity Index (Pearce, K. N. and J. E. Kinsella. 1978. J. Agric. Food Chem. 26:716-723), a measure commonly used for comparing protein emulsifiers.

This thesis is a genetic study of the cytoplasmically- inherited determinant [psi] of Saccharomyces cerevisiae . [psi] is a potentiator of ochre suppression. The molecular basis of [psi] was investigated using mutagenesis as a probe. The psi⁺ phenotype (efficient suppression) can be mutated to psi^- phenotype (loss of suppression) by ultra-violet light (UV) and nitrosoguanidine (NTG) . The UV-induced mutation was a single-hit event and the pre-mutational lesion was partly photoreactivable . Repair or expression of UV-induced mutation to the [psi] determinant was under the same genetic control as for nuclear mutation. It was concluded that [psi] has a DNA genome. The 'extrachromosomal mutagens' thymidylate starvation, 5-fluorouracil, manganese chloride and cycloheximide failed to induce psi^- mutants whilst guanidine hydrochloride, dimethyl sulphoxide and potassium chloride were shown to induce this mutation at frequencies up to 100%. Several other physical and chemical agents caused a high frequency of loss of the psi⁺ phenotype. A new class of recessive nuclear mutation (pnm) was shown to cause a loss of the psi⁺ phenotype. A simple comple- mentation test was devised to distinguish them from cytoplasmic psi^- mutants. The dominant PNM^- mutation was shown not to cause a physical loss of the [psi] genome. Two mutants with a modified PNM^- phenotype were analysed. Attempts to demon- strate genetically the involvement of [psi] with the 80S ribosome were unsuccessful. The psi⁺ phenotype was conclusively demonstrated to be inherited independently' of the nucleus using a 'heterokaryon test'. Two models for the [psi] phenomena were proposed; one postulating the presence of a DMA 'plasmid' and one postulating the involvement of a stable, self-perpetuating metabolic state.

There are 30-35 copies of the retrotransposon Ty in the haploid genome of most laboratory strains of Saccharomyces cerevisiae. Ty elements are 5.9 kb long, have LTRs of 340 bp and produce a genomic RNA that is 5.7 kb in length, through which it transposes. They contain two ORFs; the TYA gene of the class I element Tyl-15 is 1,319 nucleotides long whilst its TYB gene, which possesses the consensus sequences of retroviral acid proteases, reverse transcriptases and integrases, is 3,984 nucleotides long. TYB overlaps TYA by 38 nucleotides at its 5' end and is expressed as a TYA;TYB fusion protein. In an attempt to asign some in vivo functions to the Ty encoded proteins, the whole transcriptional unit of Tyl-15 was overexpressed in yeast. Electron micrographs of the overexpressing cells revealed an abundance of cytoplasmic, ~60 nm virus-like particles (Ty-VLPs). These contain the 5.7 kb Ty RNA and have an associated reverse transcriptase activity. Their major protein is p2 (48 kD), which is a proteolytic derivative of the primary translation product of TYA, pi (50 kD), a protein that is also assembled into particles. A truncated pi, encoded by a TYA gene shortened by 60 codons at its 3' end, still aggregates into particles. Furthermore, the addition of α2-IFN (20 kD) onto its carboxy terminus, to produce a pl-IFN fusion protein of ~68 kD, does not interfere with its particle forming properties. The hybrid VLPs are easy to purify and elicit an antibody response in rabbits to the Ty and IFN epitopes. A series of fragments spanning Tyl-15 were inserted into the packaging analysis vector, pMA924. The association of the resulting PGK-Ty-IFN hybrid RNAs with Ty-VLPs, in the presence of abundant Ty encoded proteins, was analysed by Northern blotting and used to assess the packaging capabilities of Ty RNA. Unexpectedly, RNA sequences that, direct specific packaging are produced from regions throughout Tyl-15. The signals corresponding to TYA are probably located within two regions of the genomic RNA, 5' to nucleotide 236 and 3' to nucleotide 737; those corresponding to TYB are in at least two, currently undefined, areas. The TYB gene of the class II element Tyl-17 overlaps TYA by 44 nucleotides. The fusion of α2-IFN cDNA fragments into all three reading phases of TYB followed by a Western blot analysis demonstrated that, like the TYB gene of Tyl-15, it too is expressed as a TYA:TYB fusion protein.

In yeast, aging appears to be marked by a progressive impairment in cellular mechanisms, resulting in irreversible changes in physiology and morphology. To date, very little has been reported about the biochemical changes that occur in yeast as a function of individual cell aging. To investigate this further, six generations of a brewing yeast strain of Saccharomyces cerevisiae (NCYC 1239) were separated according to cellular age using continuous phased culturing and biotin-streptavidin magnetic cell sorting. / To obtain cells with no bud scars (virgin cells), a concentrated yeast slurry was layered onto sucrose density gradients and centrifuged. The uppermost band from the gradients was collected and cells were biotinylated with biotinamidocaproate- N-hydroxysuccinimide ester, that covalently binds to lysine residues on the yeast cell wall. For continuous phased culturing, biotinylated cells were added to a carbon-limited nutrient medium and growth was synchronized using the doubling time of the cells. Harvested cells were incubated with streptavidin superparamagnetic beads and sorted with a strong permanent magnet. In total, approximately 75% of the biotinylated cells were recovered. Viability testing was conducted using vital staining and plate counts, with >98% viability reported with the vital stain and 37% viability with the agar plates. / In conclusion, continuous phased culture, together with magnetic cell sorting has the potential to become a powerful tool for the study of age-related biochemical changes in yeast. Further studies will focus on ensuring the reproducibility of the method and using the recovered cells to study biochemical changes occurring during yeasts' replicative lifespan.

Prp20, the Saccharomyces cerevisiae homolog to the mammalian regulator of chromosome condensation, RCC1, binds to double-stranded (ds) DNA in vitro through a multi-component complex. Three members of this complex bind GTP in vitro. The Prp20 complex specifically loses its ability to bind dsDNA during DNA replication as determined by an in vitro assay using cell extracts from arrested cdc mutants. This loss of dsDNA-binding activity does not affect the proper organization of the nucleoplasm as was the case for the prp20-1 and prp20-7 mutants, suggesting a segregation exists in the biochemical activities of the Prp20 protein. Detailed analysis of Prp20 demonstrates that specific and highly conserved amino acids coordinate these distinct activities of the Prp20 complex. These essential residues are mainly located in the second and the third repeats of the amino terminus and the last two repeats of the carboxyl terminus of Prp20. Furthermore, mutations in the last two repeats are suppressed by Gsp1, one of the GTP-binding components of the Prp20 complex, but not the mutations in the amino-terminus of Prp20.

Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

University, Diss., 2006--Frankfurt (Main). / Zsfassung in engl. und dt. Sprache.