Browsing by Subject "Saccharomyces cerevisiae"
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Item A method for identifying proteins involved in heat shock protein secretion in Saccharomyces cerevisiae(Texas Tech University, 2001-12) Crum, Charles DouglasYeasts have made a massive contribution to our lives in a variety of ways. They have been used for brewing, baking, pharmaceuticals and other industrial processes for a number of reasons. They possess relatively simple growth requirements and can be cultured easiy. The conservation of most biochemical activities throughout a wide range of organisms has allowed for the use of S. cerevisiae as a model eukaryote which has contributed immensely to our understanding of cell biology. S. cerevisiae exists in a haploid or diploid state and can reproduce both asexually and sexually, having both the a and a mating types. It has a genome that has sixteen linear chromosomes and has been classified in the ascomycetes group of fungi. It is generally considered nonpathogenic except in a very small number of susceptible individuals. The entire genome of S. cerevisiae has been sequenced and is available on various public databases. The Saccharomyces proteome is currently being mapped, which covers the entire range of proteins and thier function. All of these factors allow us to use S. cerevisiae as a model for studying many processes of cellular function including secretion through temperature sensitive mutants, cellular and nuclear organization as well as protein function, for example, one model has utilized the Saccharomyces invertase protein as a marker for the external localization of a gene fused to a hybrid transcript (51). Being a nonpathogenic model organism allows us to utilize the qualities of S. cerevisiae to learn about other pathogenic organism including Candida albicans, C. dublienensis. Cryptococcoccus neoformans and other eukaryotic pathogenic fungi.Item Catalytic mechanism of Saccharomyces cerevisiae NAD+-dependent 5,10-methylenetetrahydrofolate dehydrogenase(2004) Wagner, Wendi Suzanne; Robertus, Jon D.5, 10-Methylenetetrahydrofolate dehydrogenase (yMTD) is an NAD+ - dependent enzyme located in the cytosol of Saccharomyces cerevisiae. It catalyzes the conversion of 5, 10-methylenetetrahydrofolate to 5, 10- methenyltetrahydrofolate, which then spontaneously oxidizes to 10- formyltetrahydrofolate within the cell. yMTD is one of three yeast isozymes, the other two of which have additional cyclohydrolase and synthetase activities, and is thought to be responsible for flux in the oxidative direction. To date, yMTD is the only known monofunctional eukaryotic MTD. The catalytic mechanism of yMTD was explored with site-directed mutagenesis of active site residues Glu121, Cys150, and T151 and with X-ray crystallography. Results showed Glu121 to be essential for catalysis, with mutations E121A and E121Q eliminating all measurable activity. E121D, which retains the carboxylic acid of Glu121, changed the kcat/Km to 10.1% of wild-type and the Km of substrate five-fold higher than wild-type. Mutations C150A, C150S and T151V also abolished activities. T151A reduced kcat/Km to 28.1% of wild-type. Crystal structures of E121A, C150A, and T151A did not show any conformational change from wild-type, indicating that mutations did not affect protein folding. Results suggest a catalytic mechanism in which Glu121 binds the substrate near carbon C2 to orient it for the hydride transfer to NAD+ . Cys150 contributes to the electron-rich environment of the substrate as well. Thr151 binds NAD+ with a hydrogen bond to the carbonyl of the nicotinamide ring and may help to stabilize the transition state. Mutations of active site residues A50S, T57K, and Y98Q were made in the hope of restoring cyclohydrolase activity to yMTD. These efforts were unsuccessful, although the cyclohydrolase assay is too crude to measure small amounts of activity. Various folate analogs were also tried against wild-type yMTD and pteroic acid, 5-formyltetrahydrofolate, and methotrexate were identified as inhibitors. These inhibitors were used in crystal screens against wild-type, E121A, and C150A yMTD to grow crystals of yMTD with a substrate analog. Although crystals were formed, they only grew under wild-type conditions, had the same unit cell as apoenzyme, and did not show density for a substrate analog in the refined structure.Item Characterization of cobalmin-independent methionine synthase from Candida albicans and Saccharomyces cerevisiae(2006) Suliman, Huda Sirageldin; Robertus, Jon D.Item Combinatorial engineering of Saccharomyces cerevisiae for efficient pentose catabolism(2014-08) Lee, Sun-Mi; Alper, Hal S.; Contreras, Lydia; Georgiou, George; Maynard, Jennifer; Whiteley, MarvinThe efficient fermentation of lignocellulosic biomass would enable more economically and environmentally friendly production of biofuels and biochemicals. Yet, Saccharomyces cerevisiae, a platform organism for biofuels and biochemicals production, is unable to convert all of the sugars in lignocellulosic biomass into biofuels and biochemicals mainly due to the lack of a pentose catabolic pathway. Though the advance of genetic engineering enabled S. cerevisiae to utilize pentose sugars, the efficiency of pentose sugar catabolism in S. cerevisiae is still limited. Here, the goal of this research was to confer efficient pentose sugar catabolism to S. cerevisiae by combinatorial and evolutionary engineering. To this end, pentose catabolic pathways were 1) constructed by heterologous expression of pentose catabolic genes, 2) optimized through rational engineering, and 3) further improved through evolutionary engineering. Through these efforts, we reported the highest ethanol yield (0.45 g ethanol / g xylose) and the second highest xylose consumption and ethanol production rates (0.98 g xylose g cell⁻¹ h⁻¹ and 0.44 g ethanol g cell⁻¹ h⁻¹, respectively) in xylose fermentation reported to date. The high performance in xylose fermentation was achieved based on the mutant xylose isomerase (xylA3), which showed 77% increased enzyme activity, engineered through directed evolution. In addition, we have established the first cells capable of growing on arabinose in mimimal medium and demonstrated ethanol production from xylan in minimal medium. The arabinose and xylan catabolic pathways were constructed in S. cerevisiae by expressing novel pentose catabolic genes from a strain with remarkable pentose catabolic potential that we isolated and named Ustilago bevomyces. In doing so, a complete workflow of bioprospecting to pathway engineering and evolution was detailed as an effective way to transfer a desired phenotype from a non-model organism to a model organism. This study substantially improved the prospect of biofuels and biochemicals production from lignocellulosic biomass by developing efficient pentose utilizing strains, finding new pentose catabolic genes, and suggesting alternative pentose catabolic pathway. Furthermore, the general tools for metabolic engineering demonstrated in this study would also advance microbial strain engineering.Item Exploring Iron Metabolism and Regulation in Saccharomyces cerevisiae Using an Integrative Biophysical and Bioanalytical Approach(2013-12-03) Park, JinkyuFe metabolism in budding yeast Saccharomyces cerevisiae was studied using an integrative systems-level approach involving M?ssbauer, EPR, UV-Vis spectroscopy and LC-ICP-MS, combined with conventional biochemical techniques. Wild-type cells growing exponentially on rich and minimal media were well-regulated in terms of cellular Fe homeostasis, while post-exponentially grown cells were unregulated. Such cells became overloaded with Fe^(III) oxyhydroxide nanoparticles and nonheme high spin (NHHS) Fe^(III). Fe overloading probably arose from a mismatch between growth rate and Fe uptake rate. A mathematical model that describes iron trafficking and regulation in these cells was developed. The speciation of Fe in cells also depended on the nutrient composition of the growth media. Adenine deficiency induced a transient reduction of vacuolar Fe^(III) to Fe^(II) which probably accumulated in the cytosol. The concentration of glucose impacted the Fe import rate but had little effect on Fe speciation. The concentration of amino acids and nucleotide bases impacted the level of Fe accumulation and shifted the Fe distribution toward NHHS Fe^(II). A thermodynamic model which correlated nutrient-dependent Fe transformations with vacuolar pH and redox status was developed. The effect of deleting the MTM1 gene, which encodes a transport carrier on the mitochondrial inner membrane, was investigated. Deleting MTM1 caused Fe to accumulate in mitochondria and the Mn superoxide dismutase 2 (SOD2) activity to decline. Previous studies had concluded that this inactivation arose from the misincorporation of Fe into apo-Sod2p. Most of the accumulated Fe was found to be Fe^(III) nanoparticles which are unlikely to misincorporate into apo-Sod2p. Soluble extracts from WT and ?mtm1 mitochondria were subjected to size-exclusion and anion-exchange liquid chromatography interfaced with an on-line ICP-MS. Two major Mn peaks were observed, one due to MnSod2p and the other to a Mn species with a molecular mass of 2 - 3 kDa. None of the Fe traces comigrated precisely with MnSod2p, contrary to the Fe-misincorporation hypothesis. Deleting MTM1 probably diminishes SOD2 activity by failing to metallate apo-Sod2 protein. The low-molecular-mass Mn species may function to install Mn into apo-Sod2p during maturation in the mitochondrial matrix, using some maturation factor imported by Mtm1p.Item Genetic interactors of the Cdc42 GTPase effectors Gic1 and Gic2: their identification and functions in budding yeast cell polarity(2004) Gandhi, Meghal Kanaiyalal; Chan, Clarence S. M.Gic1 and Gic2 are structurally and functionally related effectors of the evolutionarily conserved Cdc42 GTPase in Saccharomyces cerevisiae. Like many other effectors of Cdc42, Gic1 and Gic2 function in the process of polarized cell growth. In the absence of both Gic1 and Gic2, yeast cells exhibit depolarized actin cytoskeleton and polarized growth defects at elevated temperatures. To obtain further insight into the biological role of Gic1 and Gic2, genetic approaches were used to identify functionally interacting partners of these proteins. A screen for multi-copy suppressors of the temperature-sensitivity of gic1 gic2 cells identified many genes (including AXL2, BNI1, CLN2, MSB1, MSB2, RSR1 and STE20) that have known roles in polarized cell growth. In addition, two pairs of structurally related genes - VHS2 and MLF3, MGC1 and TOS2 - with no previously reported functions were also identified. Functional characterization of VHS2 and MLF3 revealed their role in a pathway that affects the actin cytoskeleton organization and cell wall integrity. This pathway is functionally redundant to that mediated by GIC1 and GIC2. Functional characterization of MGC1 and TOS2 indicated that these genes function in the process of polarized growth, particularly in the process of cytokinesis. A genome-wide Synthetic Genetic Analysis identified more than 30 nonessential genes as those whose function overlaps with that of GIC1 and GIC2. Mutation in each of these genes exacerbates the growth defect of gic1 gic2 cells. As expected, some of these genes are involved in polarity-related functions, such as actin cytoskeleton organization, bud-site selection and cell wall biosynthesis. Others participate in a variety of biological processes, including organelle biogenesis, secretion and vesicular transport. The latter finding suggests that GIC1 and GIC2 may have function outside the scope of actin cytoskeleton organization. Taken together, the work presented here has uncovered the function of four previously uncharacterized genes in polarized cell growth. It has also provided hints to additional potential functions of GIC1 and GIC2. Further exploration of these functions might provide important links between Cdc42 signaling and cellular processes such as organelle biogenesis, secretion and vesicular transport, all of which need to be executed coordinately during polarized cell growth.Item Growth parameters for Saccharomyces cerevisiae in glucose containing media(Texas Tech University, 1985-08) Boyd, Glenn CraigNot availableItem In vitro polyketide biocatalysis : triketide building-blocks and enzymology(2013-05) Harper, Andrew David; Keatinge-Clay, Adrian TristanPolyketide products are useful compounds to research and industry but can be difficult to access due to their richness in stereogenic centers. Type I polyketide synthases offer unique engineering opportunities to access natural stereocontrol and resultant complex compounds. The development of a controlled in vitro platform based around type I polyketide synthases is described. It has been used to produce a small library of polyketide fragments on an unprecedented and synthetically-relevant scale and explore polyketide synthase enzymology.Item Investigation of yeast mitochondrial transcription initiation factor Mtf1p(2007-08) Klein, Dawn Elizabeth; Yin, Yuhui WhitneyBeing the first step in gene expression, transcription is a tightly regulated process in the cell cycle. In humans, mitochondrial transcription defects have been associated with numerous diseases. In order to gain a greater understanding of mitochondrial transcription initiation in higher eukaryotes, the mitochondrial transcription initiation factor, Mtf1p, from Saccharomyces cerevisiae was studied. This protein, which has homologues in all other eukaryotes, is known to interact with Rpo41p, the core yeast mitochondrial RNA polymerase, prior to promoter-DNA binding. Four mutants were created, Mtf1p Y54F, C192A, C192F, and C192M, and then expressed in Escherichia coli cells. Initially, two mutants (C192F and C192M) were successfully expressed with an N-terminal GST-tag, and Mtf1p C192F was used to optimize an overall purification scheme. Gel filtration analysis revealed that after purification, Mtf1p C192F is in an aggregated state, and as a result, Mtf1p C192F was N-terminally His-tagged in an alternative vector. However, expression was of the His-tagged protein was again unsuccessful. At the time this thesis was written, experiments to express the four Mtf1p mutants are ongoing.Item Novel Insights into the Regulation of Autophagy in Saccharomyces Cerevisiae(2011-12-15) Wu, Xi; Tu, BenjaminAutophagy is an evolutionarily conserved pathway for the degradation of intracellular contents. How autophagy is regulated, especially upon changes in metabolic and nutritional state, remains poorly understood. In Saccharomyces cerevisiae, autophagy is normally triggered by nutrient starvation. However, by using a prototrophic strain, I discovered that autophagy can be strongly induced upon switch from a rich medium (YPL) to a minimal medium (SL) without nutrient starvation. This new autophagy-inducing condition was termed SL-induced autophagy. Growth measurement confirmed that SL-induced autophagy was important for cellular homeostasis and growth following medium switch. A genetic screen uncovered IML1, NPR2, NPR3 and PBP1, which are all required for SL-induced autophagy, but not for nitrogen-starvation-induced autophagy. Iml1p, Npr2p and Npr3p function in the same complex and regulate autophagosome formation. During SL-induced autophagy, Iml1p can localize to the pre-autophagosomal structures, consistent with the role of the Iml1p complex in autophagosome formation. Moreover, a conserved domain in Iml1p was identified to be required for SL-induced autophagy as well as complex formation. I discovered that sulfur containing amino acids, but not non-sulfur containing amino acids, can specifically inhibit SL-induced autophagy. I further demonstrated that cysteine is a key metabolite that inhibits SL-induced autophagy by regulating cellular processes related to cysteine metabolism. Cysteine does not suppress SL-induced autophagy by regulating oxidative stress, protein urmylation and thiolation of cytosolic tRNAs. Future studies will be required to reveal the exact mechanism through which cysteine inhibits SL-induced autophagy. I also discovered that autophagy can be significantly induced upon depletion of a Fe-S cluster containing protein, Rli1p, and other factors that are also involved in rRNA processing and translation initiation. Interestingly, IML1, NPR2, NPR3 and PBP1 are also important for Rli1p-depletion-induced mitophagy. These results strongly suggest the mechanistic link between SL-induced autophagy and ribosome biogenesis or translation regulation. Collectively, my studies have demonstrated the existence of additional mechanisms that regulate autophagy in response to relatively more subtle changes in metabolic and nutritional state.Item Optimizing gene expression in saccharomyces cerevisiae for metabolic engineering applications(2014-05) Curran, Kathleen Anne; Alper, Hal S.; Contreras, Lydia; Georgiou, George; Iyer, Vishwanath; Otero, Jose MMetabolic engineering has enabled the advancement of biotechnology over the past few decades through the use of cells as biochemical factories. Cellular factories have now provided many new safe and sustainable routes to fuels, pharmaceuticals, polymers, and specialty chemicals. Many of these successes have been achieved using the yeast Saccharomyces cerevisiae, which has been used and shaped by humans for millennia for the production of food and drink. Consequently, S. cerevisiae is one of the most studied eukaryotic organisms in existence, and has established genetic tools for engineering efforts. However, despite the many achievements in metabolic engineering of S. cerevisiae, it is still significantly more difficult to engineer than its prokaryotic counterpart, Escherichia coli. As a result, there is an unmet need to further develop genetic tools in yeast and to do so in the context of metabolic pathway engineering. The work presented here addresses this need through the study and engineering of heterologous gene expression. First, a new biosynthetic pathway is engineered for the production of muconic acid in yeast. Muconic acid is a dicarboxylic acid that can serve as a platform chemical for the production of several bio-polymers. The final muconic acid producing strain was developed through significant metabolic engineering efforts and reached a titer of nearly 141 mg/L muconic acid, a value nearly 24-fold higher than the initial strain. Second, a new method of engineering promoters is presented that allows for increased expression of native promoters and the de novo design of synthetic promoters. The highest expression synthetic promoter is within the top 6th percentile of native yeast promoters. Third, a study of native and synthetic terminators for heterologous gene expression is completed for the first time. This study demonstrates that terminators can tune heterologous expression by as much as an order of magnitude. Fourth, a comparative study of plasmid components dissects the different contributions to plasmid burden, copy number, and gene expression level. Collectively, this work represents a significant step forward in the metabolic engineering of yeast through the establishment of a new pathway and the study and engineering of new tools for heterologous gene expression.Item The regulation of chromosome segregation by Aurora kinase, protein phosphatase 1 and nucleolar protein UTp7(2007-08) Jwa, Miri; Chan, Clarence S. M.The Sli15-Ipl1-Bir1 chromosomal passenger complex is essential for proper kinetochore-microtubule attachment and spindle stability in the budding yeast Saccharomyces cerevisiae. Subcellular localization of this complex during anaphase is regulated by the Cdc14 protein phosphatase, which is kept inactive in the nucleolus until anaphase onset. I show here that the predominantly nucleolar ribosome biogenesis protein Utp7 is also present at kinetochores and is required for normal organization of kinetochore proteins and proper chromosome segregation. Utp7 associates with and regulates the localization of Sli15 and Cdc14. It prevents the abnormal localization of Sli15 on cytoplasmic microtubules, the premature concentration of Sli15 on the pre-anaphase spindle, and the premature nucleolar release of Cdc14 before anaphase onset. Utp7 regulates Sli15 localization not entirely through its effect on Cdc14. Furthermore, the mitotic exit block caused by Cdc14 inactivation is relieved partially by the simultaneous inactivation of Utp7. Thus, Utp7 is a multifunctional protein that plays essential roles in the vital cellular processes of ribosome biogenesis, chromosome segregation and cell cycle control. Protein phosphatase 1, Glc7 opposes in vivo functions of the Ipl1-Sli15-Bir1 kinase complex in budding yeast. I show here Scd5- a targeting subunit of Glc7 that regulates endocytosis/cortical actin organization and undergoes nuclear-cytoplasmic shuttling- is present at kinetochores. Ipl1 associates with both Glc7 and Scd5. The scd5-PP1[Delta]2 mutation, which disrupts the association between Glc7 and Scd5, also disrupts the association between Ipl1 and Scd5-Glc7 without affecting the kinetochore localization of these proteins. Genetic studies suggest that Scd5 may positively regulate both Glc7 phosphatase and the Ipl1 kinase complex. In accordance, Scd5 stimulates in vitro kinase activity of Ipl1. scd5-PP1[Delta]2 cells missegregate chromosomes severely due to several defects: i) at least one of sister kinetochores appears not attached to microtubule. ii) sister chromatids are persistently cohesed through anaphase. iii) Sli15 is hyperphosphorylated and less abundant on the anaphase spindle resulting in unstable mitotic spindle. These results together suggest that Scd5 functions in diverse processes that are essential for faithful chromosome segregation. How Scd5 coordinately regulates two apparently antagonistic enzymatic activities of Ipl1 and Glc7 remains to be determined.Item A regulatory mechanism for Rsp5, a multifunctional ubiquitin ligase in Saccharomyces cerevisiae: characterization of its interaction with a deubiquitinating enzyme(2006) Kee, Younghoon; Huibregtse, Jon M.HECT E3 ubiquitin ligases are widely distributed from yeast to human cells and play important roles in many physiological processes. Rsp5, an essential HECT E3 ligase in Saccharomyces cerevisiae, is involved in many biological processes, including transcriptional activation, endocytic trafficking, mitochondrial inheritance, and RNA export pathways. Although Rsp5 has been shown to regulate multiple pathways targeting multiple substrates, mechanisms for regulating the biochemical activity of Rsp5 are largely uncharacterized (121, 199). To gain further insight into the regulation of this enzyme, I identified proteins that copurified with epitope-tagged Rsp5. Ubp2, a deubiquitinating enzyme, was a prominent copurifying protein. Rup1, a previously uncharacterized UBA domain protein, was required for binding of Rsp5 to Ubp2 both in vitro and in vivo. Biochemical and genetic evidence are consistent with a model that Ubp2and Rup1 antagonizes Rsp5-catalyzed substrate ubiquitination. In vivo and in vitro experiments showed that Rsp5 and Ubp2 display strong preferences for assembly and disassembly of K63-linked polyubiquitination, respectively. A large fraction of the K63 conjugates in ubp2∆ cells bound to Rsp5, and a proteomics approach was therefore used to identify Rsp5 substrates subject to Ubp2 regulation. Two proteins implicated in cell wall integrity, Csr2 and Ecm21, were identified and both proteins were efficiently K63- polyubiquitinated by Rsp5 and deubiquitinated by Ubp2. I have also shown that cell wall integrity is impaired in rsp5-1 cells and this can be rescued by either ubp2∆ or rup1∆ mutation, suggesting that the Ubp2/Rup1 complex negatively regulates Rsp5-mediated cell wall homeostasis. Together, these data represent a novel regulatory mechanism for Rsp5 and suggest that similar mechanisms might be utilized by its mammalian homologues. Furthermore, this work provides a basis for studying the mechanism for differential polyubiquitin chain type synthesis by HECT E3 ligases.Item The ribosome biogenesis factor Arx1p: characterization of its recycling mechanism and its role in ribosome export(2007) Hung, Nai-Jung, 1976-; Johnson, Arlen W.Translation is an essential and fundamental process that coverts genetic codes into functional polypeptides by an apparatus called ribosome. In eukaryotic cells, ribosomes are composed of two subunits: the large (60S) subunit and small (40S) subunits. In Saccharomyces cerevisiae, ribosome biogenesis is complex and requires the involvement of over ~170 trans-acting factors. As a growing number of factors were identified related to this essential metabolic pathway, our lab has contributed to functional characterization of the late 60S subunit biogenesis pathway that centers on Nmd3p. This work particularly focuses on characterizing of the nuclear shuttling trans-acting factor Arx1p found in the Nmd3p-60S subunit particle. A working model that describes how Rei1p, another cytosolic trans-acting factor, recycles Arx1p is presented. This work also shows a similar mode of Arx1p recycling by the Hsp40 J-protein, Jjj1p. Furthermore, I have investigated functional interplay between Arx1p and Rpl25p, a 60S ribosomal protein at the polypeptide exit tunnel. These findings further reveal the involvement of Arx1p at the polypeptide exit tunnel in mediating association of other factors with 60S subunits. Beyond its function at the polypeptide exit tunnel, this work also focuses on a function for Arx1p in the export of 60S subunits. In yeast and higher eukaryotes, 60S subunit export depends on the export adaptor Nmd3p via Crm1-dependent pathway. I show that ARX1 interacts with the NES of Nmd3p and nucleoporins. From these results, I propose that Arx1p acts as another export receptor to facilitate 60S subunit export.Item The role of Ipl1 kinase in chromosome segregation in Saccharomyces cerevisiae(2003-05) Kang, Jungseog; Chan, Clarence S. M.Item Selfishness in moderation for self-propagation : the yeast plasmid purloins the host mitotic apparatus for its segregation(2003-12) Mehta, Shwetal Vatsal, 1973-; Jayaram, MakkuniThe 2 micron circle of Saccharomyces cerevisiae is a high-copy, ‘selfish’ extrachromosomal DNA element that resides in the nucleus and propagates itself stably in the cell population. The plasmid segregates as a single cluster with the assistance of an active partitioning machinery, consisting of just two plasmid encoded proteins Rep1p and Rep2p and a cis-acting element STB. The work presented in this thesis reveals the molecular strategies by which the plasmid channels the chromosome segregation machinery towards its own partitioning. The yeast cohesin complex, a multiprotein molecular glue that keeps duplicated sister chromatids together until they are ready to be separated, plays an important role in plasmid segregation. During the cell cycle, the partitioning proteins mediate the recruitment of cohesin to STB. The timely association of cohesin with the plasmid during S phase as well as the timely dissociation of cohesin from it during anaphase are essential for equal partitioning of the plasmid to daughter cells. The plasmid exploits the host mitotic machinery in a second and quite unexpected manner. The mitotic spindle specifies the precise nuclear localization of the plasmid cluster, facilitates its compact organization, and is essential for the enlistment of the cohesin complex. When the spindle is restored from an initially depolymerized state of the microtubules, the cohesin complex can be assembled at STB, but is unable to support equal segregation of the plasmid. This finding underscores the importance of cell cycle timing in the functional association between cohesin and the plasmid. We propose that the cohesin complex plays analogous roles in chromosome and plasmid segregation: to pair and unpair sister chromatids in the former case, and sister clusters in the latter. In one plausible model, cohesin mediated pairing occurs between two clusters containing roughly equal numbers of replicated plasmid molecules. In the second, cohesin pairs each molecule in one cluster with its sister molecule in the second cluster, concomitant with DNA replication. One might look upon the segregation entity consisting of the sixty or so plasmid copies as “the plasmosome”, a non-essential yeast chromosome, in the manner in which binary partitioning units are segregated one to one.Item Studies on the enzymology of sterol mehtyl transferase from Saccharomyces cerevisiae(Texas Tech University, 2001-05) Marshall, Julie A.As an approach to understanding the enzymological details of sterol methylation catalysis in ergosterol biosynthesis, a kinetic analysis as well as an active site mapping study of the active center of the sterol methyl transferase (SMT) was pursued using a recombinant SMT from Saccharomyces cerevisiae. The following experiments were performed: i. Using initial velocity conditions and equilibrium dialysis to establish the kinetic constants, Km, Vmax, and KItem Study of two proteins involved in protein disulphide formation : molecular cloning and characterization of a full-length flavin-dependent monooxygenase from Saccharomyces cerevisiae & preliminary structure analysis on DsbC from Haemophilus influenzae(2003-08) Zhang, Man, 1972-; Robertus, Jon D.This work includes biochemistry studies on yeast FMO and structure analysis on a prokaryotic protein disulphide isomerase - H. influenzae DsbC. The yeast FMO (yFMO) gene was cloned, expressed and characterized in this lab previously. Deletion experiments suggested that yFMO was involved in folding proteins with disulfide bonds. In continuation of study on yeast FMO, we detected two nucleotide errors in the GenBank sequences of the yFMO gene. These errors had been incorporated into the initial gene engineering, and as a consequence, the protein studied initially is a truncated version of yFMO. To be consistent with previous work, the N-terminal his-tagged full-length yFMO was compared to the truncated enzyme in vitro. Using an oxygen uptake assay, the full-length and the truncated FMOs showed similar pH profiles, Km, Kcat and Vmax values using glutathione as a substrate, indicating that they share similar enzymatic character. Therefore, we conclude that the hypotheses proposed earlier concerning the enzyme’s functions are likely to be still valid for the full-length yeast FMO. The second project aimed for determining the structures of DsbC from several species using X-ray crystallography to improve our knowledge on the structure-function relationship of DsbC, and subsequently provide guidance for engineering DsbC to improve E. coli as a better protein expression system for proteins with multiple disulphide bonds. DsbC proteins from Haemophilus influenzae, Pseudomonas aeruginosa, Erwinia chrysanthemi, Vibrio cholerae and Yersinia pseudotuberculosis were subjected to crystallization efforts, but only crystals of DsbC from H. inf and Y.pse have been obtained. A data set at the resolution of 2.5 Å was collected from a single H. inf DsbC crystal. Molecular replacement strategy was applied, using the known structure of E. coli DsbC, to solve the structure of H. inf DsbC. Due to a flexible protein conformation, many residue side chains in the N-terminal domain of the current structural model are missing. Comparison between the current H. inf DsbC model and E. coli DsbC structure shows that the H. inf DsbC has a similar catalytic domain and a larger, more open cleft within the V-shaped dimer.Item Synthesis of sterol biosynthesis inhibitors and metabolism of isotopically labeled sterols by Saccharomyces cerevisiae(Texas Tech University, 1998-05) Zhou, WenThe dissertation focuses on the chemistry and mechanism of sterol biomethylation catalyzed by (S)-adenosyl-L-methionine:sterol methyl transferase (SMT) enzyme. The research is composed of two parts: synthesis of sterol methylation inhibitors and isotopically labeled substrates and mechanism studies of the SMT enzyme. Part 1 describes preparation of ^H and ^^C-isotopically labeled sterols, and the design, synthesis and enzymatic evaluation of three types of sterol methylation inhibitors: (1) substrate analogs which act as product inhibitors of the reaction; (2) substrate analogs which act as mechanism-based inactivators; and (3) transition state analogs. The synthetic work focused on modification of the side chains of cholesterol, zymosterol, lanosterol, and cycloartenol which are natural substrates in C-methylation reactions. The sterol side chain was modified at C-20, C-21, and C-22 positions to study the effect of configuration and conformation of the sterol side chain on biomethylation. A series of novel sterol methylation inhibitors containing aza, aziridine, and ammonium groups at positions C-22 to C-25 were synthesized to serve as transition state analogs. Substrate analogs with the 26, 27-cyclopropylidene functional group were also synthesised and discovered to be potent irreversible mechanism-based inhibitors of the SMT enzyme. Several new 24, 25-ethano and 24, 28-methano sterols, 24- inyl and 29', 29"-cyclopropylidene lanosterols, and 24(25), 26(26')-diene and 24(25)-en-25-thylnyl sterols have been synthesized. All the inhibitors were characterized by gas chromatography-mass spectrometry, high pressure liquid chromatography, and 'H and ^^C nuclear magnetic resonance spectroscopy. Part 2 describes research on the coupled methylenation-deprotonation of C-24 of the sterol side chain in plant and fungal sterol C-methylation reactions. These studies involved determination of the stereochemistry of hydrogen migration from C-24 to C-25 during biomethylation and of C-28 deprotonation. To accomplish our aims, [27-'^C], [24-^H] and [28-^H2] labeled sterols were prepared and assayed with SMT enzymes from a fungus {Saccharomyces cerevisiae) and a plant (Arabidopsis thaliana). As a result, migration of the hydrogen from C-24 to C-25 was found to be introduced from Re-facQ of the 24, 25-double bond of the sterol side chain to generate the similar 25R stereochemistry in S. cerevisiae and A. thaliana, suggesting a similar topography of the SMT active site.Item The effects of paraquat and arsenic acid on the growth of distiller's yeast(Texas Tech University, 1982-08) Sullivan, Hanna LowryThe effects of the defoliants arsenic trioxide and paraquat on the growth of Saccharomyces cerevisiae were studied. It was found that 10 and 10 M concentrations of the defoliants did not significantly inhibit the growth of the distiller's yeast, while inhibition -3 -4 -3 was observed at 10 and 10 M concentrations. At 10 M concentrations of both defoliants, the growth of the yeast was inhibited completely in both tryptic soy broth and in a semi-synthetic medium containing glucose as the carbon source. Growth curves revealed an immediate toxic effect on yeasts grown in paraquat-treated media, while the number of viable cells remained constant for a short period of time in media containing arsenic trioxide.