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: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:Among the collection of chromatin modifications that influence its function and structure, the substitution of canonical histones by the so-called histone variants is one of the most prominent actions. Since crucial meiotic transactions are modulated by chromatin, here we investigate the functional contribution of the H2A.Z histone variant during both unperturbed meiosis and upon challenging conditions where the meiotic recombination checkpoint is triggered in budding yeast by the absence of the synaptonemal complex component Zip1 We have found that H2A.Z localizes to meiotic chromosomes in an SWR1-dependent manner. Although meiotic recombination is not substantially altered, the htz1 mutant (lacking H2A.Z) shows inefficient meiotic progression, impaired sporulation, and reduced spore viability. These phenotypes are likely accounted for by the misregulation of meiotic gene expression landscape observed in htz1 In the zip1 mutant, the absence of H2A.Z results in a tighter meiotic arrest imposed by the meiotic recombination checkpoint. We have found that Mec1-dependent Hop1-T318 phosphorylation and the ensuing Mek1 activation are not significantly altered in zip1 htz1; however, downstream checkpoint targets, such as the meiosis I-promoting factors Ndt80, Cdc5, and Clb1, are drastically downregulated. The study of the checkpoint response in zip1 htz1 has also allowed us to reveal the existence of an additional function of the Swe1 kinase, independent of CDK inhibitory phosphorylation, which is relevant to restrain meiotic cell cycle progression. In summary, our study shows that the H2A.Z histone variant impacts various aspects of meiotic development adding further insight into the relevance of chromatin dynamics for accurate gametogenesis.
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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: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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