Journal articles on the topic 'Saccharomyces cerevisiae – Genetic aspects'
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OBERNAUEROVÁ, M., and J. ŠUBÍK. "Biochemical-genetic aspects of saccharose utilization by yeast of Saccharomyces cerevisiae." Kvasny Prumysl 33, no. 4 (April 1, 1987): 108–10. http://dx.doi.org/10.18832/kp1987022.
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Dorer, Russell, Charles Boone, Tyler Kimbrough, Joshua Kim, and Leland H. Hartwell. "Genetic Analysis of Default Mating Behavior in Saccharomyces cerevisiae." Genetics 146, no. 1 (May 1, 1997): 39–55. http://dx.doi.org/10.1093/genetics/146.1.39.
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Haploid Saccharomyces cerevisiae cells find each other during conjugation by orienting their growth toward each other along pheromone gradients (chemotropism). However, when their receptors are saturated for pheromone binding, yeast cells must select a mate by executing a default pathway in which they choose a mating partner at random. We previously demonstrated that this default pathway requires the SPA2 gene. In this report we show that the default mating pathway also requires the AXL1, FUS1, FUS2, FUS3, PEAZ, RVS161, and BNI1 genes. These genes, including SPA2, are also important for efficient cell fusion during chemotropic mating. Cells containing null mutations in these genes display defects in cell fusion that subtly affect mating efficiency. In addition, we found that the defect in default mating caused by mutations in SPA2 is partially suppressed by multiple copies of two genes, FUS2 and MFA2. These findings uncover a molecular relationship between default mating and cell fusion. Moreover, because axl1 mutants secrete reduced levels of a-factor and are defective at both cell fusion and default mating, these results reveal an important role for a-factor in cell fusion and default mating. We suggest that default mating places a more stringent requirement on some aspects of cell fusion than does chemotropic mating.
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REED, LESTER J., KAREN S. BROWNING, XIAO-DA NIU, ROBERT H. BEHAL, and DAVID J. UHLINGER. "Biochemical and Molecular Genetic Aspects of Pyruvate Dehydrogenase Complex from Saccharomyces cerevisiae." Annals of the New York Academy of Sciences 573, no. 1 Alpha-Keto Ac (December 1989): 155–67. http://dx.doi.org/10.1111/j.1749-6632.1989.tb14993.x.
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Pâques, Frédéric, and James E. Haber. "Multiple Pathways of Recombination Induced by Double-Strand Breaks in Saccharomyces cerevisiae." Microbiology and Molecular Biology Reviews 63, no. 2 (June 1, 1999): 349–404. http://dx.doi.org/10.1128/mmbr.63.2.349-404.1999.
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SUMMARY The budding yeast Saccharomyces cerevisiae has been the principal organism used in experiments to examine genetic recombination in eukaryotes. Studies over the past decade have shown that meiotic recombination and probably most mitotic recombination arise from the repair of double-strand breaks (DSBs). There are multiple pathways by which such DSBs can be repaired, including several homologous recombination pathways and still other nonhomologous mechanisms. Our understanding has also been greatly enriched by the characterization of many proteins involved in recombination and by insights that link aspects of DNA repair to chromosome replication. New molecular models of DSB-induced gene conversion are presented. This review encompasses these different aspects of DSB-induced recombination in Saccharomyces and attempts to relate genetic, molecular biological, and biochemical studies of the processes of DNA repair and recombination.
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Babudri, Nora, Angela Lucaccioni, and Alessandro Achilli. "ADAPTIVE MUTAGENESIS IN THE YEAST SACCHAROMYCES CEREVISIAE." Ecological genetics 4, no. 3 (September 15, 2006): 20–28. http://dx.doi.org/10.17816/ecogen4320-28.
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The nature of mutation in microorganisms has been debated for a long time. Two theories have been at odds: random spontaneous mutagenesis vs. adaptive mutagenesis. "random mutagenesis" means that mutations occur in proliferating cells before they encountered the selective agent. "adaptive mutagenesis" means that advantageous mutations form in the environment where they have been selected, in non-replicating or poorly replicating cells even though other, non-selected, mutations occur at the same time. In the last 20 years it has been definitely shown that random as well as adaptive mutagenesis occur in bacteria and yeast. microorganisms in nature do not divide or divide poorly because of adverse environmental conditions; therefore adaptive mutations could provide cells with a selective advantage and allow evolution of populations. Here we will focus on some fundamental aspects of adaptive mutagenesis in the yeast Saccharomyces cerevisiae. We begin with a historical overview on the nature of mutation. We then focus on experimental systems aimed at proving or disproving adaptive mutagenesis. We have briefly summarized the results obtained in this field, with particular attention to genetic and molecular mechanisms.
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Heude, M., and F. Fabre. "a/alpha-control of DNA repair in the yeast Saccharomyces cerevisiae: genetic and physiological aspects." Genetics 133, no. 3 (March 1, 1993): 489–98. http://dx.doi.org/10.1093/genetics/133.3.489.
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Abstract It has long been known that diploid strains of yeast are more resistant to gamma-rays than haploid cells, and that this is in part due to heterozygosity at the mating type (MAT) locus. It is shown here that the genetic control exerted by the MAT genes on DNA repair involves the a1 and alpha 2 genes, in a RME1-independent way. In rad18 diploids, affected in the error-prone repair, the a/alpha effects are of a very large amplitude, after both UV and gamma-rays, and also depends on a1 and alpha 2. The coexpression of a and alpha in rad18 haploids suppresses the sensitivity of a subpopulation corresponding to the G2 phase cells. Related to this, the coexpression of a and alpha in RAD+ haploids depresses UV-induced mutagenesis in G2 cells. For srs2 null diploids, also affected in the error-prone repair pathway, we show that their G1 UV sensitivity, likely due to lethal recombination events, is partly suppressed by MAT homozygosity. Taken together, these results led to the proposal that a1-alpha 2 promotes a channeling of some DNA structures from the mutagenic into the recombinational repair process.
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Jacobus, Ana Paula, Jeferson Gross, John H. Evans, Sandra Regina Ceccato-Antonini, and Andreas Karoly Gombert. "Saccharomyces cerevisiae strains used industrially for bioethanol production." Essays in Biochemistry 65, no. 2 (July 2021): 147–61. http://dx.doi.org/10.1042/ebc20200160.
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Abstract Fuel ethanol is produced by the yeast Saccharomyces cerevisiae mainly from corn starch in the United States and from sugarcane sucrose in Brazil, which together manufacture ∼85% of a global yearly production of 109.8 million m3 (in 2019). While in North America genetically engineered (GE) strains account for ∼80% of the ethanol produced, including strains that express amylases and are engineered to produce higher ethanol yields; in South America, mostly (>90%) non-GE strains are used in ethanol production, primarily as starters in non-aseptic fermentation systems with cell recycling. In spite of intensive research exploring lignocellulosic ethanol (or second generation ethanol), this option still accounts for <1% of global ethanol production. In this mini-review, we describe the main aspects of fuel ethanol production, emphasizing bioprocesses operating in North America and Brazil. We list and describe the main properties of several commercial yeast products (i.e., yeast strains) that are available worldwide to bioethanol producers, including GE strains with their respective genetic modifications. We also discuss recent studies that have started to shed light on the genes and traits that are important for the persistence and dominance of yeast strains in the non-aseptic process in Brazil. While Brazilian bioethanol yeast strains originated from a historical process of domestication for sugarcane fermentation, leading to a unique group with significant economic applications, in U.S.A., guided selection, breeding and genetic engineering approaches have driven the generation of new yeast products for the market.
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Kartasheva, N. N., S. V. Kuchin, and S. V. Benevolensky. "Genetic aspects of carbon catabolite repression of the STA2 glucoamylase gene in Saccharomyces cerevisiae." Yeast 12, no. 13 (October 1996): 1297–300. http://dx.doi.org/10.1002/(sici)1097-0061(199610)12:13<1297::aid-yea13>3.0.co;2-u.
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Kunz, Bernard A., Karthikeyan Ramachandran, and Edward J. Vonarx. "DNA Sequence Analysis of Spontaneous Mutagenesis in Saccharomyces cerevisiae." Genetics 148, no. 4 (April 1, 1998): 1491–505. http://dx.doi.org/10.1093/genetics/148.4.1491.
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Abstract To help elucidate the mechanisms involved in spontaneous mutagenesis, DNA sequencing has been applied to characterize the types of mutation whose rates are increased or decreased in mutator or antimutator strains, respectively. Increased spontaneous mutation rates point to malfunctions in genes that normally act to reduce spontaneous mutation, whereas decreased rates are associated with defects in genes whose products are necessary for spontaneous mutagenesis. In this article, we survey and discuss the mutational specificities conferred by mutator and antimutator genes in the budding yeast Saccharomyces cerevisiae. The implications of selected aspects of the data are considered with respect to the mechanisms of spontaneous mutagenesis.
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Spencer, F., S. L. Gerring, C. Connelly, and P. Hieter. "Mitotic chromosome transmission fidelity mutants in Saccharomyces cerevisiae." Genetics 124, no. 2 (February 1, 1990): 237–49. http://dx.doi.org/10.1093/genetics/124.2.237.
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Abstract We have isolated 136 independent mutations in haploid yeast strains that exhibit decreased chromosome transmission fidelity in mitosis. Eighty-five percent of the mutations are recessive and 15% are partially dominant. Complementation analysis between MATa and MAT alpha isolates identifies 11 chromosome transmission fidelity (CTF) complementation groups, the largest of which is identical to CHL1. For 49 independent mutations, no corresponding allele has been recovered in the opposite mating type. The initial screen monitored the stability of a centromere-linked color marker on a nonessential yeast chromosome fragment; the mitotic inheritance of natural yeast chromosome III is also affected by the ctf mutations. Of the 136 isolates identified, seven were inviable at 37 degrees and five were inviable at 11 degrees. In all cases tested, these temperature conditional lethalities cosegregated with the chromosome instability phenotype. Five additional complementation groups (ctf12 through ctf16) have been defined by complementation analysis of the mutations causing inviability at 37 degrees. Twenty-three of the 136 isolates exhibited growth defects at concentrations of benomyl permissive for the parent strain, and nine appeared to be tolerant of inhibitory levels of benomyl. All of the mutant strains showed normal sensitivity to ultraviolet and gamma-irradiation. Further characterization of these mutant strains will describe the functions of gene products crucial to the successful execution of processes required for aspects of the chromosome cycle that are important for chromosome transmission fidelity in mitosis.
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Lau, W.-T. Walter, Ken R. Schneider, and Erin K. O’Shea. "A Genetic Study of Signaling Processes for Repression of PHO5 Transcription in Saccharomyces cerevisiae." Genetics 150, no. 4 (December 1, 1998): 1349–59. http://dx.doi.org/10.1093/genetics/150.4.1349.
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Abstract In the yeast Saccharomyces cerevisiae, transcription of a secreted acid phosphatase, PHO5, is repressed in response to high concentrations of extracellular inorganic phosphate. To investigate the signal transduction pathway leading to transcriptional regulation of PHO5, we carried out a genetic selection for mutants that express PHO5 constitutively. We then screened for mutants whose phenotypes are also dependent on the function of PHO81, which encodes an inhibitor of the Pho80p-Pho85p cyclin/cyclin-dependent kinase complex. These mutations are therefore likely to impair upstream functions in the signaling pathway, and they define five complementation groups. Mutations were found in a gene encoding a plasma membrane ATPase (PMA1), in genes required for the in vivo function of the phosphate transport system (PHO84 and PHO86), in a gene involved in the fatty acid synthesis pathway (ACC1), and in a novel, nonessential gene (PHO23). These mutants can be classified into two groups: pho84, pho86, and pma1 are defective in high-affinity phosphate uptake, whereas acc1 and pho23 are not, indicating that the two groups of mutations cause constitutive expression of PHO5 by distinct mechanisms. Our observations suggest that these gene products affect different aspects of the signal transduction pathway for PHO5 repression.
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Jordá, Tania, and Sergi Puig. "Regulation of Ergosterol Biosynthesis in Saccharomyces cerevisiae." Genes 11, no. 7 (July 15, 2020): 795. http://dx.doi.org/10.3390/genes11070795.
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Ergosterol is an essential component of fungal cell membranes that determines the fluidity, permeability and activity of membrane-associated proteins. Ergosterol biosynthesis is a complex and highly energy-consuming pathway that involves the participation of many enzymes. Deficiencies in sterol biosynthesis cause pleiotropic defects that limit cellular proliferation and adaptation to stress. Thereby, fungal ergosterol levels are tightly controlled by the bioavailability of particular metabolites (e.g., sterols, oxygen and iron) and environmental conditions. The regulation of ergosterol synthesis is achieved by overlapping mechanisms that include transcriptional expression, feedback inhibition of enzymes and changes in their subcellular localization. In the budding yeast Saccharomyces cerevisiae, the sterol regulatory element (SRE)-binding proteins Upc2 and Ecm22, the heme-binding protein Hap1 and the repressor factors Rox1 and Mot3 coordinate ergosterol biosynthesis (ERG) gene expression. Here, we summarize the sterol biosynthesis, transport and detoxification systems of S. cerevisiae, as well as its adaptive response to sterol depletion, low oxygen, hyperosmotic stress and iron deficiency. Because of the large number of ERG genes and the crosstalk between different environmental signals and pathways, many aspects of ergosterol regulation are still unknown. The study of sterol metabolism and its regulation is highly relevant due to its wide applications in antifungal treatments, as well as in food and pharmaceutical industries.
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Opalek, Monika, and Dominika Wloch-Salamon. "Aspects of Multicellularity in Saccharomyces cerevisiae Yeast: A Review of Evolutionary and Physiological Mechanisms." Genes 11, no. 6 (June 24, 2020): 690. http://dx.doi.org/10.3390/genes11060690.
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The evolutionary transition from single-celled to multicellular growth is a classic and intriguing problem in biology. Saccharomyces cerevisiae is a useful model to study questions regarding cell aggregation, heterogeneity and cooperation. In this review, we discuss scenarios of group formation and how this promotes facultative multicellularity in S. cerevisiae. We first describe proximate mechanisms leading to aggregation. These mechanisms include staying together and coming together, and can lead to group heterogeneity. Heterogeneity is promoted by nutrient limitation, structured environments and aging. We then characterize the evolutionary benefits and costs of facultative multicellularity in yeast. We summarize current knowledge and focus on the newest state-of-the-art discoveries that will fuel future research programmes aiming to understand facultative microbial multicellularity.
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Huang, K. N., S. A. Odinsky, and F. R. Cross. "Structure-function analysis of the Saccharomyces cerevisiae G1 cyclin Cln2." Molecular and Cellular Biology 17, no. 8 (August 1997): 4654–66. http://dx.doi.org/10.1128/mcb.17.8.4654.
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We have generated 50 new alleles of the yeast CLN2 gene by using site-directed mutagenesis. With the recently obtained crystal structure of cyclin A as a guide, a peptide linker sequence was inserted at 13 sites within the cyclin box of Cln2 to determine if the architecture of Cln2 is similar to that of cyclin A. Linkers inserted in what are predicted to be helices 1, 2, 3, and 5 of the cyclin box resulted in nonfunctional Cln2 molecules. Linkers inserted between these putative helix sites and in the region believed to contain a fourth helix did not have significant effects upon Cln2 function. A series of deletions in the region between the third and fifth helices indicate that the putative fourth helix may lie at the C-terminal end of this region yet is not essential for function. Two residues that are predicted to form a buried salt bridge important for interaction of two helices of the cyclin box were also mutated, and an additional set of 31 mutant alleles was generated by clustered-charge-to-alanine scanning mutagenesis. All of the mutant CLN2 alleles made in this study were tested in a variety of genetic and functional assays previously demonstrated to differentiate specific cyclin functions. Some alleles demonstrated restricted patterns of defects, suggesting that these mutations may interfere with specific aspects of Cln2 function.
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Johanson, Kelly, Patricia L. Allen, Fawn Lewis, Luis A. Cubano, Linda E. Hyman, and Timothy G. Hammond. "Saccharomyces cerevisiae gene expression changes during rotating wall vessel suspension culture." Journal of Applied Physiology 93, no. 6 (December 1, 2002): 2171–80. http://dx.doi.org/10.1152/japplphysiol.01087.2001.
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This study utilizes Saccharomyces cerevisiae to study genetic responses to suspension culture. The suspension culture system used in this study is the high-aspect-ratio vessel, one type of the rotating wall vessel, that provides a high rate of gas exchange necessary for rapidly dividing cells. Cells were grown in the high-aspect-ratio vessel, and DNA microarray and metabolic analyses were used to determine the resulting changes in yeast gene expression. A significant number of genes were found to be up- or downregulated by at least twofold as a result of rotational growth. By using Gibbs promoter alignment, clusters of genes were examined for promoter elements mediating these genetic changes. Candidate binding motifs similar to the Rap1p binding site and the stress-responsive element were identified in the promoter regions of differentially regulated genes. This study shows that, as in higher order organisms, S. cerevisiae changes gene expression in response to rotational culture and also provides clues for investigations into the signaling pathways involved in gravitational response.
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Dresser, M. E., D. J. Ewing, S. N. Harwell, D. Coody, and M. N. Conrad. "Nonhomologous synapsis and reduced crossing over in a heterozygous paracentric inversion in Saccharomyces cerevisiae." Genetics 138, no. 3 (November 1, 1994): 633–47. http://dx.doi.org/10.1093/genetics/138.3.633.
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Abstract Homologous chromosome synapsis ("homosynapsis") and crossing over are well-conserved aspects of meiotic chromosome behavior. The long-standing assumption that these two processes are causally related has been challenged recently by observations in Saccharomyces cerevisiae of significant levels of crossing over (1) between small sequences at nonhomologous locations and (2) in mutants where synapsis is abnormal or absent. In order to avoid problems of local sequence effects and of mutation pleiotropy, we have perturbed synapsis by making a set of isogenic strains that are heterozygous and homozygous for a large chromosomal paracentric inversion covering a well marked genetic interval and then measured recombination. We find that reciprocal recombination in the marked interval in heterozygotes is reduced variably across the interval, on average to approximately 55% of that in the homozygotes, and that positive interference still modulates crossing over. Cytologically, stable synapsis across the interval is apparently heterologous rather than homologous, consistent with the interpretation that stable homosynapsis is required to initiate or consummate a large fraction of the crossing over observed in wild-type strains. When crossing over does occur in heterozygotes, dicentric and acentric chromosomes are formed and can be visualized and quantitated on blots though not demonstrated in viable spores. We find that there is no loss of dicentric chromosomes during the two meiotic divisions and that the acentric chromosome is recovered at only 1/3 to 1/2 of the expected level.
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Vinh, D. B., M. D. Welch, A. K. Corsi, K. F. Wertman, and D. G. Drubin. "Genetic evidence for functional interactions between actin noncomplementing (Anc) gene products and actin cytoskeletal proteins in Saccharomyces cerevisiae." Genetics 135, no. 2 (October 1, 1993): 275–86. http://dx.doi.org/10.1093/genetics/135.2.275.
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Abstract We describe here genetic interactions between mutant alleles of Actin-NonComplementing (ANC) genes and actin (ACT1) or actin-binding protein (SAC6, ABP1, TPM1) genes. The anc mutations were found to exhibit allele-specific noncomplementing interactions with different act1 mutations. In addition, mutant alleles of four ANC genes (ANC1, ANC2, ANC3 and ANC4) were tested for interactions with null alleles of actin-binding protein genes. An anc1 mutant allele failed to complement null alleles of the SAC6 and TPM1 genes that encode yeast fimbrin and tropomyosin, respectively. Also, synthetic lethality between anc3 and sac6 mutations, and between anc4 and tpm1 mutations was observed. Taken together, the above results strongly suggest that the ANC gene products contribute to diverse aspects of actin function. Finally, we report the results of tests of two models previously proposed to explain extragenic noncomplementation.
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Haber, James E. "Learning Yeast Genetics from Miro Radman." Cells 10, no. 4 (April 20, 2021): 945. http://dx.doi.org/10.3390/cells10040945.
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Miroslav Radman’s far-sighted ideas have penetrated many aspects of our study of the repair of broken eukaryotic chromosomes. For over 35 years my lab has studied different aspects of the repair of chromosomal breaks in the budding yeast, Saccharomyces cerevisiae. From the start, we have made what we thought were novel observations that turned out to have been predicted by Miro’s extraordinary work in the bacterium Escherichia coli and then later in the radiation-resistant Dienococcus radiodurans. In some cases, we have been able to extend some of his ideas a bit further.
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Kwon, Min Jin, Mark Arentshorst, Markus Fiedler, Florence L. M. de Groen, Peter J. Punt, Vera Meyer, and Arthur F. J. Ram. "Molecular genetic analysis of vesicular transport in Aspergillus niger reveals partial conservation of the molecular mechanism of exocytosis in fungi." Microbiology 160, no. 2 (February 1, 2014): 316–29. http://dx.doi.org/10.1099/mic.0.074252-0.
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The filamentous fungus Aspergillus niger is an industrially exploited protein expression platform, well known for its capacity to secrete high levels of proteins. To study the process of protein secretion in A. niger, we established a GFP-v-SNARE reporter strain in which the trafficking and dynamics of secretory vesicles can be followed in vivo. The biological role of putative A. niger orthologues of seven secretion-specific genes, known to function in key aspects of the protein secretion machinery in Saccharomyces cerevisiae, was analysed by constructing respective gene deletion mutants in the GFP-v-SNARE reporter strain. Comparison of the deletion phenotype of conserved proteins functioning in the secretory pathway revealed common features but also interesting differences between S. cerevisiae and A. niger. Deletion of the S. cerevisiae Sec2p orthologue in A. niger (SecB), encoding a guanine exchange factor for the GTPase Sec4p (SrgA in A. niger), did not have an obvious phenotype, while SEC2 deletion in S. cerevisiae is lethal. Similarly, deletion of the A. niger orthologue of the S. cerevisiae exocyst subunit Sec3p (SecC) did not result in a lethal phenotype as in S. cerevisiae, although severe growth reduction of A. niger was observed. Deletion of secA, secH and ssoA (encoding SecA, SecH and SsoA the A. niger orthologues of S. cerevisiae Sec1p, Sec8p and Sso1/2p, respectively) showed that these genes are essential for A. niger, similar to the situation in S. cerevisiae. These data demonstrate that the orchestration of exocyst-mediated vesicle transport is only partially conserved in S. cerevisiae and A. niger.
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Stratford, M. "Genetic aspects of yeast flocculation: in particular, the role of FLO genes in the flocculation of Saccharomyces cerevisiae." Colloids and Surfaces B: Biointerfaces 2, no. 1-3 (March 1994): 151–58. http://dx.doi.org/10.1016/0927-7765(94)80029-4.
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Ayoub, Marie-José, Jean-Luc Legras, Pierre Abi-Nakhoul, Huu-Vang Nguyen, Rachad Saliba, and Claude Gaillardin. "Lebanon’s Native Oenological Saccharomyces cerevisiae Flora: Assessment of Different Aspects of Genetic Diversity and Evaluation of Winemaking Potential." Journal of Fungi 7, no. 8 (August 23, 2021): 678. http://dx.doi.org/10.3390/jof7080678.
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A total of 296 isolates of Saccharomyces cerevisiae sampled from naturally fermenting grape musts from various locations in Lebanon were typed by interdelta fingerprinting. Of these, 88 isolates were compared with oenological strains originating from various countries, using microsatellite characterization at six polymorphic loci. These approaches evidenced a large diversity of the natural oenological Lebanese flora over the territory as well as in individual spontaneous fermentations. Several cases of dominance and perenniality of isolates were observed in the same wineries, where fermentations appeared to involve lineages of sibling isolates. Our work thus evidenced a “winery effect” on strains’ relatedness. Similarly, related or identical strains were also detected in vicinal wineries, suggesting strain circulation within small geographical areas and a further “vicinity effect”. Moreover, and despite its diversity, the Lebanese flora seemed interrelated, on the basis of microsatellite loci analysis, in comparison to worldwide communities. We finally tested the ability of 21 indigenous strains to act as potential starters for winemaking. Seven of them passed our pre-selection scheme and two of them at least may be good candidates for use provided pilot-scale assays confirm their suitability.
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Ng, Davis T. W., Eric D. Spear, and Peter Walter. "The Unfolded Protein Response Regulates Multiple Aspects of Secretory and Membrane Protein Biogenesis and Endoplasmic Reticulum Quality Control." Journal of Cell Biology 150, no. 1 (July 10, 2000): 77–88. http://dx.doi.org/10.1083/jcb.150.1.77.
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The unfolded protein response (UPR) is an intracellular signaling pathway that relays signals from the lumen of the ER to activate target genes in the nucleus. We devised a genetic screen in the yeast Saccharomyces cerevisiae to isolate mutants that are dependent on activation of the pathway for viability. Using this strategy, we isolated mutants affecting various aspects of ER function, including protein translocation, folding, glycosylation, glycosylphosphatidylinositol modification, and ER-associated protein degradation (ERAD). Extending results gleaned from the genetic studies, we demonstrate that the UPR regulates trafficking of proteins at the translocon to balance the needs of biosynthesis and ERAD. The approach also revealed connections of the UPR to other regulatory pathways. In particular, we identified SON1/RPN4, a recently described transcriptional regulator for genes encoding subunits of the proteasome. Our genetic strategy, therefore, offers a powerful means to provide insight into the physiology of the UPR and to identify novel genes with roles in many aspects of secretory and membrane protein biogenesis.
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Yarrington, Robert M., Yaxin Yu, Chao Yan, Lu Bai, and David J. Stillman. "A Role for Mediator Core in Limiting Coactivator Recruitment in Saccharomyces cerevisiae." Genetics 215, no. 2 (April 23, 2020): 407–20. http://dx.doi.org/10.1534/genetics.120.303254.
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Mediator is an essential, multisubunit complex that functions as a transcriptional coactivator in yeast and other eukaryotic organisms. Mediator has four conserved modules, Head, Middle, Tail, and Kinase, and has been implicated in nearly all aspects of gene regulation. The Tail module has been shown to recruit the Mediator complex to the enhancer or upstream activating sequence (UAS) regions of genes via interactions with transcription factors, and the Kinase module facilitates the transition of Mediator from the UAS/enhancer to the preinitiation complex via protein phosphorylation. Here, we analyze expression of the Saccharomyces cerevisiae HO gene using a sin4 Mediator Tail mutation that separates the Tail module from the rest of the complex; the sin4 mutation permits independent recruitment of the Tail module to promoters without the rest of Mediator. Significant increases in recruitment of the SWI/SNF and SAGA coactivators to the HO promoter UAS were observed in a sin4 mutant, along with increased gene activation. These results are consistent with recent studies that have suggested that the Kinase module functions negatively to inhibit activation by the Tail. However, we found that Kinase module mutations did not mimic the effect of a sin4 mutation on HO expression. This suggests that at HO the core Mediator complex (Middle and Head modules) must play a role in limiting Tail binding to the promoter UAS and gene activation. We propose that the core Mediator complex helps modulate Mediator binding to the UAS regions of genes to limit coactivator recruitment and ensure proper regulation of gene transcription.
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Shu, Y., H. Yang, E. Hallberg, and R. Hallberg. "Molecular genetic analysis of Rts1p, a B' regulatory subunit of Saccharomyces cerevisiae protein phosphatase 2A." Molecular and Cellular Biology 17, no. 6 (June 1997): 3242–53. http://dx.doi.org/10.1128/mcb.17.6.3242.
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The Saccharomyces cerevisiae gene RTS1 encodes a protein homologous to a variable B-type regulatory subunit of the mammalian heterotrimeric serine/threonine protein phosphatase 2A (PP2A). We present evidence showing that Rts1p assembles into similar heterotrimeric complexes in yeast. Strains in which RTS1 has been disrupted are temperature sensitive (ts) for growth, are hypersensitive to ethanol, are unable to grow with glycerol as their only carbon source, and accumulate at nonpermissive temperatures predominantly as large-budded cells with a 2N DNA content and a nondivided nucleus. This cell cycle arrest can be overcome and partial suppression of the ts phenotype of rts1-null cells occurs if the gene CLB2, encoding a Cdc28 kinase-associated B-type cyclin, is expressed on a high-copy-number plasmid. However, CLB2 overexpression has no suppressive effects on other aspects of the rts1-null phenotype. Expression of truncated forms of Rts1p can also partially suppress the ts phenotype and can fully suppress the inability of cells to grow on glycerol and the hypersensitivity of cells to ethanol. By contrast, the truncated forms do not suppress the accumulation of large-budded cells at high temperatures. Coexpression of truncated Rts1p and high levels of Clb2p fully suppresses the ts phenotype, indicating that the inhibition of growth of rts1-null cells at high temperatures is due to both stress-related and cell cycle-related defects. Genetic analyses show that the role played by Rts1p in PP2A regulation is distinctly different from that played by the other known variable B regulatory subunit, Cdc55p, a protein recently implicated in checkpoint control regulation.
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Vandermeulen, Matthew D., and Paul J. Cullen. "New Aspects of Invasive Growth Regulation Identified by Functional Profiling of MAPK Pathway Targets in Saccharomyces cerevisiae." Genetics 216, no. 1 (July 14, 2020): 95–116. http://dx.doi.org/10.1534/genetics.120.303369.
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MAPK pathways are drivers of morphogenesis and stress responses in eukaryotes. A major function of MAPK pathways is the transcriptional induction of target genes, which produce proteins that collectively generate a cellular response. One approach to comprehensively understand how MAPK pathways regulate cellular responses is to characterize the individual functions of their transcriptional targets. Here, by examining uncharacterized targets of the MAPK pathway that positively regulates filamentous growth in Saccharomyces cerevisiae (fMAPK pathway), we identified a new role for the pathway in negatively regulating invasive growth. Specifically, four targets were identified that had an inhibitory role in invasive growth: RPI1, RGD2, TIP1, and NFG1/YLR042c. NFG1 was a highly induced unknown open reading frame that negatively regulated the filamentous growth MAPK pathway. We also identified SFG1, which encodes a transcription factor, as a target of the fMAPK pathway. Sfg1p promoted cell adhesion independently from the fMAPK pathway target and major cell adhesion flocculin Flo11p, by repressing genes encoding presumptive cell-wall-degrading enzymes. Sfg1p also contributed to FLO11 expression. Sfg1p and Flo11p regulated different aspects of cell adhesion, and their roles varied based on the environment. Sfg1p also induced an elongated cell morphology, presumably through a cell-cycle delay. Thus, the fMAPK pathway coordinates positive and negative regulatory proteins to fine-tune filamentous growth resulting in a nuanced response. Functional analysis of other pathways’ targets may lead to a more comprehensive understanding of how signaling cascades generate biological responses.
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Dela Cruz, FE, DR Kirsch, and JN Heinrich. "Transcriptional activity of Drosophila melanogaster ecdysone receptor isoforms and ultraspiracle in Saccharomyces cerevisiae." Journal of Molecular Endocrinology 24, no. 2 (April 1, 2000): 183–91. http://dx.doi.org/10.1677/jme.0.0240183.
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The Drosophila melanogaster ecdysone receptor (EcR) is produced in three isoforms, which mediate developmental processes such as metamorphosis. These isoforms were expressed in Saccharomyces cerevisiae to elucidate aspects of receptor transcription activity in a highly defined genetic model system. All three EcR isoforms showed ligand-independent transcriptional activation of an ecdysone reporter gene and the amount of activation correlated with the size of the N-terminal A/B (transactivation) domain present in the isoform: EcR-B1>EcR-A>>EcR-B2. Upon co-expression with ultraspiracle (Usp), transcriptional activation was further increased with EcR-B1 or EcR-A, but was unchanged with EcR-B2 or a truncated EcR lacking the A/B N-terminal domain (EcRDeltaA/B). Thus, the enhanced activity from Usp may depend on the presence of an N-terminal domain of EcR. Co-expression with Usp of several chimeric receptors of the EcR and the mouse androgen receptor (mAR) identified one chimera, composed of the mAR N-terminus and the remainder from EcR (mAR inverted question markEcR-CDEF) that was transcriptionally silent and inducible by Usp. In contrast, the vertebrate homologue, human retinoic acid receptor (RXRalpha), showed ligand-independent transcription when co-expressed with EcRDeltaA/B but not mAR inverted question mark EcR-CDEF. Therefore, RXRalpha does not require its partner to possess an N-terminal domain, yet is intolerant of a heterologous N-terminus. Similarly, the human vitamin D receptor, which has a short N-terminal region, showed greater ligand-independent transcription in the presence of RXRalpha than in the presence of Usp. These results reveal a mechanistic basis for the differential activities among the EcR isoforms, and between Usp and RXRalpha. Furthermore, they provided the foundation for a genetic screen to identify potential insecticides as well as accessory proteins for Usp and EcR.
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Forsberg, Hanna, Mårten Hammar, Claes Andréasson, Annalena Molinér, and Per O. Ljungdahl. "Suppressors of ssy1 and ptr3 Null Mutations Define Novel Amino Acid Sensor-Independent Genes in Saccharomyces cerevisiae." Genetics 158, no. 3 (July 1, 2001): 973–88. http://dx.doi.org/10.1093/genetics/158.3.973.
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Abstract Ssy1p and Ptr3p are components of the yeast plasma membrane SPS amino acid sensor. In response to extracellular amino acids this sensor initiates metabolic signals that ultimately regulate the functional expression of several amino acid-metabolizing enzymes and amino acid permeases (AAPs). As a result of diminished leucine uptake capabilities, ssy1Δ leu2 and ptr3Δ leu2 mutant strains are unable to grow on synthetic complete medium (SC). Genes affecting the functional expression of AAPs were identified by selecting spontaneous suppressing mutations in amino acid sensor-independent (ASI) genes that restore growth on SC. The suppressors define 11 recessive (asi) complementation groups and 5 dominant (ASI) linkage groups. Strains with mutations in genes assigned to these 16 groups fall into two phenotypic classes. Mutations in the class I genes (ASI1, ASI2, ASI3, TUP1, SSN6, ASI13) derepress the transcription of AAP genes. ASI1, ASI2, and ASI3 encode novel membrane proteins, and Asi1p and Asi3p are homologous proteins that have conserved ubiquitin ligase-like RING domains at their extreme C termini. Several of the class II genes (DOA4, UBA1, BRO1, BUL1, RSP5, VPS20, VPS36) encode proteins implicated in controlling aspects of post-Golgi endosomal-vacuolar protein sorting. The results from genetic and phenotypic analysis indicate that SPS sensor-initiated signals function positively to facilitate amino acid uptake and that two independent ubiquitin-mediated processes negatively modulate amino acid uptake.
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Shor, Erika, Rocio Garcia-Rubio, Lucius DeGregorio, and David S. Perlin. "A Noncanonical DNA Damage Checkpoint Response in a Major Fungal Pathogen." mBio 11, no. 6 (December 15, 2020): e03044-20. http://dx.doi.org/10.1128/mbio.03044-20.
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ABSTRACTDNA damage checkpoints are key guardians of genome integrity. Eukaryotic cells respond to DNA damage by triggering extensive phosphorylation of Rad53/CHK2 effector kinase, whereupon activated Rad53/CHK2 mediates further aspects of checkpoint activation, including cell cycle arrest and transcriptional changes. Budding yeast Candida glabrata, closely related to model eukaryote Saccharomyces cerevisiae, is an opportunistic pathogen characterized by high genetic diversity and rapid emergence of drug-resistant mutants. However, the mechanisms underlying this genetic variability are unclear. We used Western blotting and mass spectrometry to show that, unlike S. cerevisiae, C. glabrata cells exposed to DNA damage did not induce C. glabrata Rad53 (CgRad53) phosphorylation. Furthermore, flow cytometry analysis showed that, unlike S. cerevisiae, C. glabrata cells did not accumulate in S phase upon DNA damage. Consistent with these observations, time-lapse microscopy showed C. glabrata cells continuing to divide in the presence of DNA damage, resulting in mitotic errors and cell death. Finally, transcriptome sequencing (RNAseq) analysis revealed transcriptional rewiring of the DNA damage response in C. glabrata and identified several key protectors of genome stability upregulated by DNA damage in S. cerevisiae but downregulated in C. glabrata, including proliferating cell nuclear antigen (PCNA). Together, our results reveal a noncanonical fungal DNA damage response in C. glabrata, which may contribute to rapidly generating genetic change and drug resistance.IMPORTANCE In order to preserve genome integrity, all cells must mount appropriate responses to DNA damage, including slowing down or arresting the cell cycle to give the cells time to repair the damage and changing gene expression, for example to induce genes involved in DNA repair. The Rad53 protein kinase is a conserved central mediator of these responses in eukaryotic cells, and its extensive phosphorylation upon DNA damage is necessary for its activation and subsequent activity. Interestingly, here we show that in the opportunistic fungal pathogen Candida glabrata, Rad53 phosphorylation is not induced by DNA damage, nor do these cells arrest in S phase under these conditions, in contrast to the closely related yeast Saccharomyces cerevisiae. Instead, C. glabrata cells continue to divide in the presence of DNA damage, resulting in significant cell lethality. Finally, we show that a number of genes involved in DNA repair are strongly induced by DNA damage in S. cerevisiae but repressed in C. glabrata. Together, these findings shed new light on mechanisms regulating genome stability in fungal pathogens.
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Giannattasio, Michele, and Dana Branzei. "DNA Replication Through Strand Displacement During Lagging Strand DNA Synthesis in Saccharomyces cerevisiae." Genes 10, no. 2 (February 21, 2019): 167. http://dx.doi.org/10.3390/genes10020167.
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This review discusses a set of experimental results that support the existence of extended strand displacement events during budding yeast lagging strand DNA synthesis. Starting from introducing the mechanisms and factors involved in leading and lagging strand DNA synthesis and some aspects of the architecture of the eukaryotic replisome, we discuss studies on bacterial, bacteriophage and viral DNA polymerases with potent strand displacement activities. We describe proposed pathways of Okazaki fragment processing via short and long flaps, with a focus on experimental results obtained in Saccharomyces cerevisiae that suggest the existence of frequent and extended strand displacement events during eukaryotic lagging strand DNA synthesis, and comment on their implications for genome integrity.
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Blackburn, Alexandra S., and Simon V. Avery. "Genome-Wide Screening of Saccharomyces cerevisiae To Identify Genes Required for Antibiotic Insusceptibility of Eukaryotes." Antimicrobial Agents and Chemotherapy 47, no. 2 (February 2003): 676–81. http://dx.doi.org/10.1128/aac.47.2.676-681.2003.
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ABSTRACT The adverse reactions provoked by many antibiotics in humans are well documented but are generally poorly understood at the molecular level. To elucidate potential genetic defects that could give rise to susceptibility to prokaryote-specific antibiotics in eukaryotes, we undertook genome-wide screens using the yeast Saccharomyces cerevisiae as a model of eukaryotes; our previous work with a small number of yeast mutants revealed some specific gene functions required for oxytetracycline resistance. Here, the complete yeast deletion strain collection was tested for growth in the presence of a range of antibiotics. The sensitivities of mutants revealed by these screens were validated in independent tests. None of the ∼4,800 defined deletion strains tested were found to be sensitive to amoxicillin, penicillin G, rifampin, or vancomycin. However, two of the yeast mutants were tetracycline sensitive and four were oxytetracycline sensitive; encompassed among the latter were mutants carrying deletions in the same genes that we had characterized previously. Seventeen deletion strains were found to exhibit growth defects in the presence of gentamicin, with MICs for the strains being as low as 32 μg ml−1 (the wild type exhibited no growth defects at any gentamicin concentration tested up to 512 μg ml−1). Strikingly, 11 of the strains that were most sensitive to gentamicin carried deletions in genes whose products are all involved in various aspects of vacuolar and Golgi complex (or endoplasmic reticulum) function. Therefore, these and analogous organelles, which are also the principal sites of gentamicin localization in human cells, appear to be essential for normal resistance to gentamicin in eukaryotes. The approach and data described here offer a new route to gaining insight into the potential genetic bases of antibiotic insusceptibilities in eukaryotes.
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Griffith, Jacqulyn L., Laura E. Coleman, Adam S. Raymond, Summer G. Goodson, William S. Pittard, Circe Tsui, and Scott E. Devine. "Functional Genomics Reveals Relationships Between the Retrovirus-Like Ty1 Element and Its Host Saccharomyces cerevisiae." Genetics 164, no. 3 (July 1, 2003): 867–79. http://dx.doi.org/10.1093/genetics/164.3.867.
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Abstract Retroviruses and their relatives, the long terminal repeat (LTR) retrotransposons, carry out complex life cycles within the cells of their hosts. We have exploited a collection of gene deletion mutants developed by the Saccharomyces Genome Deletion Project to perform a functional genomics screen for host factors that influence the retrovirus-like Ty1 element in yeast. A total of 101 genes that presumably influence many different aspects of the Ty1 retrotransposition cycle were identified from our analysis of 4483 homozygous diploid deletion strains. Of the 101 identified mutants, 46 had significantly altered levels of Ty1 cDNA, whereas the remaining 55 mutants had normal levels of Ty1 cDNA. Thus, approximately half of the mutants apparently affected the early stages of retrotransposition leading up to the assembly of virus-like particles and cDNA replication, whereas the remaining half affected steps that occur after cDNA replication. Although most of the mutants retained the ability to target Ty1 integration to tRNA genes, 2 mutants had reduced levels of tRNA gene targeting. Over 25% of the gene products identified in this study were conserved in other organisms, suggesting that this collection of host factors can serve as a starting point for identifying host factors that influence LTR retroelements and retroviruses in other organisms. Overall, our data indicate that Ty1 requires a large number of cellular host factors to complete its retrotransposition cycle efficiently.
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Xu, Q., G. C. Johnston, and R. A. Singer. "The Saccharomyces cerevisiae Cdc68 transcription activator is antagonized by San1, a protein implicated in transcriptional silencing." Molecular and Cellular Biology 13, no. 12 (December 1993): 7553–65. http://dx.doi.org/10.1128/mcb.13.12.7553.
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The CDC68 gene (also called SPT16) encodes a transcription factor for the expression of a diverse set of genes in the budding yeast Saccharomyces cerevisiae. To identify other proteins that are functionally related to the Cdc68 protein, we searched for genetic suppressors of a cdc68 mutation. Four suppressor genes in which mutations reverse the temperature sensitivity imposed by the cdc68-1 mutation were found. We show here that one of the suppressor genes is the previously reported SAN1 gene; san1 mutations were originally identified as suppressors of a sir4 mutation, implicated in the chromatin-mediated transcriptional silencing of the two mating-type loci HML and HMR. Each san1 mutation, including a san1 null allele, reversed all aspects of the cdc68 mutant phenotype. Conversely, increased copy number of the wild-type SAN1 gene lowered the restrictive temperature for the cdc68-1 mutation. Our findings suggest that the San1 protein antagonizes the transcriptional activator function of the Cdc68 protein. The identification of san1 mutations as suppressors of cdc68 mutations suggests a role for Cdc68 in chromatin structure.
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Xu, Q., G. C. Johnston, and R. A. Singer. "The Saccharomyces cerevisiae Cdc68 transcription activator is antagonized by San1, a protein implicated in transcriptional silencing." Molecular and Cellular Biology 13, no. 12 (December 1993): 7553–65. http://dx.doi.org/10.1128/mcb.13.12.7553-7565.1993.
Full textAbstract:
The CDC68 gene (also called SPT16) encodes a transcription factor for the expression of a diverse set of genes in the budding yeast Saccharomyces cerevisiae. To identify other proteins that are functionally related to the Cdc68 protein, we searched for genetic suppressors of a cdc68 mutation. Four suppressor genes in which mutations reverse the temperature sensitivity imposed by the cdc68-1 mutation were found. We show here that one of the suppressor genes is the previously reported SAN1 gene; san1 mutations were originally identified as suppressors of a sir4 mutation, implicated in the chromatin-mediated transcriptional silencing of the two mating-type loci HML and HMR. Each san1 mutation, including a san1 null allele, reversed all aspects of the cdc68 mutant phenotype. Conversely, increased copy number of the wild-type SAN1 gene lowered the restrictive temperature for the cdc68-1 mutation. Our findings suggest that the San1 protein antagonizes the transcriptional activator function of the Cdc68 protein. The identification of san1 mutations as suppressors of cdc68 mutations suggests a role for Cdc68 in chromatin structure.
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Aronova, Sofia, Karen Wedaman, Scott Anderson, John Yates, and Ted Powers. "Probing the Membrane Environment of the TOR Kinases Reveals Functional Interactions between TORC1, Actin, and Membrane Trafficking in Saccharomyces cerevisiae." Molecular Biology of the Cell 18, no. 8 (August 2007): 2779–94. http://dx.doi.org/10.1091/mbc.e07-03-0274.
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The TOR kinases are regulators of growth in eukaryotic cells that assemble into two distinct protein complexes, TORC1 and TORC2, where TORC1 is inhibited by the antibiotic rapamycin. Present models favor a view wherein TORC1 regulates cell mass accumulation, and TORC2 regulates spatial aspects of growth, including organization of the actin cytoskeleton. Here, we demonstrate that in yeast both TORC1 and TORC2 fractionate with a novel form of detergent-resistant membranes that are distinct from detergent-resistant plasma membrane “rafts.” Proteomic analysis of these TOR-associated membranes revealed the presence of regulators of endocytosis and the actin cytoskeleton. Genetic analyses revealed a significant number of interactions between these components and TORC1, demonstrating a functional link between TORC1 and actin/endocytosis-related genes. Moreover, we found that inhibition of TORC1 by rapamycin 1) disrupted actin polarization, 2) delayed actin repolarization after glucose starvation, and 3) delayed accumulation of lucifer yellow within the vacuole. By combining our genetic results with database mining, we constructed a map of interactions that led to the identification of additional genetic interactions between TORC1 and components involved in membrane trafficking. Together, these results reveal the broad scope of cellular processes influenced by TORC1, and they underscore the functional overlap between TORC1 and TORC2.
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Reim, Natalia I., James Chuang, Dhawal Jain, Burak H. Alver, Peter J. Park, and Fred Winston. "The conserved elongation factor Spn1 is required for normal transcription, histone modifications, and splicing in Saccharomyces cerevisiae." Nucleic Acids Research 48, no. 18 (September 17, 2020): 10241–58. http://dx.doi.org/10.1093/nar/gkaa745.
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Abstract Spn1/Iws1 is a conserved protein involved in transcription and chromatin dynamics, yet its general in vivo requirement for these functions is unknown. Using a Spn1 depletion system in Saccharomyces cerevisiae, we demonstrate that Spn1 broadly influences several aspects of gene expression on a genome-wide scale. We show that Spn1 is globally required for normal mRNA levels and for normal splicing of ribosomal protein transcripts. Furthermore, Spn1 maintains the localization of H3K36 and H3K4 methylation across the genome and is required for normal histone levels at highly expressed genes. Finally, we show that the association of Spn1 with the transcription machinery is strongly dependent on its binding partner, Spt6, while the association of Spt6 and Set2 with transcribed regions is partially dependent on Spn1. Taken together, our results show that Spn1 affects multiple aspects of gene expression and provide additional evidence that it functions as a histone chaperone in vivo.
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Hoyt, M. A., L. He, L. Totis, and W. S. Saunders. "Loss of function of Saccharomyces cerevisiae kinesin-related CIN8 and KIP1 is suppressed by KAR3 motor domain mutations." Genetics 135, no. 1 (September 1, 1993): 35–44. http://dx.doi.org/10.1093/genetics/135.1.35.
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Abstract The kinesin-related products of the CIN8 and KIP1 genes of Saccharomyces cerevisiae redundantly perform an essential function in mitosis. The action of either gene-product is required for an outwardly directed force that acts upon the spindle poles. We have selected mutations that suppress the temperature-sensitivity of a cin8-temperature-sensitive kip1-delta strain. The extragenic suppressors analyzed were all found to be alleles of the KAR3 gene. KAR3 encodes a distinct kinesin-related protein whose action antagonizes Cin8p/Kip1p function. All seven alleles analyzed were altered within the region of KAR3 that encodes the putative force-generating (or "motor") domain. These mutations also suppressed the inviability associated with the cin8-delta kip1-delta genotype, a property not shared by a deletion of KAR3. Other properties of the suppressing alleles revealed that they were not null for function. Six of the seven were unaffected for the essential karyogamy and meiosis properties of KAR3 and the seventh was dominant for the suppressing trait. Our findings suggest that despite an antagonistic relationship between Cin8p/Kip1p and Kar3p, aspects of their mitotic roles may be similar.
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Schrick, Kathrin, Barbara Garvik, and Leland H. Hartwell. "Mating in Saccharomyces cerevisiae: The Role of the Pheromone Signal Transduction Pathway in the Chemotropic Response to Pheromone." Genetics 147, no. 1 (September 1, 1997): 19–32. http://dx.doi.org/10.1093/genetics/147.1.19.
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Abstract The mating process in yeast has two distinct aspects. One is the induction and activation of proteins required for cell fusion in response to a pheromone signal; the other is chemotropism, i.e., detection of a pheromone gradient and construction of a fusion site available to the signaling cell. To determine whether components of the signal transduction pathway necessary for transcriptional activation also play a role in chemotropism, we examined strains with null mutations in components of the signal transduction pathway for diploid formation, prezygote formation and the chemotropic process of mating partner discrimination when transcription was induced downstream of the mutation. Cells mutant for components of the mitogen-activated protein (MAP) kinase cascade (ste5, ste20, ste11, ste7 or fus3 kss1) formed diploids at a frequency 1% that of the wild-type control, but formed prezygotes as efficiently as the wild-type control and showed good mating partner discrimination, suggesting that the MAP kinase cascade is not essential for chemotropism. In contrast, cells mutant for the receptor (ste2) or the β or γ subunit (ste4 and stel8) of the G protein were extremely defective in both diploid and prezygote formation and discriminated poorly between signaling and nonsignaling mating partners, implying that these components are important for chemotropism.
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Ydenberg, Casey A., Richard A. Stein, and Mark D. Rose. "Cdc42p and Fus2p act together late in yeast cell fusion." Molecular Biology of the Cell 23, no. 7 (April 2012): 1208–18. http://dx.doi.org/10.1091/mbc.e11-08-0723.
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Cell fusion is the key event of fertilization that gives rise to the diploid zygote and is a nearly universal aspect of eukaryotic biology. In the yeast Saccharomyces cerevisiae, several mutants have been identified that are defective for cell fusion, and yet the molecular mechanism of this process remains obscure. One obstacle has been that genetic screens have mainly focused on mating-specific factors, whereas the process likely involves housekeeping proteins as well. Here we implicate Cdc42p, an essential protein with roles in multiple aspects of morphogenesis, as a core component of the yeast cell fusion pathway. We identify a point mutant in the Rho-insert domain of CDC42, called cdc42-138, which is specifically defective in cell fusion. The cell fusion defect is not a secondary consequence of ineffective signaling or polarization. Genetic and morphological data show that Cdc42p acts at a late stage in cell fusion in concert with a key cell fusion regulator, Fus2p, which contains a Dbl-homology domain. We find that Fus2p binds specifically with activated Cdc42p, and binding is blocked by the cdc42-138 mutation. Thus, in addition to signaling and morphogenetic roles in mating, Cdc42p plays a role late in cell fusion via activation of Fus2p.
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de la Cruz, Jesús, Thierry Lacombe, Olivier Deloche, Patrick Linder, and Dieter Kressler. "The Putative RNA Helicase Dbp6p Functionally Interacts With Rpl3p, Nop8p and the Novel trans-acting Factor Rsa3p During Biogenesis of 60S Ribosomal Subunits in Saccharomyces cerevisiae." Genetics 166, no. 4 (April 1, 2004): 1687–99. http://dx.doi.org/10.1093/genetics/166.4.1687.
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Abstract Ribosome biogenesis requires at least 18 putative ATP-dependent RNA helicases in Saccharomyces cerevisiae. To explore the functional environment of one of these putative RNA helicases, Dbp6p, we have performed a synthetic lethal screen with dbp6 alleles. We have previously characterized the nonessential Rsa1p, whose null allele is synthetically lethal with dbp6 alleles. Here, we report on the characterization of the four remaining synthetic lethal mutants, which reveals that Dbp6p also functionally interacts with Rpl3p, Nop8p, and the so-far-uncharacterized Rsa3p (ribosome assembly 3). The nonessential Rsa3p is a predominantly nucleolar protein required for optimal biogenesis of 60S ribosomal subunits. Both Dbp6p and Rsa3p are associated with complexes that most likely correspond to early pre-60S ribosomal particles. Moreover, Rsa3p is co-immunoprecipitated with protA-tagged Dbp6p under low salt conditions. In addition, we have established a synthetic interaction network among factors involved in different aspects of 60S-ribosomal-subunit biogenesis. This extensive genetic analysis reveals that the rsa3 null mutant displays some specificity by being synthetically lethal with dbp6 alleles and by showing some synthetic enhancement with the nop8-101 and the rsa1 null allele.
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Cleves, A. E., P. J. Novick, and V. A. Bankaitis. "Mutations in the SAC1 gene suppress defects in yeast Golgi and yeast actin function." Journal of Cell Biology 109, no. 6 (December 1, 1989): 2939–50. http://dx.doi.org/10.1083/jcb.109.6.2939.
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The budding mode of Saccharomyces cerevisiae cell growth demands that a high degree of secretory polarity be established and directed toward the emerging bud. We report here our demonstration that mutations in SAC1, a gene identified by virtue of its allele-specific genetic interactions with yeast actin defects, were also capable of suppressing sec14 lethalities associated with yeast Golgi defects. Moreover, these sac1 suppressor properties also extended to sec6 and sec9 secretory vesicle defects. The genetic data are consistent with the notion that SAC1p modulates both secretory pathway and actin cytoskeleton function. On this basis, we suggest that SAC1p may represent one aspect of the mechanism whereby secretory and cytoskeletal activities are coordinated, so that proper spatial regulation of secretion might be achieved.
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Styles, Erin, Ji-Young Youn, Mojca Mattiazzi Usaj, and Brenda Andrews. "Functional genomics in the study of yeast cell polarity: moving in the right direction." Philosophical Transactions of the Royal Society B: Biological Sciences 368, no. 1629 (November 5, 2013): 20130118. http://dx.doi.org/10.1098/rstb.2013.0118.
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The budding yeast Saccharomyces cerevisiae has been used extensively for the study of cell polarity, owing to both its experimental tractability and the high conservation of cell polarity and other basic biological processes among eukaryotes. The budding yeast has also served as a pioneer model organism for virtually all genome-scale approaches, including functional genomics, which aims to define gene function and biological pathways systematically through the analysis of high-throughput experimental data. Here, we outline the contributions of functional genomics and high-throughput methodologies to the study of cell polarity in the budding yeast. We integrate data from published genetic screens that use a variety of functional genomics approaches to query different aspects of polarity. Our integrated dataset is enriched for polarity processes, as well as some processes that are not intrinsically linked to cell polarity, and may provide new areas for future study.
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Vyas, Valmik K., Sergei Kuchin, Cristin D. Berkey, and Marian Carlson. "Snf1 Kinases with Different β-Subunit Isoforms Play Distinct Roles in Regulating Haploid Invasive Growth." Molecular and Cellular Biology 23, no. 4 (February 15, 2003): 1341–48. http://dx.doi.org/10.1128/mcb.23.4.1341-1348.2003.
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ABSTRACT The Snf1 protein kinase of Saccharomyces cerevisiae has been shown to have a role in regulating haploid invasive growth in response to glucose depletion. Cells contain three forms of the Snf1 kinase, each with a different β-subunit isoform, either Gal83, Sip1, or Sip2. We present evidence that different Snf1 kinases play distinct roles in two aspects of invasive growth, namely, adherence to the agar substrate and filamentation. The Snf1-Gal83 form of the kinase is required for adherence, whereas either Snf1-Gal83 or Snf1-Sip2 is sufficient for filamentation. Genetic evidence indicates that Snf1-Gal83 affects adherence by antagonizing Nrg1- and Nrg2-mediated repression of the FLO11 flocculin and adhesin gene. In contrast, the mechanism(s) by which Snf1-Gal83 and Snf1-Sip2 affect filamentation is independent of FLO11. Thus, the Snf1 kinase regulates invasive growth by at least two distinct mechanisms.
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Nickens, Sausen, and Bochman. "The Biochemical Activities of the Saccharomyces cerevisiae Pif1 Helicase Are Regulated by Its N-Terminal Domain." Genes 10, no. 6 (May 28, 2019): 411. http://dx.doi.org/10.3390/genes10060411.
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: Pif1 family helicases represent a highly conserved class of enzymes involved in multiple aspects of genome maintenance. Many Pif1 helicases are multi-domain proteins, but the functions of their non-helicase domains are poorly understood. Here, we characterized how the N-terminal domain (NTD) of the Saccharomyces cerevisiae Pif1 helicase affects its functions both in vivo and in vitro. Removal of the Pif1 NTD alleviated the toxicity associated with Pif1 overexpression in yeast. Biochemically, the N-terminally truncated Pif1 (Pif1ΔN) retained in vitro DNA binding, DNA unwinding, and telomerase regulation activities, but these activities differed markedly from those displayed by full-length recombinant Pif1. However, Pif1ΔN was still able to synergize with the Hrq1 helicase to inhibit telomerase activity in vitro, similar to full-length Pif1. These data impact our understanding of Pif1 helicase evolution and the roles of these enzymes in the maintenance of genome integrity.
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Fernandes, Ticiana, Flávia Silva-Sousa, Fábio Pereira, Teresa Rito, Pedro Soares, Ricardo Franco-Duarte, and Maria João Sousa. "Biotechnological Importance of Torulaspora delbrueckii: From the Obscurity to the Spotlight." Journal of Fungi 7, no. 9 (August 30, 2021): 712. http://dx.doi.org/10.3390/jof7090712.
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Torulaspora delbrueckii has attracted interest in recent years, especially due to its biotechnological potential, arising from its flavor- and aroma-enhancing properties when used in wine, beer or bread dough fermentation, as well as from its remarkable resistance to osmotic and freezing stresses. In the present review, genomic, biochemical, and phenotypic features of T. delbrueckii are described, comparing them with other species, particularly with the biotechnologically well-established yeast, Saccharomyces cerevisiae. We conclude about the aspects that make this yeast a promising biotechnological model to be exploited in a wide range of industries, particularly in wine and bakery. A phylogenetic analysis was also performed, using the core proteome of T. delbrueckii, to compare the number of homologous proteins relative to the most closely related species, understanding the phylogenetic placement of this species with robust support. Lastly, the genetic tools available for T. delbrueckii improvement are discussed, focusing on adaptive laboratorial evolution and its potential.
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White, Peter J., Rhona H. Borts, and Mark C. Hirst. "Stability of the Human Fragile X (CGG)n Triplet Repeat Array inSaccharomyces cerevisiae Deficient in Aspects of DNA Metabolism." Molecular and Cellular Biology 19, no. 8 (August 1, 1999): 5675–84. http://dx.doi.org/10.1128/mcb.19.8.5675.
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ABSTRACT Expanded trinucleotide repeats underlie a growing number of human diseases. The human FMR1 (CGG) n array can exhibit genetic instability characterized by progressive expansion over several generations leading to gene silencing and the development of the fragile X syndrome. While expansion is dependent upon the length of uninterrupted (CGG) n , instability occurs in a limited germ line and early developmental window, suggesting that lineage-specific expression of other factors determines the cellular environment permissive for expansion. To identify these factors, we have established normal- and premutation-length human FMR1 (CGG) n arrays in the yeast Saccharomyces cerevisiae and assessed the frequency of length changes greater than 5 triplets in cells deficient in various DNA repair and replication functions. In contrast to previous studies withEscherichia coli, we observed a low frequency of orientation-dependent large expansions in arrays carrying long uninterrupted (CGG) n arrays in a wild-type background. This frequency was unaffected by deletion of several DNA mismatch repair genes or deletion of the EXO1 andDIN7 genes and was not enhanced through meiosis in a wild-type background. Array contraction occurred in an orientation-dependent manner in most mutant backgrounds, but loss of the Sgs1p resulted in a generalized increase in array stability in both orientations. In contrast, FMR1 arrays had a 10-fold-elevated frequency of expansion in a rad27 background, providing evidence for a role in lagging-strand Okazaki fragment processing in (CGG) n triplet repeat expansion.
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Longhese, M. P., L. Jovine, P. Plevani, and G. Lucchini. "Conditional mutations in the yeast DNA primase genes affect different aspects of DNA metabolism and interactions in the DNA polymerase alpha-primase complex." Genetics 133, no. 2 (February 1, 1993): 183–91. http://dx.doi.org/10.1093/genetics/133.2.183.
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Abstract Different pri1 and pri2 conditional mutants of Saccharomyces cerevisiae altered, respectively, in the small (p48) and large (p58) subunits of DNA primase, show an enhanced rate of both mitotic intrachromosomal recombination and spontaneous mutation, to an extent which is correlated with the severity of their defects in cell growth and DNA synthesis. These effects might be attributable to the formation of nicked and gapped DNA molecules that are substrates for recombination and error-prone repair, due to defective DNA replication in the primase mutants. Furthermore, pri1 and pri2 mutations inhibit sporulation and affect spore viability, with the unsporulated mutant cells arresting with a single nucleus, suggesting that DNA primase plays a critical role during meiosis. The observation that all possible pairwise combinations of two pri1 and two pri2 alleles are lethal provides further evidence for direct interaction of the primase subunits in vivo. Immunopurification and immunoprecipitation studies on wild-type and mutant strains suggest that the small subunit has a major role in determining primase activity, whereas the large subunit directly interacts with DNA polymerase alpha, and either mediates or stabilizes association of the p48 polypeptide in the DNA polymerase alpha-primase complex.
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Kim, Jae-Hong, Md Habibur Rahman, Donghwi Park, Myungjin Jo, Hyung-Jun Kim, and Kyoungho Suk. "Identification of Genetic Modifiers of TDP-43: Inflammatory Activation of Astrocytes for Neuroinflammation." Cells 10, no. 3 (March 18, 2021): 676. http://dx.doi.org/10.3390/cells10030676.
Full textAbstract:
Transactive response DNA-binding protein 43 (TDP-43) is a ubiquitously expressed DNA/RNA-binding protein linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP-43 has been implicated in numerous aspects of the mRNA life cycle, as well as in cell toxicity and neuroinflammation. In this study, we used the toxicity of the TDP-43 expression in Saccharomyces cerevisiae as an assay to identify TDP-43 genetic interactions. Specifically, we transformed human TDP-43 cDNAs of wild-type or disease-associated mutants (M337V and Q331K) en masse into 4653 homozygous diploid yeast deletion mutants and then used next-generation sequencing readouts of growth to identify yeast toxicity modifiers. Genetic interaction analysis provided a global view of TDP-43 pathways, some of which are known to be involved in cellular metabolic processes. Selected putative loci with the potential of genetic interactions with TDP-43 were assessed for associations with neurotoxicity and inflammatory activation of astrocytes. The pharmacological inhibition of succinate dehydrogenase flavoprotein subunit A (SDHA) and voltage-dependent anion-selective channel 3 (VDAC3) suppressed TDP-43-induced expression of proinflammatory cytokines in astrocytes, indicating the critical roles played by SDHA and VDAC3 in TDP-43 pathways during inflammatory activation of astrocytes and neuroinflammation. Thus, the findings of our TDP-43 genetic interaction screen provide a global landscape of TDP-43 pathways and may help improve our understanding of the roles of glia and neuroinflammation in ALS and FTD pathogenesis.
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Rossouw, Debra, Roberto Olivares-Hernandes, Jens Nielsen, and Florian F. Bauer. "Comparative Transcriptomic Approach To Investigate Differences in Wine Yeast Physiology and Metabolism during Fermentation." Applied and Environmental Microbiology 75, no. 20 (August 21, 2009): 6600–6612. http://dx.doi.org/10.1128/aem.01251-09.
Full textAbstract:
ABSTRACT Commercial wine yeast strains of the species Saccharomyces cerevisiae have been selected to satisfy many different, and sometimes highly specific, oenological requirements. As a consequence, more than 200 different strains with significantly diverging phenotypic traits are produced globally. This genetic resource has been rather neglected by the scientific community because industrial strains are less easily manipulated than the limited number of laboratory strains that have been successfully employed to investigate fundamental aspects of cellular biology. However, laboratory strains are unsuitable for the study of many phenotypes that are of significant scientific and industrial interest. Here, we investigate whether a comparative transcriptomics and phenomics approach, based on the analysis of five phenotypically diverging industrial wine yeast strains, can provide insights into the molecular networks that are responsible for the expression of such phenotypes. For this purpose, some oenologically relevant phenotypes, including resistance to various stresses, cell wall properties, and metabolite production of these strains were evaluated and aligned with transcriptomic data collected during alcoholic fermentation. The data reveal significant differences in gene regulation between the five strains. While the genetic complexity underlying the various successive stress responses in a dynamic system such as wine fermentation reveals the limits of the approach, many of the relevant differences in gene expression can be linked to specific phenotypic differences between the strains. This is, in particular, the case for many aspects of metabolic regulation. The comparative approach therefore opens new possibilities to investigate complex phenotypic traits on a molecular level.
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Foreman, P. K., and R. W. Davis. "Point mutations that separate the role of Saccharomyces cerevisiae centromere binding factor 1 in chromosome segregation from its role in transcriptional activation." Genetics 135, no. 2 (October 1, 1993): 287–96. http://dx.doi.org/10.1093/genetics/135.2.287.
Full textAbstract:
Abstract Centromere binding factor 1 (Cbf1p or CP1) binds to the CDEI region of Saccharomyces cerevisiae centromeres and is a member of the basic helix-loop-helix (bHLH) class of proteins. Deletion of the gene encoding Cbf1p results in an increased frequency of chromosome loss, hypersensitivity to low levels of microtubule disrupting drugs (such as thiabendazole and benomyl) and methionine auxotrophy. By polymerase chain reaction-based random mutagenesis of the CBF1 gene we have obtained a number of mutant alleles that make full-length protein with impaired function. The mutations in these alleles are clustered in or just downstream from the bHLH domain. Among the alleles obtained was a class that was more compromised for transcriptional activation and a class that was more compromised for chromosome loss and thiabendazole hypersensitivity. These results indicate that at least some aspects of the role of Cbf1p in chromosome segregation and transcriptional activation are distinct. In contrast, increased chromosome loss and thiabendazole hypersensitivity were not separated in any of the alleles, suggesting that these phenotypes reflect the same mechanistic defect. These observations are consistent with a model that suggests that one role of Cbf1p in chromosome segregation may be to improve the efficiency with which contact between the kinetochore and spindle microtubules is established or maintained.
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Hanlon, Sean E., David N. Norris, and Andrew K. Vershon. "Depletion of H2A-H2B Dimers in Saccharomyces cerevisiae Triggers Meiotic Arrest by Reducing IME1 Expression and Activating the BUB2-Dependen Branch of the Spindle Checkpoint." Genetics 164, no. 4 (August 1, 2003): 1333–44. http://dx.doi.org/10.1093/genetics/164.4.1333.
Full textAbstract:
Abstract In the yeast Saccharomyces cerevisiae, diploid strains carrying homozygous hta1-htb1Δ mutations express histone H2A-H2B dimers at a lower level than do wild-type cells. Although this mutation has only minor effects on mitotic growth, it causes an arrest in sporulation prior to the first meiotic division. In this report, we show that the hta1-htb1Δ mutant exhibits reduced expression of early and middle-sporulation-specific genes and that the meiotic arrest of the hta1-htb1Δ mutant can be partially bypassed by overexpression of IME1. Additionally, deletions of BUB2 or BFA1, components of one branch of the spindle checkpoint pathway, bypass the meiotic arrest. Mutations in the other branch of the pathway or in the pachytene checkpoint are unable to suppress the meiotic block. These observations indicate that depletion of the H2A-H2B dimer blocks sporulation by at least two mechanisms: disruption of the expression of meiotic regulatory genes and activation of the spindle checkpoint. Our results show that the failure to progress through the meiotic pathway is not the result of global chromosomal alterations but that specific aspects of meiosis are sensitive to depletion of the H2A-H2B dimer.