Improving bgl1 gene expression in Saccharomyces cerevisiae through meiosis in an isogenic triploid.
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ABSTRACT:
SUBMITTER: Yang H
PROVIDER: S-EPMC4000627 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mutations accumulate during all stages of growth, but only germ line mutations contribute to evolution. While meiosis contributes to evolution by reassortment of parental alleles, we show here that the process itself is inherently mutagenic. We have previously shown that the DNA synthesis associated with repair of a double-strand break is about 1000-fold less accurate than S-phase synthesis. Since the process of meiosis involves many programmed DSBs, we reasoned that this repair might also be mutagenic. Indeed, in the early 1960's Magni and Von Borstel observed elevated reversion of recessive alleles during meiosis, and found that the revertants were more likely to be associated with a crossover than non-revertants, a process that they called "the meiotic effect." Here we use a forward mutation reporter (CAN1 HIS3) placed at either a meiotic recombination coldspot or hotspot near the MAT locus on Chromosome III. We find that the increased mutation rate at CAN1 (6 to 21 -fold) correlates with the underlying recombination rate at the locus. Importantly, we show that the elevated mutation rate is fully dependent upon Spo11, the protein that introduces the meiosis specific DSBs. To examine associated recombination we selected for random spores with or without a mutation in CAN1. We find that the mutations isolated this way show an increased association with recombination (crossovers, loss of crossover interference and/or increased gene conversion tracts). Pol? appears to contribute about half of the mutations induced during meiosis, but is not the only source of mutations for the meiotic effect. We see no difference in either the spectrum or distribution of mutations between mitosis and meiosis. The correlation of hotspots with elevated mutagenesis provides a mechanism for organisms to control evolution rates in a gene specific manner.
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Project description:Two signals are required for meiosis and spore formation in the yeast Saccharomyces cerevisiae: starvation and the MAT products a1 and alpha 2, which determine the a/alpha cell type. These signals lead to increased expression of the IME1 (inducer of meiosis) gene, which is required for sporulation and sporulation-specific gene expression. We report here the sequence of the IME1 gene and the consequences of IME1 expression from the GAL1 promoter. The deduced IME1 product is a 360-amino-acid protein with a tyrosine-rich C-terminal region. Expression of PGAL1-IME1 in vegetative a/alpha cells led to moderate accumulation of four early sporulation-specific transcripts (IME2, SPO11, SPO13, and HOP1); the transcripts accumulated 3- to 10-fold more after starvation. Two sporulation-specific transcripts normally expressed later (SPS1 and SPS2) did not accumulate until PGAL1-IME1 strains were starved, and the intact IME1 gene was not activated by PGAL1-IME1 expression. In a or alpha cells, which lack alpha 2 or a1, expression of PGAL1-IME1 led to the same pattern of IME2 and SPO13 expression as in a/alpha cells, as measured with ime2::lacZ and spo13::lacZ fusions. Thus, in wild-type strains, the increased expression of IME1 in starved a/alpha cells can account entirely for cell type control, but only partially for nutritional control, of early sporulation-specific gene expression. PGAL1-IME1 expression did not cause growing cells to sporulate but permitted efficient sporulation of amino acid-limited cells, which otherwise sporulated poorly. We suggest that IME1 acts primarily as a positive regulator of early sporulation-specific genes and that growth arrest is an independent prerequisite for execution of the sporulation program.
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Project description:Understanding the evolutionary forces that influence patterns of gene expression variation will provide insights into the mechanisms of evolutionary change and the molecular basis of phenotypic diversity. To date, studies of gene expression evolution have primarily been made by analyzing how gene expression levels vary within and between species. However, the fundamental unit of heritable variation in transcript abundance is the underlying regulatory allele, and as a result it is necessary to understand gene expression evolution at the level of DNA sequence variation. Here we describe the evolutionary forces shaping patterns of genetic variation for 1206 cis-regulatory QTL identified in a cross between two divergent strains of Saccharomyces cerevisiae. We demonstrate that purifying selection against mildly deleterious alleles is the dominant force governing cis-regulatory evolution in S. cerevisiae and estimate the strength of selection. We also find that essential genes and genes with larger codon bias are subject to slightly stronger cis-regulatory constraint and that positive selection has played a role in the evolution of major trans-acting QTL.
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Project description:DNA double-strand break (DSB) repair by homologous recombination (HR) uses a DNA template with similar sequence to restore genetic identity. Allelic DNA repair templates can be found on the sister chromatid or homologous chromosome. During meiotic recombination, DSBs preferentially repair from the homologous chromosome, with a proportion of HR events generating crossovers. Nevertheless, regions of similar DNA sequence exist throughout the genome, providing potential DNA repair templates. When DSB repair occurs at these non-allelic loci (termed ectopic recombination), chromosomal duplications, deletions and rearrangements can arise. Here, we characterize in detail ectopic recombination arising between a dispersed pair of inverted repeats in wild-type Saccharomyces cerevisiae at both a local and a chromosomal scale-the latter identified via gross chromosomal acentric and dicentric chromosome rearrangements. Mutation of the DNA damage checkpoint clamp loader Rad24 and the RecQ helicase Sgs1 causes an increase in ectopic recombination. Unexpectedly, additional mutation of the RecA orthologues Rad51 and Dmc1 alters-but does not abolish-the type of ectopic recombinants generated, revealing a novel class of inverted chromosomal rearrangement driven by the single-strand annealing pathway. These data provide important insights into the role of key DNA repair proteins in regulating DNA repair pathway and template choice during meiosis.
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Project description:Gene expression noise is an evolvable property of biological systems that describes differences in expression among genetically identical cells in the same environment. Prior work has shown that expression noise is heritable and can be shaped by selection, but the impact of variation in expression noise on organismal fitness has proven difficult to measure. Here, we quantify the fitness effects of altering expression noise for the TDH3 gene in Saccharomyces cerevisiae. We show that increases in expression noise can be deleterious or beneficial depending on the difference between the average expression level of a genotype and the expression level maximizing fitness. We also show that a simple model relating single-cell expression levels to population growth produces patterns consistent with our empirical data. We use this model to explore a broad range of average expression levels and expression noise, providing additional insight into the fitness effects of variation in expression noise.
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Project description:Translation initiation is a complex and highly regulated process that represents an important mechanism, controlling gene expression. eIF2A was proposed as an alternative initiation factor, however, its role and biological targets remain to be discovered. To further gain insight into the function of eIF2A in Saccharomyces cerevisiae, we identified mRNAs associated with the eIF2A complex and showed that 24% of the most enriched mRNAs encode proteins related to cell wall biogenesis and maintenance. In agreement with this result, we showed that an eIF2A deletion sensitized cells to cell wall damage induced by calcofluor white. eIF2A overexpression led to a growth defect, correlated with decreased synthesis of several cell wall proteins. In contrast, no changes were observed in the transcriptome, suggesting that eIF2A controls the expression of cell wall-related proteins at a translational level. The biochemical characterization of the eIF2A complex revealed that it strongly interacts with the RNA binding protein, Ssd1, which is a negative translational regulator, controlling the expression of cell wall-related genes. Interestingly, eIF2A and Ssd1 bind several common mRNA targets and we found that the binding of eIF2A to some targets was mediated by Ssd1. Surprisingly, we further showed that eIF2A is physically and functionally associated with the exonuclease Xrn1 and other mRNA degradation factors, suggesting an additional level of regulation. Altogether, our results highlight new aspects of this complex and redundant fine-tuned regulation of proteins expression related to the cell wall, a structure required to maintain cell shape and rigidity, providing protection against harmful environmental stress.
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Project description:Cyclophilin A is conserved from yeast to humans and mediates the ability of cyclosporine to perturb signal transduction cascades via inhibition of calcineurin. Cyclophilin A also catalyzes cis-trans peptidyl-prolyl isomerization during protein folding or conformational changes; however, cyclophilin A is not essential in yeast or human cells, and the true biological functions of this highly conserved enzyme have remained enigmatic. In Saccharomyces cerevisiae, cyclophilin A becomes essential in cells compromised for the nuclear prolyl-isomerase Ess1, and cyclophilin A physically interacts with two nuclear histone deacetylase complexes, Sin3-Rpd3 and Set3C, which both control meiosis. Here we show that cyclophilin A is localized to the nucleus in yeast cells and governs the meiotic gene program to promote efficient sporulation. The prolyl-isomerase activity of cyclophilin A is required for this meiotic function. We document that cyclophilin A physically associates with the Set3C histone deacetylase and analyze in detail the structure of this protein-protein complex. Genetic studies support a model in which cyclophilin A controls meiosis via Set3C and an additional target. Our findings reveal a novel nuclear role for cyclophilin A in governing the transcriptional program required for the vegetative to meiotic developmental switch in budding yeast.
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Project description:Meiotic recombination plays a key role in sexual reproduction as it generates crossovers that, in combination with sister chromatid cohesion, physically connect homologous chromosomes, thereby promoting their proper segregation at the first meiotic division. Meiotic recombination is initiated by programmed double strand breaks (DSBs) catalyzed by the evolutionarily conserved, topoisomerase-like protein Spo11. Repair of these DSBs is highly regulated to create crossovers between homologs that are distributed throughout the genome. This repair requires the presence of the mitotic recombinase, Rad51, as well as the strand exchange activity of the meiosis-specific recombinase, Dmc1. A key regulator of meiotic DSB repair in Saccharomyces cerevisiae is the meiosis-specific kinase Mek1, which promotes interhomolog strand invasion and is required for the meiotic recombination checkpoint and the crossover/noncrossover decision. Understanding how Mek1 regulates meiotic recombination requires the identification of its substrates. Towards that end, an unbiased phosphoproteomic approach utilizing Stable Isotope Labeling by Amino Acids in Cells (SILAC) was utilized to generate a list of potential Mek1 substrates, as well as proteins containing consensus phosphorylation sites for cyclin-dependent kinase, the checkpoint kinases, Mec1/Tel1, and the polo-like kinase, Cdc5. These experiments represent the first global phosphoproteomic dataset for proteins in meiotic budding yeast.
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Project description:We describe the identification of a new meiosis-specific gene of Saccharomyces cerevisiae, NDT80. The ndt80 null and point mutants arrest at the pachytene stage of meiosis, with homologs connected by full-length synaptonemal complexes and spindle pole bodies duplicated but unseparated. Meiotic recombination in an ndt80 delta mutant is relatively normal, although commitment to heteroallelic recombination is elevated two- to threefold and crossing over is decreased twofold compared with those of the wild type. ndt80 arrest is not alleviated by mutations in early recombination genes, e.g., SPO11 or RAD50, and thus cannot be attributed to an intermediate block in prophase chromosome metabolism like that observed in several other mutants. The ndt80 mutant phenotype during meiosis most closely resembles that of a cdc28 mutant, which contains a thermolabile p34, the catalytic subunit of maturation-promoting factor. Cloning and molecular analysis reveal that the NDT80 gene maps on the right arm of chromosome VIII between EPT1 and a Phe-tRNA gene, encodes a 627-amino-acid protein which exhibits no significant homology to other known proteins, and is transcribed specifically during middle meiotic prophase. The NDT80 gene product could be a component of the cell cycle regulatory machinery involved in the transition out of pachytene, a participant in an unknown aspect of meiosis sensed by a pachytene checkpoint, or a SPO11- and RAD50-independent component of meiotic chromosomes that is the target of cell cycle signaling.
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Project description:Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals and reduces phosphorus pollution of animal waste. Our objectives were to express an Aspergillus niger phytase gene (phyA) in Saccharomyces cerevisiae and to determine the effects of glycosylation on the phytase's activity and thermostability. A 1.4-kb DNA fragment containing the coding region of the phyA gene was inserted into the expression vector pYES2 and was expressed in S. cerevisiae as an active, extracellular phytase. The yield of total extracellular phytase activity was affected by the signal peptide and the medium composition. The expressed phytase had two pH optima (2 to 2.5 and 5 to 5.5) and a temperature optimum between 55 and 60 degrees C, and it cross-reacted with a rabbit polyclonal antibody against the wild-type enzyme. Due to the heavy glycosylation, the expressed phytase had a molecular size of approximately 120 kDa and appeared to be more thermostable than the commercial enzyme. Deglycosylation of the phytase resulted in losses of 9% of its activity and 40% of its thermostability. The recombinant phytase was effective in hydrolyzing phytate phosphorus from corn or soybean meal in vitro. In conclusion, the phyA gene was expressed as an active, extracellular phytase in S. cerevisiae, and its thermostability was affected by glycosylation.
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