Browsing by Subject "Saccharomyces cerevisiae"
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Item Aerobic Uptake of Cholesterol by Ergosterol Auxotrophic Strains in Candida glabrata & Random and Site-Directed Mutagenesis of ERG25 in Saccharomyces cerevisiae(2012-09-27) Whybrew, Jennafer Marie; Bard, Martin; Lees, N. Douglas; Blacklock, BrendaCandida albicans and Candida glabrata are opportunistic human pathogens that are the leading cause of fungal infections, which are increasingly becoming the leading cause of sepsis in immunosuppressed individuals. C. glabrata in particular has become a significant concern due to the increase in clinical isolates that demonstrate resistance to triazole antifungal drugs, the most prevalent treatment for such infections. Triazole drugs target the ERG11 gene product and prevent C-14 demethylation of the first sterol intermediate, lanosterol, preventing the production of the pathways end product ergosterol. Ergosterol is required by yeast for cell membrane fluidity and cell signaling. Furthermore, C. glabrata, and not C. albicans, has been reported to utilize cholesterol as a supplement for growth. Although drug resistance is known to be caused by an increase in expression of drug efflux pumps, we hypothesize a second mechanism: that the overuse of triazole drugs has lead to the increase of resistance by C. glabrata through a 2-step process: 1) the accumulation of ergosterol auxotrophic mutations and 2) mutants able to take up exogenous cholesterol anaerobically in the body acquire a second mutation allowing uptake of cholesterol aerobically. Two groups of sterol auxotrophic C. glabrata clinical isolates have been reported to take up sterol aerobically but do not produce a sterol precursor. Sterol auxotrophs have been created in C. glabrata by disrupting different essential genes (ERG1, ERG7, ERG11, ERG25, and ERG27) in the ergosterol pathway to assess which ergosterol mutants will take up sterols aerobically. Random and site-directed mutagenesis was also completed in ERG25 of Saccharmoyces cerevisiae. The ERG25 gene encodes a sterol C-4 methyloxidase essential for sterol biosynthesis in plants, animals, and yeast. This gene functions in turn with ERG26, a sterol C-3 dehydrogenase, and ERG27, a sterol C-3 keto reductase, to remove two methyl groups at the C-4 position on the sterol A ring. In S. cerevisiae, ERG25 has four putative histidine clusters, which bind non-heme iron and a C-terminal KKXX motif, which is a Golgi to ER retrieval motif. We have conducted site-directed and random mutagenesis in the S. cerevisiae wild-type strain SCY876. Site-Directed mutagenesis focused on the four histidine clusters, the KKXX C-terminal motif and other conserved amino acids among various plant, animal, and fungal species. Random mutagenesis was completed with a procedure known as gap repair and was used in an effort to find novel changes in enzyme function outside of the parameters utilized for site-directed mutagenesis. The four putative histidine clusters are expected to be essential for gene function by acting as non-heme iron binding ligands bringing in the oxygen required for the oxidation-reduction in the C-4 demethylation reaction.Item Assessment of Trinidad community stakeholder perspectives on the use of yeast interfering RNA-baited ovitraps for biorational control of Aedes mosquitoes(PLOS, 2021-06-29) Winter, Nikhella; Stewart, Akilah T.M.; Igiede, Jessica; Wiltshire, Rachel M.; Hapairai, Limb K.; James, Lester D.; Mohammed, Azad; Severson, David W.; Duman-Scheel, Molly; Medical and Molecular Genetics, School of MedicineDengue, Zika, chikungunya and yellow fever viruses continue to be a major public health burden. Aedes mosquitoes, the primary vectors responsible for transmitting these viral pathogens, continue to flourish due to local challenges in vector control management. Yeast interfering RNA-baited larval lethal ovitraps are being developed as a novel biorational control tool for Aedes mosquitoes. This intervention circumvents increasing issues with insecticide resistance and poses no known threat to non-target organisms. In an effort to create public awareness of this alternative vector control strategy, gain stakeholder feedback regarding product design and acceptance of the new intervention, and build capacity for its potential integration into existing mosquito control programs, this investigation pursued community stakeholder engagement activities, which were undertaken in Trinidad and Tobago. Three forms of assessment, including paper surveys, community forums, and household interviews, were used with the goal of evaluating local community stakeholders' knowledge of mosquitoes, vector control practices, and perceptions of the new technology. These activities facilitated evaluation of the hypothesis that the ovitraps would be broadly accepted by community stakeholders as a means of biorational control for Aedes mosquitoes. A comparison of the types of stakeholder input communicated through use of the three assessment tools highlighted the utility and merit of using each tool for assessing new global health interventions. Most study participants reported a general willingness to purchase an ovitrap on condition that it would be affordable and safe for human health and the environment. Stakeholders provided valuable input on product design, distribution, and operation. A need for educational campaigns that provide a mechanism for educating stakeholders about vector ecology and management was highlighted. The results of the investigation, which are likely applicable to many other Caribbean nations and other countries with heavy arboviral disease burdens, were supportive of supplementation of existing vector control strategies through the use of the yeast RNAi-based ovitraps.Item Break-Induced Replication is a Source of Mutation Clusters Underlying Kataegis(Elsevier B.V., 2014-06) Sakofsky, Cynthia J.; Roberts, Steven A.; Malc, Ewa; Mieczkowski, Piotr A.; Resnick, Michael A.; Gordenin, Dmitry A.; Malkova, Anna; Department of Biology, School of ScienceClusters of simultaneous multiple mutations can be a source of rapid change during carcinogenesis and evolution. Such mutation clusters have been recently shown to originate from DNA damage within long single-strand (ss) DNA formed at resected double-strand breaks and dysfunctional replication forks. We identify here double-strand break (DSB)-induced replication (BIR) as another powerful source of mutation clusters that formed in nearly half of wild-type yeast cells undergoing BIR in the presence of alkylating damage. Clustered mutations were primarily formed along the track of DNA synthesis and were frequently associated with additional breakage and rearrangements. Moreover, the base specificity, strand coordination and strand bias of the mutation spectrum was consistent with mutations arising from damage in persistent ssDNA stretches within unconventional replication intermediates. Together, these features closely resemble kataegic events in cancers, suggesting that replication intermediates during BIR may be the most prominent source of mutation clusters across species.Item A Broad-Based Mosquito Yeast Interfering RNA Pesticide Targeting Rbfox1 Represses Notch Signaling and Kills Both Larvae and Adult Mosquitoes(MDPI, 2021-09-28) Mysore, Keshava; Sun, Longhua; Hapairai, Limb K.; Wang, Chien-Wei; Roethele, Joseph B.; Igiede, Jessica; Scheel, Max P.; Scheel, Nicholas D.; Li, Ping; Wei, Na; Severson, David W.; Duman-Scheel, Molly; Medical and Molecular Genetics, School of MedicinePrevention of mosquito-borne infectious diseases will require new classes of environmentally safe insecticides and novel mosquito control technologies. Saccharomyces cerevisiae was engineered to express short hairpin RNA (shRNA) corresponding to mosquito Rbfox1 genes. The yeast induced target gene silencing, resulting in larval death that was observed in both laboratory and outdoor semi-field trials conducted on Aedes aegypti. High levels of mortality were also observed during simulated field trials in which adult females consumed yeast delivered through a sugar bait. Mortality correlated with defects in the mosquito brain, in which a role for Rbfox1 as a positive regulator of Notch signaling was identified. The larvicidal and adulticidal activities of the yeast were subsequently confirmed in trials conducted on Aedes albopictus, Anopheles gambiae, and Culex quinquefasciatus, yet the yeast had no impact on survival of select non-target arthropods. These studies indicate that yeast RNAi pesticides targeting Rbfox1 could be further developed as broad-based mosquito larvicides and adulticides for deployment in integrated biorational mosquito control programs. These findings also suggest that the species-specificity of attractive targeted sugar baits, a new paradigm for vector control, could potentially be enhanced through RNAi technology, and specifically through the use of yeast-based interfering RNA pesticides.Item CDMS Analysis of Intact 19S, 20S, 26S, and 30S Proteasomes: Evidence for Higher-Order 20S Assemblies at a Low pH†(American Chemical Society, 2023) Anthony, Adam J.; Gautam, Amit K. S.; Miller, Lohra M.; Ma, Yiran; Hardwick, Anya G.; Sharma, Anu; Ghatak, Subhadip; Matouschek, Andreas; Jarrold, Martin F.; Clemmer, David E.; Surgery, School of MedicineCharge detection mass spectrometry (CDMS) was examined as a means of studying proteasomes. To this end, the following masses of the 20S, 19S, 26S, and 30S proteasomes from Saccharomyces cerevisiae (budding yeast) were measured: m(20S) = 738.8 ± 2.9 kDa, m(19S) = 926.2 ± 4.8 kDa, m(26S) = 1,637.0 ± 7.6 kDa, and m(30S) = 2,534.2 ± 10.8 kDa. Under some conditions, larger (20S)x (where x = 1 to ∼13) assemblies are observed; the 19S regulatory particle also oligomerizes, but to a lesser extent, forming (19S)x complexes (where x = 1 to 4, favoring the x = 3 trimer). The (20S)x oligomers are favored in vitro, as the pH of the solution is lowered (from 7.0 to 5.4, in a 20 mM ammonium acetate solution) and may be related to in vivo proteasome storage granules that are observed under carbon starvation. From measurements of m(20S)x (x = 1 to ∼13) species, it appears that each multimer retains all 28 proteins of the 20S complex subunit. Several types of structures that might explain the formation of (20S)x assemblies are considered. We stress that each structural type [hypothetical planar, raft-like geometries (where individual proteasomes associate through side-by-side interactions); elongated, rodlike geometries (where subunits are bound end-to-end); and geometries that are roughly spherical (arising from aggregation through nonspecific subunit interactions)] is highly speculative but still interesting to consider, and a short discussion is provided. The utility of CDMS for characterizing proteasomes and related oligomers is discussed.Item Characterization of a broad-based mosquito yeast interfering RNA larvicide with a conserved target site in mosquito semaphorin-1a genes(Springer Nature, 2019-05-22) Mysore, Keshava; Li, Ping; Wang, Chien-Wei; Hapairai, Limb K.; Scheel, Nicholas D.; Realey, Jacob S.; Sun, Longhua; Severson, David W.; Wei, Na; Duman-Scheel, Molly; Medical and Molecular Genetics, School of MedicineBACKGROUND: RNA interference (RNAi), which has facilitated functional characterization of mosquito neural development genes such as the axon guidance regulator semaphorin-1a (sema1a), could one day be applied as a new means of vector control. Saccharomyces cerevisiae (baker's yeast) may represent an effective interfering RNA expression system that could be used directly for delivery of RNA pesticides to mosquito larvae. Here we describe characterization of a yeast larvicide developed through bioengineering of S. cerevisiae to express a short hairpin RNA (shRNA) targeting a conserved site in mosquito sema1a genes. RESULTS: Experiments conducted on Aedes aegypti larvae demonstrated that the yeast larvicide effectively silences sema1a expression, generates severe neural defects, and induces high levels of larval mortality in laboratory, simulated-field, and semi-field experiments. The larvicide was also found to induce high levels of Aedes albopictus, Anopheles gambiae and Culex quinquefasciatus mortality. CONCLUSIONS: The results of these studies indicate that use of yeast interfering RNA larvicides targeting mosquito sema1a genes may represent a new biorational tool for mosquito control.Item Characterization of a fatty acid elongase condensing enzyme by site-directed mutagenesis and biochemical analysis(2014) Hernandez-Buquer, Selene; Long, Eric C. (Eric Charles); Blacklock, Brenda J.; Li, LeiFatty 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.Item COMPARATIVE ANALYSIS OF THE DISCORDANCE BETWEEN THE GLOBAL TRANSCRIPTIONAL AND PROTEOMIC RESPONSE OF THE YEAST SACCHAROMYCES CEREVISIAE TO DELETION OF THE F-BOX PROTEIN, GRR1(2010-05) Heyen, Joshua William; Goebl, Mark, 1958-; Roach, Peter J.; Clemmer, David E.; Wang, Mu; Chen, JakeThe Grr1 (Glucose Repression Resistant) protein in Saccharomyces cerevisiae is an F-box protein for the E3 ubiquitin ligase protein complex known as the SCFGrr1 (Skp, Cullin, F-box). F-box proteins serve as substrate receptors for this complex and in this capacity Grr1 serves to promote the ubiquitylation and subsequent proteasomal degradation of a number of intracellular protein substrates. Substrates of SCFGrr1 include the G1-S phase cyclins, Cln1 and Cln2, the Cdc42 effectors and cell polarity proteins, Gic1 and Gic2, the FCH-bar domain protein, Hof1, required for cytokinesis, the meiosis activating serine/threonine protein kinase, Ime2, the transcriptional regulators of glucose transporters, Mth1 and Std1, and the mitochondrial retrograde response inhibitor Mks1. Stabilization of these substrates lead to pleiotrophic phenotypic defects in grr1Δ strains including resistance to glucose repression, accumulation of grr1Δ cells in G2 and M phase of the cell cycle, sensitivity to osmotic stress, and resistance to divalent cations. However, many of these phenotypes are not reflected at the gene expression level. We conducted a quantitative genomic vii and proteomic comparison of 914 loci in a grr1Δ and wild-type strain grown to early log-phase in glucose media. These loci encompassed 16.7% of the Saccharomyces proteome of which 22.3% exhibited discordance between gene and protein expression. GO process enrichment analysis revealed that discordant loci were enriched in the processes of “trafficking”, “mitosis”, and “carbon/energy” metabolism. Here we show that these instances of discordance are biologically relevant and in fact reflect phenotypes of grr1Δ strains not evident at the transcriptional level. Additionally, through combined biochemical and network analysis of discordant loci among “carbon and energy metabolism” we were able to not only construct a model for central carbon metabolism in grr1Δ strains but also were able to elucidate a novel molecular event that may serve to regulate glucose repression of genes needed for respiration in response to changes in glucose concentration.Item A conserved female-specific larval requirement for MtnB function facilitates sex separation in multiple species of disease vector mosquitoes(BMC, 2021-06-26) Mysore, Keshava; Sun, Longhua; Roethele, Joseph B.; Li, Ping; Igiede, Jessica; Misenti, Joi K.; Duman‑Scheel, Molly; Medical and Molecular Genetics, School of MedicineBackground: Clusters of sex-specific loci are predicted to shape the boundaries of the M/m sex-determination locus of the dengue vector mosquito Aedes aegypti, but the identities of these genes are not known. Identification and characterization of these loci could promote a better understanding of mosquito sex chromosome evolution and lead to the elucidation of new strategies for male mosquito sex separation, a requirement for several emerging mosquito population control strategies that are dependent on the mass rearing and release of male mosquitoes. This investigation revealed that the methylthioribulose-1-phosphate dehydratase (MtnB) gene, which resides adjacent to the M/m locus and encodes an evolutionarily conserved component of the methionine salvage pathway, is required for survival of female larvae. Results: Larval consumption of Saccharomyces cerevisiae (yeast) strains engineered to express interfering RNA corresponding to MtnB resulted in target gene silencing and significant female death, yet had no impact on A. aegypti male survival or fitness. Integration of the yeast larvicides into mass culturing protocols permitted scaled production of fit adult male mosquitoes. Moreover, silencing MtnB orthologs in Aedes albopictus, Anopheles gambiae, and Culex quinquefasciatus revealed a conserved female-specific larval requirement for MtnB among different species of mosquitoes. Conclusions: The results of this investigation, which may have important implications for the study of mosquito sex chromosome evolution, indicate that silencing MtnB can facilitate sex separation in multiple species of disease vector insects.Item Correction: Mysore et al. A Broad-Based Mosquito Yeast Interfering RNA Pesticide Targeting Rbfox1 Represses Notch Signaling and Kills Both Larvae and Adult Mosquitoes. Pathogens 2021, 10, 1251(MDPI, 2022-08-23) Mysore, Keshava; Sun, Longhua; Hapairai, Limb K.; Wang, Chien-Wei; Roethele, Joseph B.; Igiede, Jessica; Scheel, Max P.; Scheel, Nicholas D.; Li, Ping; Wei, Na; Severson, David W.; Duman-Scheel, Molly; Medical and Molecular Genetics, School of MedicineIn the original publication [1], there was a mistake in Figure 1 as published. The wrong graph was inadvertently included in panel 1f (dose–response curve). Additionally, the original image for the gel shown in panel 1a is now included in the Supplementary Materials.