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Biochemical and genetic studies of mitochondrial protein synthesis in Saccharomyces cerevisiae : characterization of the AEP3 and TRM5 gene productsLee, Changkeun, 1971- 18 September 2012 (has links)
Protein synthesis in archaebacteria and the cytoplasm of eukaryotes is initiated using the initiator methionyl-tRNA (Met-tRNA[subscript i][superscript Met]). In contrast, formylated methionyltRNA (fMet-tRNA[subscript i][superscript Met][subscript f]) is found in eubacteria, and in chloroplasts and mitochondria of eukaryotes, and this formylated initiator tRNA was widely believed to be required for initiation of protein synthesis in those systems. However, the fact that initiation of protein synthesis in yeast mitochondria can occur with unformylated initiator tRNA has changed our perspective about the initiation of mitochondrial protein synthesis. This dissertation is composed of two parts. Part I describes an investigation of the yeast AEP3 gene which was isolated by a genetic screening system in Saccharomyces cerevisiae. The main goal of this part was to discover new accessory factor(s) that might be involved in initiation of protein syntheis of yeast mitochondria when there is no formylation of initiator tRNA and determine how they support the initiation process in Saccharomyces cerevisiae. The synthetic petite genetic screen identified the AEP3 gene. Protein-protein binding assays as well as protein-initiator tRNA binding assays indicate that Aep3p is associated with the initiation process in yeast mitochondrial protein synthesis. This discovery is important because it suggests the possible mechanism by which initiation of protein synthesis in yeast mitochondria occur under conditions where there is no formylation of initiator tRNA. Part II describes a study of the TRM5 gene encoding a tRNA methyltransferase in S. cerevisiae. The TRM5 gene encodes a tRNA (guanine-N1-)-methyltransferase (Trm5p) previously known to methylate guanosine at position 37 (m¹G37) in certain cytoplasmic tRNAs in S. cerevisiae. The main goal of this part was to investigate whether Trm5p is also responsible for m¹G37 modification of mitochondrial tRNAs. Full-length Trm5p, purified as a fusion protein with maltose-binding protein, exhibited robust methyltransferase activity with tRNA isolated from a [Delta]trm5 mutant strain, as well as with a synthetic mitochondrial tRNA[superscript Met][subscript f] and tRNA[superscript Phe]. High pressure liquid chromatography analysis showed the methylated product to be m¹G. Analysis of subcellular fractionation and immunoblotting revealed that the enzyme was localized to both cytoplasm and mitochondria. Our data including the analysis of N-terminal truncation mutants suggest that this tRNA modification plays an important role in reading frame maintenance in mitochondrial protein synthesis. / text
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Comprehensive phenotype analysis and characterization of molecular markers of the poles of Saccharomyces cerevisiaePage, Nicolas. January 2001 (has links)
The bipolar budding pattern of a/a Saccharomyces cerevisiae cells appears to depend on persistent spatial markers. Genetic analysis reported here indicates that BUD8 and BUD9 potentially encode components of the markers at the distal and proximal poles, respectively. Mutants deleted for BUD8 or BUD9 bud exclusively from the proximal and distal poles, respectively, and the double-mutant phenotype suggests that the bipolar budding pathway has been totally disabled. Moreover, overexpression of these genes can cause either an increased bias for budding at the distal (BUD8) or proximal (BUD9) pole or a randomization of bud position, depending on the level of expression. Both molecules are related plasma membrane glycoproteins that are both N- and O-glycosylated. Each protein was localized predominantly in the expected location, with Bud8p delivered to the presumptive bud site just before bud emergence, and Bud9p delivered to the bud side of the mother-bud neck just before cytokinesisis. Promoter-swap experiments revealed the importance of time of transcription in localization: expression of Bud8p from the BUD9 promoter leads to its localization predominantly in the sites typical for Bud9p, and vice versa. Moreover, expression of Bud8p from the BUD9 promoter fails to rescue the budding-pattern defect of a bud8 mutant but fully rescues that of a bud9 mutant. However, although expression of Bud9p from the BUD8 promoter fails to rescue a bud9 mutant, it also rescues only partially the budding-pattern defect of a bud8 mutant. / Using a collection of mutants individually deleted for almost every yeast gene, I undertook a genome-wide phenotype analysis for altered sensitivity to a yeast antifungal protein, the K1 killer toxin. Mutations in most genes have no effect on toxin sensitivity, with less than 10% having a phenotype. Only 4% of these were previously known to have a toxin phenotype. There is a markedly non-random functional distribution of mutants with a toxin phenotype. Many genes fall into a limited set of functional classes or modules, which define specific areas of cellular function. These include known pathways of cell wall synthesis and signal transduction, and offer new insights into these processes and into cell wall morphogenesis.
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N-chain glucose processing and proper -1,3-glucan biosynthesis are required for normal cell wall -1,6-glucan levels in Saccharomyces cerevisiaeDijkgraaf, Gerrit J. P. January 2001 (has links)
CWH41 is required for beta-1,6-glucan biosynthesis and encodes glucosidase I, an enzyme involved in protein N-chain glucose processing. Therefore, the effects of N-chain glucosylation and processing on beta-1,6-glucan biosynthesis were examined, and it was shown that incomplete N-chain glucose processing results in loss of beta-1,6-glucan. To explore the involvement of other N-chain-dependent events with beta-1,6-glucan synthesis, the S. cerevisiae KRE5 and CNE1 genes were investigated, which encode homologs of the 'quality control' components UDP-Glc:glycoprotein glucosyltransferase and calnexin, respectively. The essential activity of Kre5p was found to be separate from its possible role as a UDP-Glc:glycoprotein glucosyltransferase. A ∼30% decrease in beta-1,6-glucan was observed upon disruption of CNE1, a phenotype which is additive with other beta-1,6-glucan synthetic mutants. Analysis of the cell wall anchorage of alpha-agglutinin suggests the existence of two beta-1,6-glucan biosynthetic pathways, one N-chain dependent, the other involving protein glycosylphosphatidylinositol modification. / Fks1p and Fks2p are related proteins thought to be catalytic subunits of the beta-1,3-glucan synthase. The fks1Delta mutant was partial K1 killer toxin resistant and showed a 30% reduction in alkali-soluble beta-1,3-glucan that was accompanied by a modest reduction in beta-1,6-glucan. The gas1Delta mutant lacking a 1,3-beta-glucanosyltransferase displayed a similar reduction in alkali-soluble beta-1,3-glucan but did not share the beta-1,6-glucan defect, indicating that beta-1,6-glucan reduction is not a general phenotype among beta-1,3-glucan biosynthetic mutants. FKS2 overexpression suppressed the killer toxin phenotype of fks1Delta mutants, implicating Fks2p in the biosynthesis of the residual beta-1,6-glucan present in fks1Delta cells. Eight out of twelve fks1tsfks2Delta mutants had altered beta-glucan levels at the permissive temperature: the FKS1F1258Y N1520D allele was severely affected in both polymers and displayed a 55% reduction in beta-1,6-glucan, while the in vitro hyperactive FKS1T6051 M761T allele increased both beta-glucan levels. These beta-1,6-glucan phenotypes may be due to altered availability of, and structural changes in, the beta-1,3-glucan polymer, which might serve as a beta-1,6-glucan acceptor at the cell surface. Alternatively, Fks1p and Fks2p could actively participate in the biosynthesis of both polymers as beta-glucan transporters. beta-1,6-Glucan deficient mutants had reduced in vitro glucan synthase activity and mislocalized Fks1p and Fks2p, possibly contributing to the observed beta-1,6-glucan defects.
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Functional characterization of Saccharomyces cerevisiae Zeo1p, a Mid2p interacting proteinGreen, Robin G. January 2002 (has links)
We have previously demonstrated that Mid2p is required for the activation of the PKC1-MPK1 cell integrity pathway during cell exposure to mating pheromone, calcofluor white (CFW), and heat. Accumulating evidence indicates that Mid2p might regulate this pathway via the small GTPase, Rho1p. To understand the mechanism by which Mid2p signals, we initiated a two hybrid screen using the essential cytoplasmic tail of Mid2p as bait. ZEO1 (YOL109w), a previously uncharacterized open reading frame, was identified. ZEO1 encodes a 12kDa protein that co-localizes to the plasma membrane and interacts with the cytoplasmic tail of Mtl1p, a Mid2p functional homologue. Like mid2Delta mutants, cells deleted for ZEO1 are resistant to calcofluor white. In addition, ZEO1 null strains are no longer hypersensitive to calcofluor white caused by high copy expression of MID2. A role for Zeo1p in the cell integrity pathway is supported by the finding that disruption of ZEO1 leads to a Mid2p-dependent constitutive phosphorylation of Mpk1p. (Abstract shortened by UMI.)
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Functional and cell biological characterization of Saccharomyces cerevisiae Kre5pLevinson, Joshua N. January 2002 (has links)
Saccharomyces cerevisiae Kre5p is important for the biosynthesis of beta-1,6-glucan, which is required for proper cell wall assembly and architecture. A functional and cell biological analysis of Kre5p was conducted to further elucidate its role in beta-1,6-glucan synthesis. Kre5p was found to be a primarily soluble N-glycoprotein of ∼200 kD that localizes to the endoplasmic reticulum. Observation of Kre5p-deficient cells reveals a severe cell wall morphological defect, and kre5Delta cells were shown to have only residual levels of beta-1,6-glucan. KRE6 was identified as a multicopy suppressor of a temperature-sensitive kre5 allele, suggesting these proteins participate in a common pathway. An analysis of truncated versions of Kre5p indicates that it may have two independent, essential activities, or that it functions in a homodimeric state. Finally, Candida albicans KRE5 was shown to partially restore growth to kre5Delta cells, suggesting it has a function similar to that of the S. cerevisiae protein.
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Disruption of a putative calcium channel gene in Saccharomyces cerevisiaeCho, John Myung-Jae. January 1996 (has links)
A search of the Saccharomyces genome data base revealed an open reading frame of 2039 amino acids with homology to L-type calcium channels. Northern blots probed with a 540 bp PCR product of the ORF showed a transcript of 6.1 kb. Two procedures were used to disrupt the gene: the ORF was truncated by an integrative disruption after the third pore motif, or deleted in the first three pore domains using a one-step disruption construct. In most strains tested, the disruptions gave no apparent phenotype when tested under a variety of conditions. However, conspicuous phenotypes were seen in the strain YEL161-2A, a strain super-sensitive to alpha-mating factor (sst1). In most respects, truncation gave a less severe phenotype than deletion, suggesting that the truncated gene retains partial function. Calcium uptake during normal growth, as well as the increased calcium uptake in response to mating factor, were reduced progressively by the truncation and deletion respectively. Growth rate and cell viability were reduced, cell size heterogeneity increased, and recovery from mating factor arrest was delayed and abnormal. The cells became sensitive to MnCl$ sb2.$ The phenotype resulting from gene truncation was alleviated by a high-calcium medium, and exacerbated by low calcium. Complementation of the deleted strain by a Yep13 plasmid containing BAR1 (SST1) restored normal growth and viability. However, somewhat paradoxically, deletion of the putative calcium channel gene in another sst1 strain (SY1159) showed no phenotype.
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Timing is everything: The link between chromosomal mobility and homologous recombinationJoseph, Fraulin January 2021 (has links)
Chromosomes are very dynamic structures that are constantly undergoing physical changes necessary for cell survival. Studies in yeast and metazoans have shown that chromosomal loci exhibit large-scale changes in mobility in response to DNA double-strand breaks (DSBs). If left unrepaired, DSBs can lead to disease and even cell death. One of the predominant cellular pathways utilized to repair DSBs is homologous recombination (HR). DSB repair via HR requires a homologous DNA template to recover the missing genetic information lost at the break site. Our lab proposes that increased chromosome mobility (ICM) facilitates recombination by helping a broken chromosome successfully find its homolog. In support of this view, ICM is under the genetic control of the HR machinery and requires activation of the DNA damage checkpoint response. However, there is currently no consensus on the precise functional role of ICM in HR.
In Chapter 1, I describe in detail the known steps of DSB repair via the HR pathway, and discuss some of the important advancements made in the field of cell biology that has helped shape our understanding of HR. I highlight the use of in vivo cell imaging and fluorescently labeled DNA repair proteins during the study of HR. Additionally, I discuss some of the first studies that examined chromosome dynamics within the nucleus in live cells. Lastly, I describe the phenomenon of increased chromosome mobility and expand upon why it needs to be studied further.
In Chapter 2, I present in detail our method for measuring the pairing of DNA loci during HR at a site-specific DSB in Saccharomyces cerevisiae. This method utilizes live cell imaging and a chromosome tagging system in diploid yeast to visualize homologous chromosomes during HR-mediated repair. Using this method, we demonstrate that in wild type (WT) cells, homologous chromosomes come together, repair and then move apart after repair is complete. Importantly, the kinetics we observe in the pairing of homologous chromosomes match the kinetics of site-specific DSB formation and the subsequent gene conversion of that site.
In Chapter 3, I describe our study that elucidates the relationship between ICM and multiple HR steps. We find a tight temporal correlation between the recruitment of the recombination proteins, ICM, the physical pairing of homologous loci, and gene conversion. Importantly, we can shift the timing of ICM by altering the initiation of DNA end resection - an early step in the HR process. Our data highlight the importance of DNA end resection as a vital precursor to ICM and demonstrate a strong temporal linkage between ICM and HR. Taken together our data support the claim that ICM is essential to HR and mechanistically involved in the process of DNA repair.
In Chapter 4, we explore chromosome mobility in response to different forms of DNA damage such as spontaneous DSBs, collapsed replication forks, and ionizing radiation (IR). We find that spontaneous DSBs and collapsed replication forks do not induce a change in chromosome mobility. However, exposure to ionizing radiation results in a robust increase in global chromosome mobility that is dependent on activation of the DNA damage checkpoint. Overall, these findings demonstrate how ICM is tightly regulated and highly dependent on the circumstances surrounding the formation of the DSB.
Lastly, in Chapter 5, I summarize all of my findings and discuss how they relate to one another with respect to the linkage between ICM and HR. I also provide a perspective on future experiments needed to advance the field.
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Functional characterization of Saccharomyces cerevisiae Zeo1p, a Mid2p interacting proteinGreen, Robin G. January 2002 (has links)
No description available.
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Functional and cell biological characterization of Saccharomyces cerevisiae Kre5pLevinson, Joshua N. January 2002 (has links)
No description available.
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Disruption of a putative calcium channel gene in Saccharomyces cerevisiaeCho, John Myung-Jae January 1996 (has links)
No description available.
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