scholarly journals SWI/SNF antagonizes SIR heterochromatin to promote transcription of genes expressed during mitotic exit in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Mayuri Rege ◽  
Jessica L. Feldman ◽  
Nicholas L. Adkins ◽  
Craig L. Peterson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Histone Deacetylases ◽  
Genome Stability ◽  
Transcriptional Profiling ◽  
Chromatin Remodelers ◽  
Heterochromatin Formation ◽  
Sir Proteins ◽  
Genes Encoding ◽  
Mutant Phenotypes

ABSTRACTHeterochromatin is a repressive, specialized chromatin structure that is central to eukaryotic transcriptional regulation and genome stability. In the budding yeast, Saccharomyces cerevisiae, heterochromatin formation requires Sir2p, Sir3p, and Sir4p, and these Sir proteins create specialized chromatin structures at telomeres and silent mating type loci. Previously, we reported that the SWI/SNF chromatin remodeling enzyme can evict Sir3 from chromatin fibers in vitro, though whether this activity contributes to the role of SWI/SNF as a transcriptional activator at euchromatic loci is unknown. Here, we characterize genetic interactions between the SIR genes (SIR2, SIR3, and SIR4) and genes encoding subunits of the chromatin remodelers SWI/SNF and INO80C, as well genes encoding the histone deacetylases Hst3 and Hst4. We find that loss of SIR genes partially rescues the growth defects of swi2, ino80, and hst3/hst4 mutants during replication stress conditions. Interestingly, partial suppression of swi2, ino80, and hst3 hst4 mutant phenotypes is due to the pseudo-diploid state of sir mutants, but a significant portion is due to more direct functional interactions. Consistent with this view, transcriptional profiling of strains lacking Swi2 or Sir3 identifies a set of genes whose expression in the M/G1 phase of the cell cycle requires SWI/SNF to antagonize the repressive impact of Sir3.

Dominant effects of tubulin overexpression in Saccharomyces cerevisiae

1989 ◽  
Vol 9 (3) ◽  
pp. 1049-1059
Author(s):  
D Burke ◽  
P Gasdaska ◽  
L Hartwell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Loss ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Inducible Promoters ◽  
Selective Degradation ◽  
Novel Structure ◽  
Genes Encoding ◽  
Transient Overexpression ◽  
Mutant Phenotypes

The consequences of altering the levels of alpha- and beta-tubulin in Saccharomyces cerevisiae were examined by constructing fusions of the structural genes encoding the tubulins to strong galactose-inducible promoters. Overexpression of beta-tubulin (TUB2) was lethal: cells arrested in the G2 stage of the cell cycle exhibited an increased frequency of chromosome loss, were devoid of microtubules, and accumulated beta-tubulin in a novel structure. Overexpression of the major alpha-tubulin gene (TUB1) was not lethal and did not affect chromosome segregation. The rate of alpha-tubulin mRNA and protein synthesis was increased, but the protein did not accumulate. Overexpression of both alpha- and beta-tubulin together resulted in arrested cell division, and cells accumulated excess tubules that contained both alpha- and beta-tubulin. Transient overexpression of both tubulins resulted in a high frequency of chromosome loss. These data suggest that strong selective pressure exists to prevent excess accumulation of microtubules or beta-tubulin and suggest a model by which this goal may be achieved by selective degradation of unassembled alpha-tubulin. Furthermore, the phenotype of beta-tubulin overexpression is similar to the phenotype of a beta-tubulin deficiency. These results add to a number of recent studies demonstrating that mutant phenotypes generated by overexpression can be informative about the function of the gene product.

scholarly journals Identification of RIFL, a novel adipocyte-enriched insulin target gene with a role in lipid metabolism

2012 ◽  
Vol 303 (3) ◽  
pp. E334-E351 ◽  
Author(s):  
Gang Ren ◽  
Ji Young Kim ◽  
Cynthia M. Smas
Keyword(s):  
Lipid Metabolism ◽  
Transcriptional Profiling ◽  
Serum Triglyceride ◽  
Transcript Level ◽  
Wild Type ◽  
New Genes ◽  
Brown Adipose ◽  
Genes Encoding ◽  
Triglyceride Content

To identify new genes that are important in fat metabolism, we utilized the Lexicon-Genentech knockout database of genes encoding transmembrane and secreted factors and whole murine genome transcriptional profiling data that we generated for 3T3-L1 in vitro adipogenesis. Cross-referencing null models evidencing metabolic phenotypes with genes induced in adipogenesis led to identification of a new gene, which we named RIFL (refeeding induced fat and liver). RIFL-null mice have serum triglyceride levels approximately one-third of wild type. RIFL transcript is induced >100-fold during 3T3-L1 adipogenesis and is also increased markedly during adipogenesis of murine and human primary preadipocytes. siRNA-mediated knockdown of RIFL during 3T3-L1 adipogenesis results in an ∼35% decrease in adipocyte triglyceride content. Murine RIFL transcript is highly enriched in white and brown adipose tissue and liver. Fractionation of WAT reveals that RIFL transcript is exclusive to adipocytes with a lack of expression in stromal-vascular cells. Nutritional and hormonal studies are consistent with a prolipogenic function for RIFL. There is evidence of an approximately eightfold increase in RIFL transcript level in WAT in ob/ob mice compared with wild-type mice. RIFL transcript level in WAT and liver is increased ∼80- and 12-fold, respectively, following refeeding of fasted mice. Treatment of 3T3-L1 adipocytes with insulin increases RIFL transcript ≤35-fold, whereas agents that stimulate lipolysis downregulate RIFL. Interestingly, the 198-amino acid RIFL protein is predicted to be secreted and shows ∼30% overall conservation with the NH2-terminal half of angiopoietin-like 3, a liver-secreted protein that impacts lipid metabolism. In summary, our data suggest that RIFL is an important new regulator of lipid metabolism.

scholarly journals Control of Translocations between Highly Diverged Genes by Sgs1, the Saccharomyces cerevisiae Homolog of the Bloom'sSyndrome Protein

2006 ◽  
Vol 26 (14) ◽  
pp. 5406-5420 ◽  
Author(s):  
Kristina H. Schmidt ◽  
Joann Wu ◽  
Richard D. Kolodner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequences ◽  
Genome Stability ◽  
Dna Helicase ◽  
Telomere Maintenance ◽  
Template Switching ◽  
Checkpoint Kinase ◽  
Genes Encoding ◽  
Atm Protein ◽  
Break Induced Replication

ABSTRACT Sgs1 is a RecQ family DNA helicase required for genome stability in Saccharomyces cerevisiae whose human homologs BLM, WRN, and RECQL4 are mutated in Bloom's, Werner, and Rothmund Thomson syndromes, respectively. Sgs1 and mismatch repair (MMR) are inhibitors of recombination between similar but divergent (homeologous) DNA sequences. Here we show that SGS1, but not MMR, is critical for suppressing spontaneous, recurring translocations between diverged genes in cells with mutations in the genes encoding the checkpoint proteins Mec3, Rad24, Rad9, or Rfc5, the chromatin assembly factors Cac1 or Asf1, and the DNA helicase Rrm3. The S-phase checkpoint kinase and telomere maintenance factor Tel1, a homolog of the human ataxia telangiectasia (ATM) protein, prevents these translocations, whereas the checkpoint kinase Mec1, a homolog of the human ATM-related protein, and the Rad53 checkpoint kinase are not required. The translocation structures observed suggest involvement of a dicentric intermediate and break-induced replication with multiple cycles of DNA template switching.

scholarly journals Evidence that Spt2/Sin1, an HMG-Like Factor, Plays Roles in Transcription Elongation, Chromatin Structure, and Genome Stability in Saccharomyces cerevisiae

2006 ◽  
Vol 26 (4) ◽  
pp. 1496-1509 ◽  
Author(s):  
Amine Nourani ◽  
Francois Robert ◽  
Fred Winston
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genome Stability ◽  
Transcription Elongation ◽  
Coding Region ◽  
Elongation Factors ◽  
Chromatin Dynamics ◽  
Coding Regions ◽  
Genome Wide ◽  
Genes Encoding ◽  
Approaches To Study

ABSTRACT Spt2/Sin1 is a DNA binding protein with HMG-like domains that has been suggested to play a role in chromatin-mediated transcription in Saccharomyces cerevisiae. Previous studies have suggested models in which Spt2 plays an inhibitory role in the initiation of transcription of certain genes. In this work, we have taken several approaches to study Spt2 in greater detail. Our results have identified previously unknown genetic interactions between spt2Δ and mutations in genes encoding transcription elongation factors, including members of the PAF and HIR/HPC complexes. In addition, genome-wide and gene-specific chromatin immunoprecipitation analyses suggest that Spt2 is primarily associated with coding regions in a transcription-dependent fashion. Furthermore, our results show that Spt2, like other elongation factors, is required for the repression of transcription from a cryptic promoter within a coding region and that Spt2 is also required for repression of recombination within transcribed regions. Finally, we provide evidence that Spt2 plays a role in regulating the levels of histone H3 over transcribed regions. Taken together, our results suggest a direct link for Spt2 with transcription elongation, chromatin dynamics, and genome stability.

scholarly journals Elimination of ribosome inactivating factors improves the efficiency of Bacillus subtilis and Saccharomyces cerevisiae cell-free translational systems

2018 ◽  
Author(s):  
Tetiana Brodiazhenko ◽  
Marcus Johansson ◽  
Hiraku Takada ◽  
Tracy Nissan ◽  
Vasili Hauryliuk ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Bacillus Subtilis ◽  
Rna Degradation ◽  
Translation Regulation ◽  
Chain Complexes ◽  
Free Translation ◽  
Genes Encoding ◽  
Nascent Chain ◽  
Translation Systems

AbstractCell-free translational systems based on cellular lysates optimized for in vitro protein synthesis have multiple applications both in basic and applied science, ranging from studies of translation regulation to cell-free production of proteins and ribosome-nascent chain complexes. In order to achieve both high activity and reproducibility in a translational system, it is essential that the ribosomes in the cellular lysate are enzymatically active. Here we demonstrate that genomic disruption of genes encoding ribosome inactivating factors – HPF in Bacillus subtilis and Stm1 in Saccharomyces cerevisiae – robustly improve the activities of bacterial and yeast translational systems. A possible next step in developing strains for production of even more efficient cell-free translation systems could be achieved by combining a genomic disruption of the ribosome hibernation machinery with inactivation of the genes responsible for proteolysis and RNA degradation.

Ty element-induced temperature-sensitive mutations of Saccharomyces cerevisiae.

Genetics ◽  
1992 ◽  
Vol 131 (4) ◽  
pp. 821-832 ◽  
Author(s):  
K Kawakami ◽  
B K Shafer ◽  
D J Garfinkel ◽  
J N Strathern ◽  
Y Nakamura
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
High Temperatures ◽  
Insertional Mutagenesis ◽  
Trna Gene ◽  
Protein A ◽  
Temperature Sensitive ◽  
Mutant Phenotypes

Abstract Temperature-sensitive mutants of Saccharomyces cerevisiae were isolated by insertional mutagenesis using the HIS3 marked retrotransposon TyH3HIS3. In such mutants, the TyHIS3 insertions are expected to identify loci which encode genes essential for cell growth at high temperatures but dispensable at low temperatures. Five mutations were isolated and named hit for high temperature growth. The hit1-1 mutation was located on chromosome X and conferred the pet phenotype. Two hit2 mutations, hit2-1 and hit2-2, were located on chromosome III and caused the deletion of the PET18 locus which has been shown to encode a gene required for growth at high temperatures. The hit3-1 mutation was located on chromosome VI and affected the CDC26 gene. The hit4-1 mutation was located on chromosome XIII. These hit mutations were analyzed in an attempt to identify novel genes involved in the heat shock response. The hit1-1 mutation caused a defect in synthesis of a 74-kD heat shock protein. Western blot analysis revealed that the heat shock protein corresponded to the SSC1 protein, a member of the yeast hsp70 family. In the hit1-1 mutant, the TyHIS3 insertion caused a deletion of a 3-kb DNA segment between the delta 1 and delta 4 sequences near the SUP4 locus. The 1031-bp wild-type HIT1 DNA which contained an open reading frame encoding a protein of 164 amino acids and the AGG arginine tRNA gene complemented all hit1-1 mutant phenotypes, indicating that the mutant phenotypes were caused by the deletion of these genes. The pleiotropy of the HIT1 locus was analyzed by constructing a disruption mutation of each gene in vitro and transplacing it to the chromosome. This analysis revealed that the HIT1 gene essential for growth at high temperatures encodes the 164-amino acid protein. The arginine tRNA gene, named HSX1, is essential for growth on a nonfermentable carbon source at high temperatures and for synthesis of the SSC1 heat shock protein.

mRNA transcription in nuclei isolated from Saccharomyces cerevisiae

1986 ◽  
Vol 6 (5) ◽  
pp. 1633-1639
Author(s):  
J F Jerome ◽  
J A Jaehning
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase Ii ◽  
Blot Hybridization ◽  
Single Copy ◽  
Rna Polymerases ◽  
Slot Blot Hybridization ◽  
Genes Encoding ◽  
Diploid Cells ◽  
Alpha Amanitin

We developed an improved method for the isolation of transcriptionally active nuclei from Saccharomyces cerevisiae, which allows analysis of specific transcripts. When incubated with alpha-32P-labeled ribonucleoside triphosphates in vitro, nuclei isolated from haploid or diploid cells transcribed rRNA, tRNA, and mRNAs in a strand-specific manner, as shown by slot blot hybridization of the in vitro synthesized RNA to cloned genes encoding 5.8S, 18S and 28S rRNAs, tRNATyr, and GAL7, URA3, TY1 and HIS3 mRNAs. A yeast strain containing a high-copy-number plasmid which overproduced GAL7 mRNA was initially used to facilitate detection of a discrete message. We optimized conditions for the transcription of genes expressed by each of the three yeast nuclear RNA polymerases. Under optimal conditions, labeled transcripts could be detected from single-copy genes normally expressed at low levels in the cells (HIS3 and URA3). We determined that the alpha-amanitin sensitivity of transcript synthesis in the isolated nuclei paralleled the sensitivity of the corresponding purified RNA polymerases; in particular, mRNA synthesis was 50% sensitive to 1 microgram of alpha-amanitin per ml, establishing transcription of mRNA by RNA polymerase II.

scholarly journals Dominant effects of tubulin overexpression in Saccharomyces cerevisiae.

1989 ◽  
Vol 9 (3) ◽  
pp. 1049-1059 ◽  
Author(s):  
D Burke ◽  
P Gasdaska ◽  
L Hartwell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Loss ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Inducible Promoters ◽  
Selective Degradation ◽  
Novel Structure ◽  
Genes Encoding ◽  
Transient Overexpression ◽  
Mutant Phenotypes

The consequences of altering the levels of alpha- and beta-tubulin in Saccharomyces cerevisiae were examined by constructing fusions of the structural genes encoding the tubulins to strong galactose-inducible promoters. Overexpression of beta-tubulin (TUB2) was lethal: cells arrested in the G2 stage of the cell cycle exhibited an increased frequency of chromosome loss, were devoid of microtubules, and accumulated beta-tubulin in a novel structure. Overexpression of the major alpha-tubulin gene (TUB1) was not lethal and did not affect chromosome segregation. The rate of alpha-tubulin mRNA and protein synthesis was increased, but the protein did not accumulate. Overexpression of both alpha- and beta-tubulin together resulted in arrested cell division, and cells accumulated excess tubules that contained both alpha- and beta-tubulin. Transient overexpression of both tubulins resulted in a high frequency of chromosome loss. These data suggest that strong selective pressure exists to prevent excess accumulation of microtubules or beta-tubulin and suggest a model by which this goal may be achieved by selective degradation of unassembled alpha-tubulin. Furthermore, the phenotype of beta-tubulin overexpression is similar to the phenotype of a beta-tubulin deficiency. These results add to a number of recent studies demonstrating that mutant phenotypes generated by overexpression can be informative about the function of the gene product.

scholarly journals Genetic Analysis of the TOR Pathway in Aspergillus nidulans

2005 ◽  
Vol 4 (9) ◽  
pp. 1595-1598 ◽  
Author(s):  
Gregory J. Fitzgibbon ◽  
Igor Y. Morozov ◽  
Meriel G. Jones ◽  
Mark X. Caddick
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Analysis ◽  
Aspergillus Nidulans ◽  
Minor Role ◽  
Tor Signaling ◽  
Tor Pathway ◽  
Genes Encoding ◽  
Tor Kinase ◽  
Mutant Phenotypes ◽  
A Minor

ABSTRACT We identified five genes encoding components of the TOR signaling pathway within Aspergillus nidulans. Unlike the situation in Saccharomyces cerevisiae, there is only a single Tor kinase, as in plant and animal systems, and mutant phenotypes suggest that the TOR pathway plays only a minor role in regulating nitrogen metabolism.

scholarly journals The AraC-Type Transcriptional Regulator GliR (PA3027) Activates Genes of Glycerolipid Metabolism in Pseudomonas aeruginosa

2021 ◽  
Vol 22 (10) ◽  
pp. 5066
Author(s):  
Karolina Kotecka ◽  
Adam Kawalek ◽  
Kamil Kobylecki ◽  
Aneta Agnieszka Bartosik
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Transcriptional Profiling ◽  
Transcriptional Regulator ◽  
Promoter Sequence ◽  
Large Set ◽  
Mrna Levels ◽  
Cellular Functions ◽  
Promoter Regions ◽  
Genes Encoding

Pseudomonas aeruginosa encodes a large set of transcriptional regulators (TRs) that modulate and manage cellular metabolism to survive in variable environmental conditions including that of the human body. The AraC family regulators are an abundant group of TRs in bacteria, mostly acting as gene expression activators, controlling diverse cellular functions (e.g., carbon metabolism, stress response, and virulence). The PA3027 protein from P. aeruginosa has been classified in silico as a putative AraC-type TR. Transcriptional profiling of P. aeruginosa PAO1161 overexpressing PA3027 revealed a spectacular increase in the mRNA levels of PA3026-PA3024 (divergent to PA3027), PA3464, and PA3342 genes encoding proteins potentially involved in glycerolipid metabolism. Concomitantly, chromatin immunoprecipitation-sequencing (ChIP-seq) analysis revealed that at least 22 regions are bound by PA3027 in the PAO1161 genome. These encompass promoter regions of PA3026, PA3464, and PA3342, showing the major increase in expression in response to PA3027 excess. In Vitro DNA binding assay confirmed interactions of PA3027 with these regions. Furthermore, promoter-reporter assays in a heterologous host showed the PA3027-dependent activation of the promoter of the PA3026-PA3024 operon. Two motifs representing the preferred binding sites for PA3027, one localized upstream and one overlapping with the −35 promoter sequence, were identified in PA3026p and our data indicate that both motifs are required for full activation of this promoter by PA3027. Overall, the presented data show that PA3027 acts as a transcriptional regulator in P. aeruginosa, activating genes likely engaged in glycerolipid metabolism. The GliR name, from a glycerolipid metabolism regulator, is proposed for PA3027 of P. aeruginosa.

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