Browsing by Subject "Yeast"
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Item Biophysical and Bioanalytical Analysis of the Iron-ome in Mitochondria Isolated from Saccharomyces cerevisiae(2011-08-08) Garber Morales, Jessica H.An integrative biophysical and bioanalytical approach to studying the Fe distribution in isolated mitochondria was developed. This procedure involved large-scale growths, the inclusion of a chelator in isolation buffers and an anaerobic isolation protocol. Electron microscopy confirmed that mitochondrial membranes were intact and that samples were largely devoid of contaminants. The Fe-ome-the sum of all Fe species in mitochondria--was studied using a combination of EPR, Mossbauer Spectroscopy, Electron Absorption, ICP-MS and Protein analysis. Isolated mitochondria were packed prior to analysis to improve the S/N ratio. The residual buffer content of sample pellets was determined by use of a radio-labeled buffer. There was essentially no difference in the packing efficiency of mitochondria isolated from respiring and fermenting cells. The determined packing factor, 0.80, was used to calculate concentrations of individual species in neat mitochondria. The Fe-omes of mitochondria isolated from cells grown on respiring, respirofermenting and fermenting media were determined. Neat mitochondria contained ~ 750 mM Fe, regardless of whether the cells had been grown on respiring or fermenting media. The Fe distribution of respirofermenting samples (which can undergo respiration and fermentation simultaneously) was nearly identical to that of respiring mitochondria. Fermenting samples had a very different Fe-distribution. Nearly 40 % of the iron in respiring mitochondria was present in respiratory complexes including cytochrome c, cytochrome bc1, succinate dehydrogenase, and cytochrome c oxidase. Fermenting mitochondria contain an Fe-ome dominated by non-protein centers. Approximately 80 % of the Fe was present as a combination of nonheme HS Fe2+, nonheme Fe3+ and Fe3+ nanoparticles. These centers were present in roughly equal amounts. The remaining 20 % of the Fe was present as respiratory complexes which have concentrations ~ 1/2 to 1/3 that of respiring mitochondria. A model is presented in which the nonheme HS Fe2+ species serves as a feedstock for Fe/S and heme biosynthesis. When the cell is growing on respiring media, this metabolic reservoir diminishes as respiratory complexes are constantly synthesized. Under fermentative growth, the metabolic pool increases due to the reduced demand for respiration-related prosthetic groups.Item Characterization of factors involved in 3' to 5' mRNA degradation in yeast(2005) Wang, Lingna; Johnson, Arlen W.Item Effects of levucell sb yeast on average daily gain, feed intake, and morbidity of newly received cattle(Texas Tech University, 2006-05) Keyser, Sara A.; Galyean, Michael L.; Johnson, Jay W.; Albin, Robert C.Three separate loads of beef heifers (n = 277 heifers) were transported to the Texas Tech Burnett Center in New Deal, TX to examine the effects of a yeast supplement (Levucell SB yeast; Lallemand Animal Nutrition, Milwaukee, WI) on health and performance of feedlot cattle. In Load 1, 91 beef heifers (average BW = 223.5 kg) were shipped 1,403 km from an order buyer facility in Meridian, MS. On arrival, cattle were weighed and processed and assigned randomly to one of two treatments (five pens per treatment) during a 35-d receiving period: 1) Control (C) = a 65% concentrate receiving diet; or 2) Yeast (Y) = a 65% concentrate receiving diet with Levucell SB yeast added to supply 0.5 g of yeast/(heifer•d). Diets were changed to 72% concentrate on d 21 to 35. Following processing, cattle were moved to their assigned pens and fed their respective diets ad libitum once daily at 0800. Cattle were observed daily for symptoms of bovine respiratory disease (BRD) and treated as needed when rectal temperature was > 39.7 °C. Loads 2 and 3 (93 heifers each; average BW = 223.5 kg and 226.1 kg respectively) were processed and assigned to treatments and pens as described for Load 1. Averaged over the three loads, feeding Levucell SB yeast did not affect the overall (P > 0.12) dry matter intake (DMI) or average daily gain (ADG) during the 35-d study. Although, numerical advantages in ADG for the Y treatment were evident from d 0 to 14 and 0 to 28, changes in ADG were inconsistent among the three loads. As with ADG, concentrate DMI for the various measurement periods did not differ between treatments, but a trend was evident for a slight increase from d 0 to 35 in concentrate DMI for the Y vs. C treatment for Loads 1 and 3, but not with Load 2. Because treatment effects on ADG and DMI were not significant, G:F did not differ between treatments. Within loads, no differences (P = 0.21 to 0.28) were noted for the percentage of cattle treated once or more for BRD; however, a consistently smaller proportion of the cattle in the Y treatment group were treated compared with those in the C group. Thus, averaged over the three loads, an increase (P = 0.04) in the percentage of C heifers treated once or more compared with Y heifers (24.0 vs. 13.78% respectively) was observed. An odds ratio of 1.99 for C vs. Y indicated that C heifers were approximately twice as likely to be treated once or more for BRD than were Y heifers. From the results of the three loads of newly received heifers used in this experiment, the addition of 0.5 g/heifer daily of Levucell SB yeast to the diet of newly received cattle plus oral dosing of approximately 1 g/heifer at the time of arrival processing resulted in fewer heifers being treated for BRD. The feeding of Levucell SB yeast during the receiving period had limited effects on performance of the 277 heifers used in the experiment.Item Functional characterization of the recycling mechanism for the 60S nuclear export adapter Nmd3p in yeast(2004) Hedges, John Benjamin; Johnson, Arlen W.Ribosomes are essential macromolecular machines that translate, through a messenger RNA intermediate, the information encoded in the DNA sequence of all cells into proteins. Because of their fundamental role in cell survival, an enormous amount of cellular resources must be dedicated to ribosome synthesis. Eukaryotic cells must contend with transport of materials between two compartments, the nucleus and cytoplasm. During ribosome biogenesis, these cells assemble ribosomal subunits at a specific subnuclear structure called the nucleolus and only release them into the nucleoplasm upon completion of initial assembly. The subunits must then traverse the nucleoplasm before reaching the nuclear pore complex (NPC) where they are exported to the cytoplasm to act in translation. Processing of the small (40S) subunit through this pathway occurs relatively quickly. However, processing of the large subunit (60S) involves a greater number of maturation steps. One of the last of these steps being export of the large subunit through the NPC, mediated through the export adapter protein Nmd3. The large ribosomal subunit protein Rpl10 is required for 60S export and subunit joining in yeast. It is believed that the role of Rpl10p in export is to provide the 60S binding site for Nmd3p in the nucleus. Through examination of rpl10 mutant effects on the 60S export pathway, I’ve instead found that the role of Rpl10p is indirect. This work shows that disruption of either Rpl10p or the Rpl10p 60S loading factor, Sqt1p, leads to a block in export due to entrapment of Nmd3p on 60S subunits in the cytoplasm. For rpl10 mutants these effects are suppressed by specific alleles of NMD3 that restore recycling to the nucleus. To gain a better understanding of the export function of Nmd3p, this work also examines the NES and 60S binding domains of Nmd3p and, in light of the Rpl10p results shown here, establishes an assay to identify other 60S components required for this binding. From these findings, I propose the model that Rpl10p is required for the release of Nmd3p from subunits in the cytoplasm to support further rounds of 60S export and to provide a final “quality control” step in 60S maturation prior to 40S joining.Item Genetics of commitment to cell division in S. cerevisiae(Texas Tech University, 2004-12) Zhang, JianIn all eukaryotes, proliferation is regulated by cell cycle controls. Elucidating the intrinsic mechanism whereby these control mechanisms modulate proliferation is essential for understanding the role of the cell cycle in development, aging and cancer. Our laboratory uses budding yeast S. cerevisiae as a model system. In budding yeast, proliferation is dependent upon cell growth, cell size, and the expression of Gl phase cyclins (Clns). However, the relationship among these requirements is poorly understood. In this study, the relationship between cell growth, cell size, Cln expression, and proliferation was analyzed. It is found that rapidly growing cells express, and require, more Cln protein to divide than do slowly growing cells. To clarify the role of cell size, defined amounts of CLTN mRNA were expressed in cells of different sizes. It is found that a critical threshold of Cln protein was required for proliferation, and that Cln1 protein expression was strongly modulated by cell size. In addition, expression of high levels of CLNs promoted proliferation in a size-independent manner suggesting that Clns are rate-limiting. To examine the relationship between cell growth and the ability of cells to proliferate, a systematic genome-wide genetic screen was conducted to identify mutants that dramatically altered the proliferative capacity of cells. In so doing, 49 gene deletions that dramatically changed cell size were identified. Twenty of these made cells abnormally small (whi mutants), and 29 made cells abnormally large (uge mutants). Nearly all of these genes have putative human homologues. Interestingly, five uge gene products are components of Ccr4-Not transcriptional complexes. Furthermore, it is found that CCR4 positively regulates CLNl mRNA expression. In ccr4Ä strains, CLNl mRNA expression was decreased in asynchronous cultures and delayed in synchronized cultures, but restoration ofCCR4 expression induces CLNl mRNA expression and rescues the size phenotype ofccr4A. My results suggest that CCR4 modulates the ability of the Bck2 protein to induce CLNJ and CLN2 transcription. In summary, my research has identified new gene products involved in cell cycle control and has helped elucidate the mechanism whereby cells coordinate cell growth with proliferation.Item Identification of biomolecules by mechanical modulation Raman microscopy(2011-12) Hinko, Kathleen Ann; Florin, Ernst-LudwigRaman microscopy is a tool used by physicists to collect molecular information from a wide variety of samples. Biophysicists have increasingly made use of Raman microscopy in combination with optical tweezers to identify the molecular makeup of structures inside cells. There are high levels of background and noise in Raman spectra from cells, however, that obscure low intensity scattering peaks and prevent complete molecular characterization. We have designed and built a Mechanical Modulation Raman Microscope(MMRM) that is capable of background subtraction and noise reduction for Raman spectra from cells in vivo. There are two mechanisms of modulation: (1) three-axis stage modulation for objects fixed to the coverslip and (2) separate optical trap modulation for objects in solution. In both cases, objects of interest are modulated in and out of the Raman excitation volume while spectra are collected. Difference spectra are created by subtracting the spectrum without the object from the spectrum including the object. These difference spectra are averaged over the number of cycles of modulation. With the mechanical modulation technique, the background in Raman spectra is removed, and the signal-to-noise ratio is improved by two orders of magnitude. This technique was applied to fission yeast cells. Mechanical modulation Raman spectra of exponentially growing cells and starved cells were collected in three dimensions, and spatial differences were observed in the molecular composition for different metabolic states of individual yeast cells.Item Influence of yeast cell wall supplementation during the finishing phase on feedlot steer performance, carcass characteristics and post-mortem tenderness(2013-08) Aragon, Samantha Nicole; Johnson, Bradley J.; Trojan, Sara J.; Ballou, Michael A.Objectives were to evaluate benefits of yeast cell wall (YCW) supplementation on performance, carcass traits and tenderness of steers finished with zilpaterol hydrochloride (ZH). A randomized complete block design was used. British X Continental steers (n = 72; initial BW = 305±13 kg) were blocked by BW and allotted randomly to 24 pens (8 pens/treatment; 3 pens/block; 3 steers/pen). Treatments were: 1) control (CON); 2) YCW containing 100,000 IU vitamin D2/g (5.0 g•hd-1•d-1) (Y-D); 3) YCW C (5.0 g•hd-1•d-1) (Y-C). Steers were supplemented with respective treatments for 55 d, of which ZH was fed d 30-49. Cattle were weighed at d 0, 21, and 55. Carcass data was collected at the plant, and strip loins were obtained. Strips were cut into steaks and assigned to one of four aging periods (7, 14, 21 or 28 d). Tenderness was examined using Warner-Bratzler shear force (WBSF). Shrunk performance showed no differences. Carcass adjusted average daily gain (ADG) from d 21-55 was 0.29 kg greater for Y-D and 0.35 kg greater for Y-C when compared to CON (P = 0.04 and 0.01, respectively). Additionally, YCW increased G:F from d 21-55 with a 20.77% improvement for Y-D and 28.46% for Y-C over CON (P = 0.06 and 0.01, respectively). Carcass data revealed no differences, yet there was a trend for a 6 kg increase in HCW by both Y-D and Y-C compared to CON (P = 0.16 and 0.12). The treatment × aging interaction with the WBSF data was not significant (P = 0.20) and no differences were found in cooking loss (P = 0.88). Treatment Y-C displayed WBSF values 0.30 kg higher than CON and 0.29 kg greater than Y-D (P = 0.0062 and 0.0075). Within the 7 d aging period, Y-C steaks were 0.62 kg (P = 0.005) and 0.54 kg (P = 0.014) less tender than CON or Y-D, respectively. For 14 d steaks, Y-C WBSF values were 0.58 kg greater than CON (P = 0.008). No differences were found in the 21 or 28 d aging periods. The frequency distribution of WBSF values consistently displayed a trend for tougher Y-C steaks within each aging period. These data indicate yeast cell wall supplementation could increase performance of finishing steers while vitamin D2 supplementation at the current dosage yielded no beneficial effects on tenderness.Item Investigating the Roles of Vacuoles in Iron Trafficking in Saccharomyces cerevisiae(2013-11-27) Cockrell, Allison LeighTransition metals play essential roles in biological systems, but Fe can also be toxic to cells. In order to maintain this balance between necessity and toxicity mechanisms are employed for regulating and storing intracellular Fe. In Saccharomyces cerevisiae, vacuoles are responsible for sequestering, storing, and supplying Fe to the cytosol. Many of the proteins and regulatory pathways involved in Fe trafficking and storage in S. cerevisiae have been identified, but the forms of Fe which are involved in these processes have not been fully characterized. In these studies, biophysical and bioanalytical techniques were used to study intracellular Fe distributions in S. cerevisiae cells and organelles. Ultimately, Fe-containing species were biophysically characterized and absolute Fe concentrations in cells and organelles were quantified. The motivation for these studies stemmed from previous studies which revealed that the majority of the whole-cell Fe is a non-heme, high-spin (NHHS) form of Fe^(3+). This Fe is not localized to the mitochondria. The purpose of these studies was to determine if the vacuoles contained this NHHS Fe^(3+). A large-scale isolation procedure was developed to obtain purified vacuoles from S. cerevisiae and to investigate the Fe in these organelles. M?ssbauer and EPR analysis revealed that the primary form of Fe in vacuoles is a mononuclear, NHHS Fe^(3+) species. A second form of Fe was also observed as superparamagnetic ferric phosphate nanoparticles (NP). By investigating model compounds of Fe and polyphosphate we determined that a shift in vacuolar pH induces the conversion between NHHS Fe^(3+) and NP. These results showed that there are at least two forms of Fe in vacuoles, and that the ratio of these two forms is dependent upon the pH of these organelles. Biophysical analyses of whole cells also revealed the presence of low concentrations of a non-heme, high-spin Fe^(2+) species. The goal of these next projects was to determine if this NHHS Fe^(2+) species was localized to the cytosol. Genetic strains lacking or over-expressing the vacuolar Fe import protein Ccc1p were studied by M?ssbauer spectroscopy (?CCC1 and CCC1-up, respectively). ?CCC1 cells showed low vacuolar Fe (NHHS Fe3+ and NP), and increased NHHS Fe^(2+). We hypothesize that this NHHS Fe^(2+) is cytosolic Fe. We also propose that this NHHS Fe^(2+) is involved in the regulating intracellular Fe levels. CCC1-up cells accumulated more Fe than wild-type (WT) cells, and showed elevated levels of vacuolar Fe (NHHS Fe^(3+) and NP). These cells also accumulated high levels of NHHS Fe^(2+). The CCC1-up cells exhibited an adenine deficient phenotype, where the cells developed a red color during growth. With excess adenine the levels of NHHS Fe^(2+) declined, which indicated that this Fe accumulation was related to adenine deficiency. We conclude that adenine deficiency leads to the accumulation of a sequestered (possibly vacuolar) form of NHHS Fe^(2+). Overall, we have identified two separate pools of NHHS Fe^(2+) in ?CCC1 and CCC1-up cells. In ?CCC1 cells the NHHS Fe^(2+) pool is localized to the cytosol and is sensed by the cell. In CCC1-up cells the NHHS Fe^(2+) is sequestered from the Fe regulatory mechanism- possibly in the vacuoles. These data have helped us better understand the roles of vacuoles in Fe trafficking and the dynamics of vacuolar Fe trafficking.Item Metabolic Engineering of S. cerevisiae for Carotenoid Production Optimization(2014-11-19) Olson, Michelle LIsoprenoids are naturally produced compounds in the budding yeast Saccharomyces cerevisiae. They are involved in essential cellular functions of the cell and are also further synthesized into pharmaceuticals, carotenoids, and biofuel alternatives. S. cerevisiae is a key model eukaryotic organism because it is both tractable and a nonpathogenic GRAS organism (Generally Recognized As Safe). S. cerevisiae is used extensively in metabolic engineering due to its well-curated and annotated genome and the wide range of tools available for genetic modifications. Engineering S. cerevisiae for production of heterologous isoprenoid compounds is a sustainable and cost effective alternate to production via chemical synthesis. ?-carotene, an abundant isoprenoid compound in nature, protects cells from oxidative stress and reactive oxidative species in the environment. Through a novel adaptive evolution experiment of S. cerevisiae with oxidative stress as the driving force, we obtained a carotenoid hyper-producer strain that is able to produce 18 ? 1 mg/g [dry cell weight] ?-carotene in 3 ml cultures. To test the potential for scale-up ?-carotene production in yeast, we fermented the cultures in a 7L bioreactor. Optimization of the bioreactor parameters revealed the influence of media composition, aeration, and pH on ?-carotene production. We aimed to further optimize ?-carotene production in S. cerevisiae with genetic and metabolic engineering. We reintroduced the cytosolic catalase T (CTT1) gene and overexpressed the known bottleneck of the isoprenoid biosynthesis pathway, HMG1. The reintroduction of CTT1 into SM14 (carotenoid hyper-producer) demonstrated improvement in carotenoid production, where production increased from 15 ? 3.3 mg/g [dry cell weight] to 22 ? 2.1 mg/g [dry cell weight]. The overexpression of truncated HMG1 in SM14, on the other hand, did not increase ?-carotene production. The isoprenoid pathway is of a very complex phenotype and there are many direct and indirect variables involved in pathway performance. Even with these modifications to the hyper-producer strain, there seems to be limitations on ?-carotene production. Further studies currently under investigation to increase ?-carotene production include utilizing the fatty acid ?-oxidation pathway for increasing the fatty acid content of the cell.Item MRNA degradation in the control of gene expression in yeast(2001-08) Brown, Justin Travis; Johnson, Arlen W.The pathways for eukaryotic mRNA translation and degradation are composed of numerous interconnected elements. I have characterized the general cellular roles of factors involved in mRNA degradation pathways and their part in the control of gene expression in the budding yeast Saccharomyces cerevisiae. Xrn1p is the non-essential cytoplasmic 5´ exoribonuclease required for rapid mRNA turnover. xrn1 mutants have been associated with wildly disparate phenotypes, including karyogamy and meiotic recombination. I identified two translation mutations (in eIF4E and eIF2B) from a synthetic lethal screen, supporting my assertion that aberrant gene expression contributes to xrn1 pleiotropy. I then demonstrated that mutations in capping enzyme and in eIF4G genetically interact with xrn1. My results contradict the currently held model that stabilization of mRNAs by deletion of XRN1 should suppress the inviability of upstream translation mutations. I conclude that the accumulation of messages in an xrn1 mutant is in fact lethal in combination with particular defects in translation. A previous xrn1 synthetic lethal screen identified SKI2 and SKI3. My screen also identified mutations complemented by SKI4/CSL4, SKI6/RRP41, RRP46, and SKI8. Debate persists over the primary role of the Ski proteins. PolyA-minus mRNA is not translated efficiently in wild-type eukaryotic cells, but is translated efficiently in ski mutants, perhaps due to altered translational specificity. However, as the SKI genes are required for 3´ mRNA degradation, it is possibly a consequence of inhibition of 3´ mRNA decay. I show that Ski2p, Ski3p and Ski8p form a stable complex and that Ski2p and Ski3p are cytoplasmic, not nuclear as previously maintained. To further distinguish between the Ski models and to directly assess the heretofore unexamined effects of 3´ mRNA degradation on translation in non-mutant cells, I show that an RNA can be translated efficiently in wild-type cells without polyA or Pab1p when 3´ degradation is blocked in cis. In addition, this enhanced expression phenocopies a ski mutant. Hence, functional redundancy is the simplest model to explain synthetic lethality between xrn1 and ski mutations: mRNA decay is an essential process and lethality consequently arises from inhibition of both 5´ and 3´ pathways for mRNA degradation.Item Novel approaches for metabolic engineering of yeast at multiple scales(2014-05) Crook, Nathan Charles; Alper, Hal S.; Contreras, Lydia; Georgiou, George; Maynard, Jennifer; Ellington, AndrewLiving systems contain enormous potential to solve many pressing engineering problems, including the production of usable energy, the synthesis and degradation of a variety of materials, and the treatment of disease. Metabolic engineering, as one approach to harness this potential, treats the behavior of a living system as the combined product of multiple interacting modules, each of which can be tuned to maximize performance. However, the scarcity of techniques for predictive or high-throughput engineering design of these modules, especially in eukaryotes, contributes to long strain development times and high research cost. In this work, we develop several new tools to expand our capabilities for predictive design and high-throughput engineering in yeast. At the transcriptional level, we develop a method which, for the first time, enables predictive strengthening endogenous yeast promoters and also the de novo design of strong synthetic promoters. At the translational level, we show that it is possible to exploit the context resulting from the arrangement of DNA parts in order to predictably increase or decrease gene expression. We also develop a powerful new approach for directed evolution of enzymes in yeast, termed in vivo continuous evolution, which enables the creation of library sizes orders of magnitude larger than can be obtained with the current state of the art using significantly less labor. Finally, we harness the programmatic inhibitory potential of RNA interference to optimize and demonstrate a system for rapid strain engineering with minimal genomic editing. Taken together, this work provides new techniques which enable a significant reduction in the development time of new yeast strains and informs future development of new tools for metabolic engineering.Item Nutritional value of Candida utilis produced from potato starch(Texas Tech University, 1971-05) Rentschler, NellNot availableItem 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 Search for selection pressures associated with aggregation propensity following whole genome duplication in S.cerevisiae.(2011-12) Wittig, Michael David; Press, William H.; Marcotte, Edward M.It has been theorized that most proteins are under selection pressure to be soluble in crowded cellular spaces. To maintain solubility a proteins’ aggregation propensity should be inversely proportional to their maximum likely concentration. This theory was examined by comparing the proteome of the model organism S. cerevisiae, which has previously undergone a Whole Genome Duplication (WGD) event to the proteome of the closely related yeast K. waltii, which has not undergone WGD. This comparison revealed the following: 1) Predicted aggregation propensities are higher in S. cerevisiae than K. waltii. 2) Aggregation propensity does not predict which genes reverted to a single copy after WGD. 3) In genes which were retained as duplicates in S. cerevisiae after WGD, aggregation propensities rose from the inferred common ancestral protein. 4) Genes retained as duplicates showed less of an increase relative to their homologues in K. waltii than genes which were not retained as duplicates. 5) The relationship between the log predicted aggregation propensity and log mRNA expression level or log protein abundance was not linear as previously predicted. These results suggest that while there is broad selection pressure for reduced aggregation pressure for genes which have been duplicated, the precise relationship between aggregation propensity and gene expression is more complicated than previously predicted. These results also allow speculation that the whole genome duplication in S.cerevisiae may have been made possible by a general relaxation of aggregation-related selection pressure.Item Serum cholesterol and urinary chromium levels of adults supplemented with brewer's yeast or chromium chloride(Texas Tech University, 1984-12) Wang, Minching MarinaNot availableItem Systemic protein aggregation in stress and aging restructures cytoplasmic architecture(2012-12) O'Connell, Jeremy Daniel 1982-; Marcotte, Edward M.A common maxim of protein biochemistry states, “structure is function.” This is generally just as true for an individual polypeptide chains as for multi-protein complexes. The advent of yeast tagged-protein libraries has allowed systematic screening of a protein’s local interaction partners as well as a roughly mapping its cellular location. Recently our group and others discovered hundreds proteins forming new structures in stationary phase yeast cells using the yeast GFP-tag library. That equates to well over a quarter of normally diffuse cytoplasmic proteins assembled into discrete structures that appear as foci or fibers, all of unknown function. This study provides evidence that many of these foci are formed by protein aggregation- that contrary the maxim, structure can be dysfunction. Furthermore, this study uses yeast to demonstrate the generality of cytoplasmic protein aggregation in response to a variety of stresses, provides evidence that increasing aggregation of particular cytoplasmic proteins correlates with aging even across organisms, and proposes a theoretical framework for how cellular energy levels affect protein aggregation propensity.Item The Application of LC-ICP-MS to Study Metal Ion Homeostasis in Biological Systems(2014-12-10) McCormick, Sean P.Eukaryotic cells contain low-molecular-mass metal complexes (LMMMCs), defined as having masses between 200 ? 10,000 Da, but these so-called labile or chelatable metal pools are poorly defined in terms of structures and functions. LMMMCs are thought to participate in metal-ion regulation, trafficking, storage and/or signaling in cells. These cellular processes are often dysfunctional in metal-associated diseases. The objective of these studies was to detect and characterize LMMMCs in eukaryotic cells, organelles and tissues. A novel liquid chromatography system in a cold inert-atmosphere glove box was interfaced with an in-line inductively coupled plasma mass spectrometer, and this LC-ICP-MS system was used to detect LMMMCs in yeast cells, mitochondria, and vacuoles as well as in mouse brain and liver cells and mitochondria. In each biological system, this separations technique was applied to detect numerous LMMMCs. The molecular mass and concentration of such species were estimated. In yeast, the previously reported mismetallation of MnSOD2 was examined in the mutant strain ?mtm1. A combination of SEC and AEX chromatography revealed that the degree of mismetallation of the SOD2 protein, in which Fe replace Mn in the active site, was no greater in ?mtm1 cells than in WT cells. The mitochondria of such mutant cells did exhibit an intense chromatography peak of Mn corresponding to at mass of 2000 ? 3000 Da. Mitochondria from WT cells exhibited a similar species, but at much lower intensity. This was the only Mn species present, suggesting that it was the used to metallate apo-SOD2. Mitochondria isolated from WT yeast cells contained 6 Co, 3 Cu, 2 Mn, 5 Fe and 3 Zn LMMMCs and approximately 6 P- and S- LLM species. Some of the P- and S- LMMCs probably arose from compounds like ATP, ADP, etc. Molecular masses of the LMM Cu peaks were higher (> 5 kDa) than for the LMM complexes of other transition metals. Zinc, Mn, and Fe had multiple species of interest which demonstrate the presence and labiality of the metals in pools. The same separation system was utilized to examine mice brain LMM extracts were found to contain > 30 LMMMCs. Eleven Co, 2 Cu, 5 Mn, 4 Mo, 3 Fe and 2 Zn LLM complexes were detected. Most Cu and Zn complexes appeared to be protein-bound with masses ranging from 4?20 kDa. In these systems, Co was the only metal for which the aqueous complex was reproducibly observed. A second mouse study used the LC-ICP-MS system to examine the forms of iron present in mouse plasma. Chromatograms exhibited ~6 Fe-associated peaks that were assigned to ferritin, transferrin, and hemopexin, respectively; the other 3 peaks could not be assigned. The LC-ICP-MS experiment demonstrates that numerous Fe-containing species coexist with transferrin in healthy WT mouse plasma.Item Transporter engineering as a tool for metabolic engineering(2013-08) Young, Eric Mosher; Alper, Hal S.; Appling, Dean; Contreras, Lydia; Georgiou, George; Maynard, JenniferThe purpose of metabolic engineering is to understand, design, and optimize metabolism. The objective is chemicals synthesis by microbes. To fulfill this purpose and achieve this objective, tools that control metabolism are essential. Molecular transport is a vital metabolic step yet tools to control it are underdeveloped. Therefore, this work aims to establish transporter engineering, a tool that can rewire transport. Efficient xylose utilization is a key component to economical consumption of lignocellulosic biomass, the most abundant source of sugars on the planet. Transport is a limiting step in the metabolism of xylose by the industrial yeast Saccharomyces cerevisiae. In yeast, transport proteins enabling xylose uptake also permit transit of a broad spectrum of other sugars. Furthermore, glucose is preferred as a substrate to the exclusion of xylose. Therefore, the goal of transporter engineering in this context is twofold: improve xylose uptake while reducing glucose uptake. Four strategies were used to accomplish this goal. First, we performed an iterative bioprospecting approach to explore the extant biodiversity of sugar transporters. However, the transporters tested lack efficient and exclusive xylose transport, motivating development of additional engineering strategies. Second, a directed evolution strategy increased xylose transport efficiency, demonstrating the power directed evolution has to improve transport phenotypes. Third, a targeted engineering strategy was used to analyze key residues responsible for the improved xylose transport phenotype, representing the first targeted engineering strategy to improve xylose growth and reduce glucose growth. Finally, rational engineering was explored. With all of the information collected using the previous strategies, design rules could be developed and implemented. A triple mutant of C. intermedia GXS1 was engineered that does not confer growth on glucose, but xylose growth is retained. By implementing this design rule in S. stipitis RGT2 and S. cerevisiae HXT7, additional xylose exclusive variants can be engineered. This demonstrates that a fundamental design component has been identified and can be used to rewire transport. Thus, this work builds the foundation for molecular transporter engineering.Item Use of Candida utilis for altering protein content of dry-milled grain sorghum fractions(Texas Tech University, 1969-08) Brackeen, Daniel LenNot availableItem Yeast supplementation alters the performance and health of cattle during the receiving period(2011-05) Finck, Derek N.; Johnson, Bradley J.; Rathmann, Ryan J.; Beckett, Jonathan L.The objective of this experiment was to determine the effect of yeast supplements on performance and health of steers during the receiving period. Weaned crossbred steers (n = 184; initial BW = 203 ± 1 kg) were blocked by BW and randomly assigned to pen (4 pens/block; 5-6 hd/pen). Pens within a block were randomly assigned to one of four treatments (9 pens/treatment): 1) control (CON; no yeast additive), 2) live yeast (LY; 5 g.hd-1.d-1 BIOSAF, Lesaffre Feed Additives, Milwaukee, WI), 3) yeast cell wall (YCW; 5 g.hd-1.d-1 Pronady 500, Lesaffre Feed Additives), 4) live yeast + yeast cell wall (LY+YCW; 5 g.hd-1.d-1 live yeast and 5 g.hd-1.d-1 yeast cell wall). Daily DMI was recorded and individual BW was collected every 14 d for 56 d. Data were analyzed using a randomized complete block design using the fixed effect of treatment and random effect of block (SAS Inst. Inc., Cary, NC). A subset of 24 steers was utilized after 38 d on feed to determine the effect of yeast supplementation on the response to a lipopolysaccharide (LPS) challenge. Calves were fitted with jugular catheters and indwelling rectal temperature measuring devices that measured rectal temperature at 1-min intervals, and were moved into individual stanchions. On d 39, blood samples were collected at 30-min intervals from -2 to 8 h and then at 24 h relative to administration of LPS (0.5 μg/kg BW) at 0 h. Blood samples were used to determine serum interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), cortisol concentrations, and neutrophil:lymphocyte (N:L) ratios. Data were subjected to analysis of variance specific for repeated measures using Statview (SAS Inst. Inc.) with sources of variation including treatment, time and their interactions. Specific time point comparisons within treatment group were conducted using a Paired t-test to compare pre-challenge values with specific time points post-challenge. Steers receiving LY or YCW showed a 7% numerical increase (P = 0.59) in ADG and a 7.7 ± 4.7 kg increase in BW at d 56. Cumulative DMI increased (P = 0.05) for the LY, YCW, and LY+YCW treatments compared to CON (5.47, 6.02, 5.96, and 5.89 kg/d; CON, LY, YCW, and LY+YCW, respectively). Steer morbidity and mortality were not affected by yeast supplementation (P ≥ 0.10). In response to LPS challenge, basal RT prior to LPS tended (P ≤ 0.06) to differ among groups with CON calves having higher RT compared to LY+YCW (P ≤ 0.01) and LY (P ≤ 0.04) calves. After the LPS challenge, RT remained higher in the CON calves compared to other treatments (P ≤ 0.05). By 10 h post-LPS, RT were still greater (P ≤ 0.05) in CON calves compared to all other calves, and remained numerically greater throughout the study. Serum cortisol increased in all groups post-LPS with peak concentrations observed at 1 h. Peak cortisol concentrations were 26.5 ng/mL greater (P ≤ 0.04) in CON calves compared to LY+YCW calves. Interferon-gamma (IFN-γ) concentrations tended (P ≤ 0.06) to be greater in CON calves compared to YCW calves prior to LPS exposure. Collectively, these data indicated that the use of yeast additives increased total feed consumed by the steers during the first 56 d of the feeding period, and improved health, thus allowing for enhanced performance.