Dissertations / Theses on the topic 'Saccharomyces cerevisiae – Reproduction – Research'

Ribosome biogenesis is a crucial yet poorly understood and complex process in all cells. To date, most studies on eukaryotic ribosome biogenesis have relied on yeast genetics and whole cell analysis of ribosomal RNA processing. An early and critical step in ribosome biogenesis is the post-transcriptional modification of rRNA. Pseudouridylation is the most frequently occurring modification. Pseudouridylation is catalyzed by H/ACA small nucleolar ribonucleoproteins (snoRNPs) which are one of the two major classes of snoRNPs found within eukaryotes and archaea. H/ACA snoRNPs consist of four conserved core proteins Cbf5, Gar1, Nop10, and Nhp2 (eukaryotes), and a substrate specific H/ACA snoRNA. Mutations causing the rare inherited disease Dyskeratosis congenita are found in CBF5, NOP10, and NHP2. Here I report the purification of H/ACA protein Cbf5 in the presence of detergents. Additionally, I report initial in vitro RNA binding studies using Nhp2 and the snoRNA snR34 as well as the effects of Dyskeratosis congenita substitutions within Nhp2 on this interaction.
vii, 83 leaves : ill. ; 29 cm

Thesis (Ph.D.)--University of Florida, 2004.
Typescript. Title from title page of source document. Document formatted into pages; contains 117 pages. Includes Vita. Includes bibliographical references.

Translation initiation factor 5A (IF5A) is an essential, highly conserved protein found within all eukaryotic (eIF5A) and archaeal (aIF5A) cells. The IF5A protein is unique in that it contains the amino acid hypusine; a two-step post translational modification of a single, conserved lysine residue. Although hypusination of eIF5A is vital for eukaryotic cell viability, the primary role of the protein and its hypusine side chain remain a mystery. eIF5A, initially identified as a translation initiation factor, is not required for global protein synthesis leading to the prevailing proposal that eIF5A is purely involved in the translation of a select subset of mRNAs. Recently a number of mutational studies have focused on the conserved, hypusine-containing loop region of eIF5A where specific residues have been found to be essential for activity without affecting hypusination. It has been postulated that eIF5A exists as a dimer (40 kDa) under native conditions and that these residues may be at the interface of dimerisation. The aim of this research was therefore to conduct a mutational analysis of the loop region in support of this hypothesis. A functional analysis of the Saccharomyces cerevisiae eIF5A mutant proteins K48D, G50A, H52A and K56A revealed that these substitutions impaired growth to varying degrees in vivo with G50A and K48D mutant proteins displaying the most convincing defects. Gel filtration profiles gave unexpected results determining eIF5A mutant and wild type proteins to have a native molecular weight of 30 to 31 kDa, suggesting that the eIF5A oligomeric state may be transitory and subject to certain conditions. The inconclusive results obtained from using gel filtration studies led to an investigation into the feasibility of producing native, hypusinated peptides for future structural studies using nuclear magnetic resonance. Hypusinated and unhypusinated eIF5A were successfully separated into their domains making this a possibility. Finally, this study proposes a role for eIF5A in eukaryotic IRES-driven translation initiation.

The yeast Saccharomyces cerevisiae is an important model organism for the study of cell biology. The similarity between yeast and human genes and the conservation of fundamental pathways means it can be used to investigate characteristics of healthy and diseased cells throughout the lifespan. Yeast is an equally important biotechnological tool that has long been the organism of choice for the production of alcoholic beverages, bread and a large variety of industrial products. For example, yeast is used to manufacture biofuels, lubricants, detergents, industrial enzymes, food additives and pharmaceuticals such as anti-parasitics, anti-cancer compounds, hormones (including insulin), vaccines and nutraceuticals. Its function as a cell factory is possible because of the speed with which it can be grown to high cell yields, the knowledge that it is generally recognized as safe (GRAS) and the ease with which metabolism and cellular pathways, such as translation can be manipulated. In this thesis, these two pathways are explored in the context of their biotechnological application to ageing research: (i) understanding translational processes during the high-yielding production of membrane protein drug targets and (ii) the manipulation of yeast metabolism to study the molecule, L-carnosine, which has been proposed to have anti-ageing properties. In the first of these themes, the yeast strains, spt3?, srb5?, gcn5? and yTHCBMS1, were examined since they have been previously demonstrated to dramatically increase the yields of a target membrane protein (the aquaporin, Fps1) compared to wild-type cells. The mechanisms underlying this discovery were therefore investigated. All high yielding strains were shown to have an altered translational state (mostly characterised by an initiation block) and constitutive phosphorylation of the translational initiation factor, eIF2a. The relevance of the initiation block was further supported by the finding that other strains, with known initiation blocks, are also high yielding for Fps1. A correlation in all strains between increased Fps1 yields and increased production of the transcriptional activator protein, Gcn4, suggested that yields are subject to translational control. Analysis of the 5´ untranslated region (UTR) of FPS1 revealed two upstream open reading frames (uORFs). Mutagenesis data suggest that high yielding strains may circumvent these control elements through either a leaky scanning or a re-initiation mechanism. In the second theme, the dipeptide L-carnosine (ß-alanyl-L-histidine) was investigated: it has previously been shown to inhibit the growth of cancer cells but delay senescence in cultured human fibroblasts and extend the lifespan of male fruit flies. To understand these apparently contradictory properties, the effects of L-carnosine on yeast were studied. S. cerevisiae can respire aerobically when grown on a non-fermentable carbon source as a substrate but has a respiro-fermentative metabolism when grown on a fermentable carbon source; these metabolisms mimic normal cell and cancerous cell metabolisms, respectively. When yeast were grown on fermentable carbon sources, in the presence of L-carnosine, a reduction in cell growth and viability was observed, which was not apparent for cells grown on a non-fermentable carbon source. The metabolism-dependent mechanism was confirmed in the respiratory yeast species Pichia pastoris. Further analysis of S. cerevisiae yeast strains with deletions in their nutrient-sensing pathway, which result in an increase in respiratory metabolism, confirmed the metabolism-dependent effects of L-carnosine.

El proyecto se llevara a cabo, en el laboratorio de bromatología del colegio de postgraduado, Texcoco, México; apoyado en el Laboratorio de Bromatología, perteneciente a la Facultad de Medicina Veterinaria y Zootecnia de la Universidad Autónoma del Estado de México, ubicada en el Campus el Cerrillo Piedras Blancas, Toluca, Estado de México.
La Unión Europea ha prohibido la inclusión de antibióticos e ionoforos como aditivos en la alimentación animal, la exploración de alimentos como aditivos naturales son una alternativa para modificar la fermentación ruminal, la mejora en la utilización de alimentos (Salem et al., 2014), y sobre todo reducir gases del efecto invernadero (GHG) se ha hecho más atractivo (Elghandour et al., 2017; Kholif et al., 2017). Los estudios han mostrado que la inclusión de enzimas exógenas en la alimentación de los rumiantes ha mejorado al funcionamiento productivo del animal, beneficiando la digestibilidad nutritiva y la fermentación ruminal (Valdes et al., 2015). McAllister et al. (2001) ha propuesto varias acciones en la utilización de alimentos como la solubilización de la fibra dietética, la complementación de microrganismos ruminales con sustrato fácilmente fermentable, la mejora de la actividad de enzima microbiana en el rumen, la mejora de la relación y la colonización de microorganismos ruminales a la pared celular de la planta (Chung et al., 2012). Sin embargo, Lewis et al. (1999) han observado los efectos débiles de la alimentación de enzimas exógenas para mejorar la calidad del forraje y la utilización por los rumiantes.

La croissance polarisée est un processus fondamental chez les eucaryotes, qui fait intervenir des protéines conservées de la levure à l'homme, notamment les protéines G de type Rho et leurs régulateurs. Cdc42 joue un rôle essentiel dans le contrôle de l'établissement et du maintien de la polarité cellulaire. Chez la levure Saccharomyces cerevisiae, Cdc42p possède un seul facteur d'échange, Cdc24p. Cdc42p et Cdc24p sont nécessaires pour l'orientation de la croissance pendant la conjugaison. L'objectif de ce travail de thèse est d'identifier d'autres fonctions de Cdc24p et de comprendre comment Cdc24p contrôle l'activation de Cdc42p, au cours de ce processus. A cette fin, nous avons identifié et caractérisé des mutants de cdc24 et de cdc42 spécifiquement déficients dans la conjugaison, en particulier cdc24-m6 et cdc42[V36M]. Ces mutants répondent normalement aux phéromones et orientent leur croissance en direction du partenaire, mais ont un défaut dans l'étape de fusion, ils accumulent les prézygotes. Cdc24-m6 et cdc42[V36M] sont incapables de localiser une protéine requise pour la fusion, au niveau de la zone de contact entre les deux cellules, en dépit d'une exocytose normale. Nous avons pu établir que la fonction de Cdc42p dans la fusion ne fait pas intervenir la cascade des MAPKs et que Cdc42[V36M]p est capable d'interagir avec les effecteurs à domaine CRIB. La surexpression de Cdc24p dans les cellules cdc42[V36M], et de Cdc42[C18A]p (mutant " fast-cycling ") dans cdc24-m6, permet de compenser partiellement les défauts de fusion de ces mutants. Nos résultats indiquent que l'oscillation de Cdc42p entre les formes liées au GDP et au GTP est nécessaire à la fusion cellulaire, peut-être en activant ou en localisant la machinerie de fusion au site de contact
Polarized growth is a fundamental process in eukaryotic cells, which requires proteins conserved between yeast and mammals such as Rho GTPases and their regulators. Cdc42 plays a central role in controlling the establishment and maintenance of cell polarity. In the yeast, Saccharomyces cerevisiae, Cdc42p is activated by the guanine nucleotide exchange factor, Cdc24p. Cdc42p and Cdc24p are required for oriented growth during yeast mating. The goal of this study is to identify additional functions of Cdc24p and to understand how Cdc24p controls Cdc42p activation during this process. We identified and characterized two mating specific cdc24 and cdc42 mutants, cdc24-m6 and cdc42[V36M]. These mutants respond normally to pheromone and orient their growth towards a mating partner, yet are defective in cell fusion and accumulate prezygotes. Cdc24-m6 and cdc42[V36M] cells do not correctly localize a protein required for cell fusion, to the cell contact region, despite normal exocytosis. This function of Cdc42p in cell fusion does not require the MAPK cascade, and furthermore Cdc42[V36M]p is able to bind CRIB domain containing effectors. Overexpression of Cdc24p in cdc42[V36M] cells, and overexpression of fast cycling Cdc42[C18A]p in cdc24-m6 cells, partially suppressed their fusion defects. Together, our results show that Cdc42p GDP/GTP cycling is necessary for cell fusion, perhaps in activating or localizing the fusion machinery at the site of cell contact. We have identified a new function of the Cdc42p GTPase module in polarization of the fusion machinery during yeast mating and we suggest that Cdc42p cycling might be regulated during cell fusion

The transition to multicellularity is a major step in the evolution of complex life. The first steps in this transition are poorly understood because multicellularity evolved long ago, and transitional forms have been lost to extinction. Previous studies developed a novel microbial model system in which simple multicellularity could be evolved de novo (Ratcliff et al., 2012). By evolving our snowflake yeast to undergo sexual reproduction we hypothesized that sex created variation in key multicellular traits, which spurs multicellular adaptation. In our 'snowflake yeast' model system, two traits are of central importance: cluster size, and programmed cell death (apoptosis). Apoptosis previously evolved to regulate cluster size, by acting as break points within clusters, allowing them to modify the size and number of multicellular propagules they produce. In prior experiments, this only develops after yeast have evolved to form large clusters. Prior experiments in the lab demonstrated that the longer snowflake yeast have been evolving, the greater the fitness benefit provided by sex. Here we examine whether this is due to sex creating greater amounts of diversity in the traits of post-sex offspring in more highly evolved multicellular yeast, allowing post-recombination offspring to 'fine tune' their multicellular traits. By using flow cytometry, we collected data on our multicellular traits. By gathering the biomass mean of the cluster size in each population and staining the cells with propidium iodide to determine the apoptotic tendencies of our cells we were able to compare our outcomes to the pre-sex ancestor, and we determined there was no increase in variation. Although apoptosis did not have an increase in variation due to sex, it created a variation in cluster size; the variation was seen in the population W8. This still supports our hypothesis that sex creates variation in multicellular traits, which allows for rapid adaptation.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Universidade Estadual Paulista (UNESP)
A composição nutricional da levedura a destaca como alimento funcional. O presente trabalho objetivou avaliar a ação da levedura (Saccharomyces cerevisiae) nas respostas reprodutivas de fêmeas de tilápia do Nilo. No Estudo-I 105 fêmeas da linhagem GIFT foram distribuídas em 15 aquários de 500L (três tratamentos e cinco repetições com sete peixes/aquário). No Estudo-II foram utilizados 63 fêmeas e 21 machos da linhagem GIFT distribuídos em três aquários com capacidade de 7000L de água/cada (21 fêmeas e sete machos/aquário). Em ambos os Estudos, os reprodutores foram alimentados ad libitum com dietas isoprotéica e isoenergética contendo 2,0% de levedura íntegra, 2,0% de levedura autolisada ou ausente de inclusão. O Estudo-I foi realizado na UNESP – AquaNutri, Campus de Botucatu e o Estudo-II foi conduzido no Caunesp - Tilapilcultura Campus de Jaboticabal. Avaliou-se nas fêmeas o desempenho produtivo, status nutricional e composição química das gônadas e, o desempenho reprodutivo e o desenvolvimento inicial das larvas. Os dados foram submetidos à análise de variância e quando significativo aplicou-se o teste de comparações múltiplas de médias. A suplementação de levedura íntegra ou autolisada na dieta das reprodutoras, não melhorou o desempenho produtivo e não influenciou o desempenho reprodutivo das fêmeas. Entretanto, melhorou o desenvolvimento larval 72h após eclosão, com melhor desenvolvimento nas larvas dos reprodutores alimentados com a dieta suplementada com 2,0% de levedura íntegra.
The nutritional composition yeast detach how functional food. The research present objective evaluates the action yeast (Saccharomyces cerevisiae) in the response reproductive of females of Nile tilapia. In Study-I 105 females of GIFT lineage was randomized in 15 500L-aquarius (three treatments and five repetition with seven fish/aquarium). In Study-II was utilized 63 females and 21 males of GIFT lineage was randomized in three 7000L-aquarium (21 female and seven male for aquarium). The spawning was feed ad libitum with diet isoproteic and isoenergetic with 2,0% whole yeast and autolised yeast or supplementation absent. In Study-I were realize in UNESP – AquaNutri Campus de Botucatu and Study-II in Caunesp – Tilapicultura Campus de Jaboticabal. Was evaluation in female productive performance, nutritional status and chemistry composition of gonad (Study-I) and reproductive performance of spawning and development larvae (Study-II). The data was submitted variance analyses and multiple comparison test of mean. The supplemented whole yeast or autolised yeast in diet of spawning didn’t improvement productive performance and didn’t influence reproductive performance of female. Therefore improved of development larvae at 72h after eclosion with improved development in larvae of spawning feed with diet 2,0% whole yeast.

Thesis (Ph.D.)--University of Florida, 2004.
Typescript. Title from title page of source document. Document formatted into pages; contains 100 pages. Includes Vita. Includes bibliographical references.

Indiana University-Purdue University Indianapolis (IUPUI)
DNA suffers constant damage, leading to a variety of lesions that require repair. One of the most devastating lesions is a double-strand break (DSB), which results in physical dissociation of two pieces of a chromosome. Necessarily, cells have evolved a number of DSB repair mechanisms. One mechanism of DSB repair is break-induced replication (BIR), which involves invasion of one side of the broken chromosome into a homologous template, followed by copying of the donor molecule through telomeric sequences. BIR is an important cellular process implicated in the restart of collapsed replication forks, as well as in various chromosomal instabilities. Furthermore, BIR uniquely combines processive replication involving a replication fork with DSB repair. This work employs a system in Saccharomyces cerevisiae to investigate genetic control, physical outcomes, and frameshift mutagenesis associated with BIR initiated by a controlled HO-endonuclease break in a chromosome. Mutations in POL32, which encodes a third, non-essential subunit of polymerase delta (Pol delta), as well as RAD9 and RAD24, which participate in the DNA damage checkpoint response, resulted in a BIR defect characterized by decreased BIR repair and increased loss of the broken chromosome. Also, increased incidence of chromosomal fusions determined to be half-crossover (HCO) molecules was confirmed in pol32 and rad24, as well as a rad9rad50S double mutant. HCO formation was also stimulated by addition of a replication-inhibiting drug, methyl-methane sulfonate (MMS), to cells undergoing BIR repair. Based on these data, it is proposed that interruption of BIR after it has initiated is one mechanism of HCO formation. Addition of a frameshift mutation reporter to this system allowed mutagenesis associated with BIR DNA synthesis to be measured. It is demonstrated that BIR DNA synthesis is intrinsically inaccurate over the entire path of the replication fork, as the rate of frameshift mutagenesis during BIR is up to 2800-fold higher than normal replication. Importantly, this high rate of mutagenesis was observed not only close to the DSB where BIR is less stable, but also far from the DSB where the BIR replication fork is fast and stabilized. Pol  proofreading and mismatch repair (MMR) are confirmed to correct BIR errors. Based on these data, it is proposed that a high level of DNA polymerase errors that is not fully compensated by error-correction mechanisms is largely responsible for mutagenesis during BIR. Pif1p, a helicase that is non-essential for DNA replication, and elevated dNTP levels during BIR also contributed to BIR mutagenesis. Taken together, this work characterizes BIR as an essential repair process that also poses risks to a cell, including genome destabilization and hypermutagenesis.

The thioredoxin system consisting of thioredoxin (Trx), thioredoxin reductase and NADPH plays a significant role in a large number of redox-dependent processes such as DNA synthesis and anti-oxidant defense. Elevated levels of this system have been associated with a number of diseases including cancer and HIV. Understanding the regulation of this network from a systems perspective is therefore essential. However, contradictory descriptions of thioredoxin as both an enzyme and redox couple have stifled the adoption of systems biology approaches within the field. Using kinetic modeling, this discrepancy was resolved by proposing that saturation of Trx activity could be due to the saturation of the Trx redox cycle which consequently allowed development of the first computational models of the thioredoxin system in Jurkat T-cells and Escherichia coli. While these models successfully described the network properties of the thioredoxin system in these organisms, further confirmatory studies were required before this modeling approach could be generally accepted. The aim of this study was to utilize computational and molecular methods to confirm or reject this proposed mechanism for thioredoxin activity. To determine if there is any difference in the kinetic models obtained when thioredoxin was modeled as an enzyme or as a redox couple, representative core models were developed. The data showed that when modeling Trx as a redox couple, the system was able to achieve steady state, there was a re-distribution of Trx into its oxidized form and, thioredoxin reductase affected the rates within the system. On the other hand, when Trx was modeled as an enzyme, the system could not reach a steady state, Trx remained in the reduced form and thioredoxin reductase concentration had no effect on the rates within the system. As these properties could be directly tested invitro, we sought to directly confirm which model was correct. The thioredoxin system from Saccharomyces cerevisiae was cloned, expressed and purified and substrate saturation curves were generated using insulin as a model substrate. The data showed that the system reached steady state and with increasing concentrations of insulin, the system saturated with a progressive re-distribution of the thioredoxin moiety into its oxidized form. Further, increasing the thioredoxin reductase concentration increased the flux through the system. Collectively, the results obtained through invitro analyses provided unambiguous support for the thioredoxin redox couple model. These results will enable the construction of a complete computational model of the yeast thioredoxin system and provide a basis for the analysis of this network in a number of pathologies.
Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

碩士
國立高雄應用科技大學
化學工程系碩士班
92
β-(1,3)/(1,6)-glucans are the polymers of (1,3)-β-linked glucopyranos with the branch of (1,6)-β-linked glucopyranos. These glucans have been shown to have immunopharma- cological activity in humans and animals. This study present a process for isolation of β-(1,3)/(1,6)-glucans from baker’s yeast (Saccharomyces cerevisiae), together with compositions and methods of treatment. The process of the study comprises the steps of: (a) extracting alkali soluble components from cells with alkali and heat; (b) extracting the chitins from the cell wall with acid and heat; (c) extracting lipids with ethanol and recovered the β-(1,3)/(1,6)-glucan; and (d) using the different drying methods to give microparticulate glucan. The comparison of FTIR for the products of every step in process, the significant spectral 891 cm-1 is attributed to a β-(1,3)/(1,6)-glucan which is identified the product from the fractionation procedure. Hot sodium bicarbonate and sodium hydroxide can be very efficient to remove proteins and alkali-soluble mannoprotein from yeast. The chitin dissolves in hot acetic acid. After ethanol extraction and removal of the lipids, the insoluble residue contained pure β-(1,3)/(1,6)-glucan. From the FTIR and NMR analytical data, the purified β-(1,3)/(1,6)-glucan is found to have exclusively high purity. The electron microscopy SEM and TEM show that the three different drying β-(1,3)/(1,6)-glucan products have platelet particle aggregates which form a large particle size. The average thickness of platelets is measured to be less than 100nm. The drying products contain 13% water from the TGA/DTA measurements. After heating to above 260℃, the β-(1,3)/(1,6)-glucan decomposed to CO2 and H2O. Structural studies of β-(1,3)/(1,6)-glucan from diffraction patterns recorded by X-ray and TEM have revealed that these spacings are roughly in agreement with a tetragonal unit cell of dimensions a = 0.878 nm c = 0.872 nm for ether drying products, a = 0.866 nm c = 0.844 nm for lyophilization drying products, respectively. The process is simple、fast and ease. The resulting glucan is obtained in high yields to about 10.5 % with high purity. The mannoprotein is also obtained as a byproduct.

碩士
朝陽科技大學
應用化學系碩士班
93
The purposes of the research were to find out suitable conditions for Saccharomyces cerevisiae to ferment glucose to produce ethanol, investigate the effects of adding swine feces compost to the pulps and peels of TNG No.13 and TNG No.17 pineapples, hydrolyzing the pulps and peels with diluted sulfuric acids and sterilizing the pulps and peels on the ethanol concentrations of fermented solutions inoculated with the yeast. Experimental results showed that 15 ℃, pH 4, the concentration of glucose in YM broth was 300 g/L, the yeast suspension, 9.24×107 CFU/mL, added to YM broth at a volume rate of 1:100 and fermented with seven days were suitable for producing ethanol. The pulps and peels of the pineapples hydrolyzed with 0.8 % sulfuric acid and fermented by the yeast got higher ethanol concentrations than those hydrolyzed with 0.4 % sulfuric acid. The pulp of TNG No.13 pineapple fermented by the yeast produced higher ethanol concentration than that of TNG No.17 pineapple, and vice versa for the peels of the pineapples. The pulps and peels, adjust inorganic nitrogen concentration to 0.18 g/L with aqueous extract of swine feces compost, fermented by the yeast produced more ethanol than those were not adjusted. Sterilizing the pulps and peels had positive effect on the ethanol concentrations of fermented solutions.

Heat induced protein haze is a common problem in white wine. Grape derived pathogenesis related proteins slowly denature and aggregate during wine storage and this gives rise to light dispersing haze. Protein haze formation is currently prevented by removing proteins using bentonite, an aluminium silicate clay, but this method has drawbacks. A potential alternative or complementary method is the use of haze protective factors ( HPF ), specific mannoproteins from Saccharomyces cerevisiae that visually reduce protein haze. Hpf1p was originally isolated from Muscat Gordo Blanco wine and Hpf2p from a synthetic grape juice ferment. Based on partial amino acid sequences, putative structural genes, HPF1 and HPF2, for these proteins were identified. HPF1 has a homologue, HPF1 ', ( 71 % similarity ) in S. cerevisiae. Sequence analysis suggests that Hpf1p, Hpf1 ' p and Hpf2p are localised to the cell wall or plasma membrane. This study aimed to determine the biological function of the HPF genes in S. cerevisiae. HPF overexpression and deletion strains were constructed and analysed for cell wall related phenotypes. Under a number of conditions, including cold temperature and ethanol stress, the hpf1 Δ hpf1 ' Δ strain was more tolerant than the wild type strain. However, mating efficiency of the hpf1 Δ hpf1 ' Δ strain was significantly less than the wild type strain and this was found to be correlated with the persistence of a septum between the mating partners. The decreased mating efficiency was also mating type specific, only occurring in MAT α cells. This study also aimed to establish conclusively that the HPF genes do indeed encode proteins with haze protective properties. Haze protective activity of the material from ferment supernatants was assessed. Material from the HPF deletion strains exhibited significantly less haze protective activity than the wild type. Moreover, material derived from HPF1 and HPF1 ' overexpressors was more active than material from the wild type. A 6xHis - tagged Hpf2p was expressed and purified using immobilised metal affinity chromatography. This Hpf2p had significant haze protective activity. Modification of N - glycans of 6xHis - Hpf2p by Endoglycosidase H decreased its haze protective activity.visually reduce protein haze. Hpf1p was originally isolated from Muscat Gordo Blanco wine and Hpf2p from a synthetic grape juice ferment. Based on partial amino acid sequences, putative structural genes, HPF1 and HPF2, for these proteins were identified. HPF1 has a homologue, HPF1 ', ( 71 % similarity ) in S. cerevisiae. Sequence analysis suggests that Hpf1p, Hpf1 ' p and Hpf2p are localised to the cell wall or plasma membrane. This study aimed to determine the biological function of the HPF genes in S. cerevisiae. HPF overexpression and deletion strains were constructed and analysed for cell wall related phenotypes. Under a number of conditions, including cold temperature and ethanol stress, the hpf1 Δ hpf1 ' Δ strain was more tolerant than the wild type strain. However, mating efficiency of the hpf1 Δ hpf1 ' Δ strain was significantly less than the wild type strain and this was found to be correlated with the persistence of a septum between the mating partners. The decreased mating efficiency was also mating type specific, only occurring in MAT α cells. This study also aimed to establish conclusively that the HPF genes do indeed encode proteins with haze protective properties. Haze protective activity of the material from ferment supernatants was assessed. Material from the HPF deletion strains exhibited significantly less haze protective activity than the wild type. Moreover, material derived from HPF1 and HPF1 ' overexpressors was more active than material from the wild type. A 6xHis - tagged Hpf2p was expressed and purified using immobilised metal affinity chromatography. This Hpf2p had significant haze protective activity. Modification of N - glycans of 6xHis - Hpf2p by Endoglycosidase H decreased its haze protective activity.
Thesis (Ph.D.)--School of Agriculture and Wine, 2003.

Indiana University-Purdue University Indianapolis (IUPUI)
Fatty acid elongation is the extension of de novo synthesized fatty acids through a series of four reactions analogous to those of fatty acid synthase. ELOs catalyze the first reaction in the elongation pathway through the condensation of an acyl group with a two carbon unit derived from malonyl-CoA. This study uses the condensing enzyme, EloA, from the cellular slime mold, Dictyostelium discoideum as a model for the family of ELOs. EloA has substrate specificity for monounsaturated and saturated C16 fatty acids and catalyzes the elongation of 16:1Δ9 to 18:1Δ11. Site-directed mutagenesis was used to change residues highly conserved among the ELO family to examine their potential role in the condensation reaction. Mutant EloAs were expressed in yeast and fatty acid methyl esters prepared from total cellular lipids were analyzed by gas chromatography/mass spectrometry. Sixteen out of twenty mutants had a decrease in 18:1Δ11 production when compared to the wild-type EloA with little to no activity observed in ten mutants, four mutants had within 20% of wild-type activity, and six mutants had 10-60% of wild-type activity. Immunoblot studies using anti-EloA serum were used to determine if the differences in elongation activity were related to changes in protein expression for each mutant. Analysis of immunoblots indicated that those mutants with little to no activity, with the exception of T130A and Q203A, had x comparable protein expression to the wild-type. Further research included the solubilization of the His6-ELoA fusion protein and preliminary work toward the isolation of the tagged protein and the use of a radiolabeled condensation assay to determine the activity of the eluted protein. Preliminary results indicated that the protein was solubilized but the eluted protein showed no activity when examined by a condensation assay. The work presented here contributes to a better understanding of the role of certain amino acid residues in the activity of EloA and serves as a stepping-stone for future EloA isolation work.

Indiana University-Purdue University Indianapolis (IUPUI)
Proteasome assembly is a rapid and highly sequential process that occurs through a series of intermediates. While the quest to understand the exact process of assembly is ongoing, there remains an incomplete understanding of what happens early on during the process, prior to the involvement of the β subunits. A significant feature of proteasome assembly is the property of proteasomal subunits to self-assemble. While archaeal α and β subunits from Thermoplasma acidophilum can assemble into entire 20S units in vitro, certain α subunits from divergent species have a property to self-assemble into single and double heptameric rings. In this study, we have shown that recombinant α subunits from Methanococcus maripaludis also have a tendency to self-assemble into higher order structures when expressed in E. coli. Using a novel cross-linking strategy, we were able to establish that these higher order structures were double α rings that are structurally similar to a half-proteasome (i.e. an α-β ring pair). Our experiments on M. maripaludis α subunits represent the first biochemical evidence for the orientation of rings in an α ring dimer. We also investigated self-assembly of α subunits in S. cerevisiae and attempted to characterize a highly stable and unique high molecular weight complex (HMWC) that is formed upon co-expression of α5, α6, α7 and α1 in E. coli. Using our cross-linking strategy, we were able to show that this complex is a double α ring in which, at the least, one α1 subunit is positioned across itself. We were also able to detect α1-α1 crosslinks in high molecular weight complexes that are formed when α7 and α1 are co-expressed, and when α6, α7 and α1 are co-expressed in E. coli. The fact that we able to observe α1-α1 crosslinks in higher order structures that form whenever α7 and α1 were present suggests that α1-α1 crosslinks might be able to serve as potential trackers to detect HMWCs in vivo. This would be an important step in determining if these HMWCs represent bona fide assembly intermediates, or dead-end complexes whose formation must be prevented in order to ensure efficient proteasome assembly.

Indiana University-Purdue University Indianapolis (IUPUI)
DNA double strand break (DSB) is one of the most threatening of all types of DNA damages as it leads to a complete breakage of the chromosome. The cell has evolved several mechanisms to repair DSBs, one of which is break-induced replication (BIR). BIR repair of DSBs occurs through invasion of one end of the broken chromosome into a homologous template followed by processive replication of DNA from the donor molecule. BIR is a key cellular process and is implicated in the restart of collapsed replication forks and several chromosomal instabilities. Recently, our lab demonstrated that the fidelity of DNA synthesis associated with BIR in yeast Saccharomyces Cerevisiae is extremely low. The level of frameshift mutations associated with BIR is 1000-fold higher as compared to normal DNA replication. This work demonstrates that BIR stimulates base substitution mutations, which comprise 90% of all point mutations, making them 400-1400 times more frequent than during S-phase DNA replication. We show that DNA Polymerase δ proofreading corrects many of the base substitutions in BIR. Further, we demonstrate that Pif1, a 5’-3’ DNA helicase, is responsible for making BIR efficient and also highly mutagenic. Pif1p is responsible for the majority of BIR mutagenesis not only close to the DSB site, where BIR is less stable but also at chromosomal regions far away from the DSB break site, where BIR is fast, processive and stable. This work further reveals that, at positions close to the DSB, BIR mutagenesis in the absence of Pif1 depends on Rev3, the catalytic subunit of translesion DNA Polymerase ζ. We observe that mutations promoted by Pol ζ are often complex and propose that they are generated by a Pol ζ- led template switching mechanism. These complex mutations were also found to be frequently associated with gross chromosomal rearrangements. Finally we demonstrate that BIR is carried out by unusual conservative mode of DNA synthesis. Based on this study, we speculate that the unusual mode of DNA synthesis associated with BIR leads to various kinds of genomic instability including mutations and chromosomal rearrangements.