scholarly journals Saccharomyces cerevisiae RAD2 gene: isolation, subcloning, and partial characterization.

1984 ◽  
Vol 4 (2) ◽  
pp. 290-295 ◽  
Author(s):  
L Naumovski ◽  
E C Friedberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Hybridization ◽  
Genomic Library ◽  
Normal Growth ◽  
Growth Conditions ◽  
Gene Isolation ◽  
Uv Sensitivity ◽  
Pair Fragment ◽  
Rna Transcript ◽  
Internal Fragment

A plasmid (pNF2000) containing a 9.7-kilobase pair DNA insert that complements the UV sensitivity of rad2-1, rad2-2, and rad2-4 mutants of Saccharomyces cerevisiae has been isolated from a yeast genomic library. Genetic analysis of strains derived by transformation of rad2 mutants with an integrating plasmid containing a 9.3-kilobase pair fragment from pNF2000 shows that the fragment integrates exclusively at the chromosomal rad2 gene. We therefore conclude that this plasmid contains the RAD2 gene. The 9.3-kilobase pair fragment was partially digested with Sau3A and cloned into a multicopy yeast vector designed for easy retrieval of Sau3A inserts. The smallest subclone that retains the RAD2 gene is 4.5 kilobase pairs. This fragment was partially digested with Sau3A and cloned into an integrating plasmid. These plasmids were isolated and integrated into a heterozygous rad2/RAD2 strain. Plasmids containing internal fragments of the RAD2 gene were identified because they yielded UV-sensitive transformants due to disruption of the RAD2 gene. Sporulation of diploids transformed with integrating plasmids containing internal fragments of RAD2 gave rise to four viable haploids per tetrad, indicating that unlike the RAD3 gene of S. cerevisiae, the RAD2 gene is not essential for the viability of haploid cells under normal growth conditions. Measurements of the RNA transcript by RNA-DNA hybridization with the internal fragment as the probe indicate a size of approximately 3.2 kilobases.

Saccharomyces cerevisiae RAD2 gene: isolation, subcloning, and partial characterization

1984 ◽  
Vol 4 (2) ◽  
pp. 290-295
Author(s):  
L Naumovski ◽  
E C Friedberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Hybridization ◽  
Genomic Library ◽  
Normal Growth ◽  
Growth Conditions ◽  
Gene Isolation ◽  
Uv Sensitivity ◽  
Pair Fragment ◽  
Rna Transcript ◽  
Internal Fragment

A plasmid (pNF2000) containing a 9.7-kilobase pair DNA insert that complements the UV sensitivity of rad2-1, rad2-2, and rad2-4 mutants of Saccharomyces cerevisiae has been isolated from a yeast genomic library. Genetic analysis of strains derived by transformation of rad2 mutants with an integrating plasmid containing a 9.3-kilobase pair fragment from pNF2000 shows that the fragment integrates exclusively at the chromosomal rad2 gene. We therefore conclude that this plasmid contains the RAD2 gene. The 9.3-kilobase pair fragment was partially digested with Sau3A and cloned into a multicopy yeast vector designed for easy retrieval of Sau3A inserts. The smallest subclone that retains the RAD2 gene is 4.5 kilobase pairs. This fragment was partially digested with Sau3A and cloned into an integrating plasmid. These plasmids were isolated and integrated into a heterozygous rad2/RAD2 strain. Plasmids containing internal fragments of the RAD2 gene were identified because they yielded UV-sensitive transformants due to disruption of the RAD2 gene. Sporulation of diploids transformed with integrating plasmids containing internal fragments of RAD2 gave rise to four viable haploids per tetrad, indicating that unlike the RAD3 gene of S. cerevisiae, the RAD2 gene is not essential for the viability of haploid cells under normal growth conditions. Measurements of the RNA transcript by RNA-DNA hybridization with the internal fragment as the probe indicate a size of approximately 3.2 kilobases.

Constitutive and inducible Saccharomyces cerevisiae promoters: evidence for two distinct molecular mechanisms

1986 ◽  
Vol 6 (11) ◽  
pp. 3847-3853
Author(s):  
K Struhl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Mechanisms ◽  
Normal Growth ◽  
Regulatory Elements ◽  
Growth Conditions ◽  
Physical Structure ◽  
Base Pairs ◽  
Inducible Promoters ◽  
Upstream Promoter ◽  
Selection Of

his3 and pet56 are adjacent Saccharomyces cerevisiae genes that are transcribed in opposite directions from initiation sites that are separated by 200 base pairs. Under normal growth conditions, in which his3 and pet56 are transcribed at similar basal levels, a poly(dA-dT) sequence located between the genes serves as the upstream promoter element for both. In contrast, his3 but not pet56 transcription is induced during conditions of amino acid starvation, even though the critical regulatory site is located upstream of both respective TATA regions. Moreover, only one of the two normal his3 initiation sites is subject to induction. From genetic and biochemical evidence, I suggest that the his3-pet56 intergenic region contains constitutive and inducible promoters with different properties. In particular, two classes of TATA elements, constitutive (Tc) and regulatory (Tr), can be distinguished by their ability to respond to upstream regulatory elements, by their effects on the selection of initiation sites, and by their physical structure in nuclear chromatin. Constitutive and inducible his3 transcription is mediated by distinct promoters representing each class, whereas pet56 transcription is mediated by a constitutive promoter. Molecular mechanisms for these different kinds of S. cerevisiae promoters are proposed.

scholarly journals Activation of the Mitogen-activated Protein Kinase, Slt2p, at Bud Tips Blocks a Late Stage of Endoplasmic Reticulum Inheritance in Saccharomyces cerevisiae

2010 ◽  
Vol 21 (10) ◽  
pp. 1772-1782 ◽  
Author(s):  
Xia Li ◽  
Yunrui Du ◽  
Steven Siegel ◽  
Susan Ferro-Novick ◽  
Peter Novick
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Protein Phosphatase ◽  
Normal Growth ◽  
Growth Conditions ◽  
Mitogen Activated Protein Kinase ◽  
Mitochondrial Inheritance ◽  
Daughter Cells ◽  
Late Step ◽  
Mitogen Activated Protein

Inheritance of the endoplasmic reticulum (ER) requires Ptc1p, a type 2C protein phosphatase of Saccharomyces cerevisiae . Genetic analysis indicates that Ptc1p is needed to inactivate the cell wall integrity (CWI) MAP kinase, Slt2p. Here we show that under normal growth conditions, Ptc1p inactivates Slt2p just as ER tubules begin to spread from the bud tip along the cortex. In ptc1Δ cells, the propagation of cortical ER from the bud tip to the periphery of the bud is delayed by hyperactivation of Slt2p. The pool of Slt2p that controls ER inheritance requires the CWI pathway scaffold, Spa2p, for its retention at the bud tip, and a mutation within Slt2p that prevents its association with the bud tip blocks its role in ER inheritance. These results imply that Slt2p inhibits a late step in ER inheritance by phosphorylating a target at the tip of daughter cells. The PI4P5-kinase, Mss4p, is an upstream activator of this pool of Slt2p. Ptc1p-dependant inactivation of Slt2p is also needed for mitochondrial inheritance; however, in this case, the relevant pool of Slt2p is not at the bud tip.

scholarly journals Genomewide Recruitment Analysis of Rpb4, a Subunit of Polymerase II in Saccharomyces cerevisiae, Reveals Its Involvement in Transcription Elongation

2008 ◽  
Vol 7 (6) ◽  
pp. 1009-1018 ◽  
Author(s):  
Jiyoti Verma-Gaur ◽  
Sudha Narayana Rao ◽  
Toshiki Taya ◽  
Parag Sadhale
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Normal Growth ◽  
Growth Conditions ◽  
Pol Ii ◽  
Coding Regions ◽  
The Core ◽  
A Subunit ◽  
On Chip

ABSTRACT The Rpb4/Rpb7 subcomplex of yeast RNA polymerase II (Pol II) has counterparts in all multisubunit RNA polymerases from archaebacteria to higher eukaryotes. The Rpb4/7 subcomplex in Saccharomyces cerevisiae is unique in that it easily dissociates from the core, unlike the case in other organisms. The relative levels of Rpb4 and Rpb7 in yeasts affect the differential gene expression and stress response. Rpb4 is nonessential in S. cerevisiae and affects expression of a small number of genes under normal growth conditions. Here, using a chromatin immunoprecipitation (“ChIP on-chip”) technique, we compared genomewide binding of Rpb4 to that of a core Pol II subunit, Rpb3. Our results showed that in spite of being nonessential for survival, Rpb4 was recruited on coding regions of most transcriptionally active genes, similar to the case with the core Pol II subunit, Rpb3, albeit to a lesser extent. The extent of Rpb4 recruitment increased with increasing gene length. We also observed Pol II lacking Rpb4 to be defective in transcribing long, GC-rich transcription units, suggesting a role for Rpb4 in transcription elongation. This role in transcription elongation was supported by the observed 6-azauracil (6AU) sensitivity of the rpb4Δ mutant. Unlike most phenotypes of rpb4Δ, the 6AU sensitivity of the rpb4Δ strain was not rescued by overexpression of RPB7. This report provides the first instance of a distinct role for Rpb4 in transcription, which is independent of its interacting partner, Rpb7.

Molecular cloning and nucleotide sequence analysis of the Saccharomyces cerevisiae RAD1 gene

1984 ◽  
Vol 4 (10) ◽  
pp. 2161-2169
Author(s):  
E Yang ◽  
E C Friedberg
Keyword(s):  
Genomic Library ◽  
Open Reading Frame ◽  
Nucleotide Sequence Analysis ◽  
Multicopy Plasmid ◽  
Uv Resistance ◽  
Reading Frame ◽  
Uv Sensitivity ◽  
Resistance Function ◽  
Rad Mutants ◽  
Internal Fragment

We have screened a yeast genomic library for complementation of the UV sensitivity of mutants defective in the RAD1 gene and isolated a plasmid designated pNF1000 with an 8.9-kilobase insert. This multicopy plasmid quantitatively complemented the UV sensitivity of two rad1 mutants tested but did not affect the UV resistance of other rad mutants. The location of the UV resistance function in pNF1000 was determined by deletion analysis, and an internal fragment of the putative RAD1 gene was integrated into the genome of a RAD1 strain. Genetic analysis of several integrants showed that integration occurred at the chromosomal RAD1 site, demonstrating that the internal fragment was derived from the RAD1 gene. A 3.88-kilobase region of pNF1000 was sequenced and showed the presence of a small open reading frame 243 nucleotides long that is apparently unrelated to RAD1, as well as a 2,916-nucleotide larger open reading frame presumed to encode RAD1 protein. Depending on which of two possible ATG codons initiates translation, the size of the RAD1 protein is calculated at 110 or 97 kilodaltons.

scholarly journals Pressure-Induced Endocytic Degradation of the Saccharomyces cerevisiae Low-Affinity Tryptophan Permease Tat1 Is Mediated by Rsp5 Ubiquitin Ligase and Functionally Redundant PPxY Motif Proteins

2013 ◽  
Vol 12 (7) ◽  
pp. 990-997 ◽  
Author(s):  
Asaha Suzuki ◽  
Takahiro Mochizuki ◽  
Satoshi Uemura ◽  
Toshiki Hiraki ◽  
Fumiyoshi Abe
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Pressure ◽  
Hydrostatic Pressure ◽  
Ubiquitin Ligase ◽  
High Hydrostatic Pressure ◽  
Intracellular Localization ◽  
Normal Growth ◽  
Growth Conditions ◽  
Content Type ◽  
Genes Encoding

ABSTRACT Cells of Saccharomyces cerevisiae express two tryptophan permeases, Tat1 and Tat2, which have different characteristics in terms of their affinity for tryptophan and intracellular localization. Although the high-affinity permease Tat2 has been well documented in terms of its ubiquitin-dependent degradation, the low-affinity permease Tat1 has not yet been characterized fully. Here we show that a high hydrostatic pressure of 25 MPa triggers a degradation of Tat1 which depends on Rsp5 ubiquitin ligase and the EH domain-containing protein End3. Tat1 was resistant to a 3-h cycloheximide treatment, suggesting that it is highly stable under normal growth conditions. The ubiquitination of Tat1 most likely occurs at N-terminal lysines 29 and 31. Simultaneous substitution of arginine for the two lysines prevented Tat1 degradation, but substitution of either of them alone did not, indicating that the roles of lysines 29 and 31 are redundant. When cells were exposed to high pressure, Tat1-GFP was completely lost from the plasma membrane, while substantial amounts of Tat1 K29R-K31R -GFP remained. The HPG1-1 (Rsp5 P514T ) and rsp5-ww3 mutations stabilized Tat1 under high pressure, but any one of the rsp5-ww1 , rsp5-ww2 , and bul1 Δ bul2 Δ mutations or single deletions of genes encoding arrestin-related trafficking adaptors did not. However, simultaneous loss of 9-arrestins and Bul1/Bul2 prevented Tat1 degradation at 25 MPa. The results suggest that multiple PPxY motif proteins share some essential roles in regulating Tat1 ubiquitination in response to high hydrostatic pressure.

A Saccharomyces cerevisiae mutant defines a new locus essential for resistance to the antitumour drug bleomycin

1996 ◽  
Vol 42 (8) ◽  
pp. 835-843 ◽  
Author(s):  
Dindial Ramotar ◽  
Jean-Yves Masson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Hydrogen Peroxide ◽  
Dna Repair ◽  
Chromosomal Translocation ◽  
Normal Growth ◽  
Fold Increase ◽  
Growth Conditions ◽  
Dna Lesions ◽  
Methyl Methanesulfonate ◽  
Yeast Saccharomyces Cerevisiae

The antitumor drug bleomycin can produce a variety of lesions in the cellular DNA by a free radical dependent mechanism. To understand how these DNA lesions are repaired, bleomycin-hypersensitive mutants were isolated from the yeast Saccharomyces cerevisiae. We report here the analysis of one mutant, DRY25, that showed extreme sensitivity to bleomycin. This mutant also exhibited hypersensitivity to hydrogen peroxide and t-butyl hydroperoxide, but showed no sensitivity to other DNA-damaging agents, including γ-rays, ultraviolet light, and methyl methanesulfonate. Subsequent analysis revealed that strain DRY25 was severely deficient in the repair of bleomycin-induced DNA lesions. Under normal growth conditions, DRY25 displayed a 3-fold increase in the frequency of chromosomal translocation that was further stimulated by 5- to 15-fold when the cells were treated with either bleomycin or hydrogen peroxide, but not by methyl methanesulfonate, as compared with the wild type. Genetic analysis indicated that the mutant defect was independent of the nucleotide excision, postreplication, or recombinational DNA-repair pathways. These data suggest that one conceivable defect of DRY25 is that it lacks a protein that protects the cell against oxidative damage to DNA. A clone that fully complemented DRY25 defect was isolated and the possible roles of the complementing gene are discussed.Key words: yeast, bleomycin, DNA repair, mutations.

RAD4 gene of Saccharomyces cerevisiae: molecular cloning and partial characterization of a gene that is inactivated in Escherichia coli

1987 ◽  
Vol 7 (3) ◽  
pp. 1180-1192
Author(s):  
R Fleer ◽  
C M Nicolet ◽  
G A Pure ◽  
E C Friedberg
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Insertional Mutagenesis ◽  
Genomic Library ◽  
Excision Repair ◽  
Essential Gene ◽  
Yeast Cells ◽  
E Coli ◽  
Uv Sensitivity

In contrast to other Saccharomyces cerevisiae RAD genes involved in nucleotide excision repair of DNA, the RAD4 gene could not be isolated by screening a yeast genomic library for recombinant plasmids which complement the UV sensitivity of rad4 mutants (Pure et al., J. Mol. Biol. 183:31-42, 1985). We therefore attempted to walk to RAD4 from the neighboring SPT2 gene and obtained an integrating derivative of a plasmid isolated by Roeder et al. (Mol. Cell. Biol. 5:1543-1553, 1985) which contains a 4-kilobase fragment of yeast DNA including a mutant allele of SPT2. When integrated into several different rad4 mutant strains, this plasmid (pR169) complements UV sensitivity at a frequency of approximately 10%. However, a centromeric plasmid containing rescued sequences which include flanking yeast DNA no longer complements the phenotype of rad4 mutants. Complementing activity was restored by in vivo repair of a defined gap in the centromeric plasmid. The repaired plasmid fully complements the UV sensitivity of all rad4 mutants tested when isolated directly from yeast cells, but when this plasmid is propagated in Escherichia coli complementing activity is lost. We have mapped the physical location of the RAD4 gene by insertional mutagenesis and by transcript mapping. The gene is approximately 2.3 kilobases in size and is located immediately upstream of the SPT2 gene. Both genes are transcribed in the same direction. RAD4 is not an essential gene, and no increased transcription of this gene is observed in cells exposed to the DNA-damaging agent 4-nitroquinoline-1-oxide. The site of inactivation of RAD4 in a particular plasmid propagated in E. coli was localized to a 100-base-pair region by gene disruption and gap repair experiments. In addition, we have identified the approximate locations of the chromosomal rad4-2, rad4-3, and rad4-4 mutations.

scholarly journals Overlapping Roles of the Cytoplasmic and Mitochondrial Redox Regulatory Systems in the Yeast Saccharomyces cerevisiae

2005 ◽  
Vol 4 (2) ◽  
pp. 392-400 ◽  
Author(s):  
Eleanor W. Trotter ◽  
Chris M. Grant
Keyword(s):  
Oxidative Stress ◽  
Saccharomyces Cerevisiae ◽  
Redox State ◽  
Redox Homeostasis ◽  
Thioredoxin Reductase ◽  
Normal Growth ◽  
Growth Conditions ◽  
Reduced Form ◽  
Yeast Saccharomyces Cerevisiae ◽  
Thioredoxin System

ABSTRACT Thioredoxins are small, highly conserved oxidoreductases which are required to maintain the redox homeostasis of the cell. Saccharomyces cerevisiae contains a cytoplasmic thioredoxin system (TRX1, TRX2, and TRR1) as well as a complete mitochondrial thioredoxin system, comprising a thioredoxin (TRX3) and a thioredoxin reductase (TRR2). In the present study we have analyzed the functional overlap between the two systems. By constructing mutant strains with deletions of both the mitochondrial and cytoplasmic systems (trr1 trr2 and trx1 trx2 trx3), we show that cells can survive in the absence of both systems. Analysis of the redox state of the cytoplasmic thioredoxins reveals that they are maintained independently of the mitochondrial system. Similarly, analysis of the redox state of Trx3 reveals that it is maintained in the reduced form in wild-type cells and in mutants lacking components of the cytoplasmic thioredoxin system (trx1 trx2 or trr1). Surprisingly, the redox state of Trx3 is also unaffected by the loss of the mitochondrial thioredoxin reductase (trr2) and is largely maintained in the reduced form unless cells are exposed to an oxidative stress. Since glutathione reductase (Glr1) has been shown to colocalize to the cytoplasm and mitochondria, we examined whether loss of GLR1 influences the redox state of Trx3. During normal growth conditions, deletion of TRR2 and GLR1 was found to result in partial oxidation of Trx3, indicating that both Trr2 and Glr1 are required to maintain the redox state of Trx3. The oxidation of Trx3 in this double mutant is even more pronounced during oxidative stress or respiratory growth conditions. Taken together, these data indicate that Glr1 and Trr2 have an overlapping function in the mitochondria.

scholarly journals RAD4 gene of Saccharomyces cerevisiae: molecular cloning and partial characterization of a gene that is inactivated in Escherichia coli.

1987 ◽  
Vol 7 (3) ◽  
pp. 1180-1192 ◽  
Author(s):  
R Fleer ◽  
C M Nicolet ◽  
G A Pure ◽  
E C Friedberg
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Insertional Mutagenesis ◽  
Genomic Library ◽  
Excision Repair ◽  
Essential Gene ◽  
Yeast Cells ◽  
E Coli ◽  
Uv Sensitivity

In contrast to other Saccharomyces cerevisiae RAD genes involved in nucleotide excision repair of DNA, the RAD4 gene could not be isolated by screening a yeast genomic library for recombinant plasmids which complement the UV sensitivity of rad4 mutants (Pure et al., J. Mol. Biol. 183:31-42, 1985). We therefore attempted to walk to RAD4 from the neighboring SPT2 gene and obtained an integrating derivative of a plasmid isolated by Roeder et al. (Mol. Cell. Biol. 5:1543-1553, 1985) which contains a 4-kilobase fragment of yeast DNA including a mutant allele of SPT2. When integrated into several different rad4 mutant strains, this plasmid (pR169) complements UV sensitivity at a frequency of approximately 10%. However, a centromeric plasmid containing rescued sequences which include flanking yeast DNA no longer complements the phenotype of rad4 mutants. Complementing activity was restored by in vivo repair of a defined gap in the centromeric plasmid. The repaired plasmid fully complements the UV sensitivity of all rad4 mutants tested when isolated directly from yeast cells, but when this plasmid is propagated in Escherichia coli complementing activity is lost. We have mapped the physical location of the RAD4 gene by insertional mutagenesis and by transcript mapping. The gene is approximately 2.3 kilobases in size and is located immediately upstream of the SPT2 gene. Both genes are transcribed in the same direction. RAD4 is not an essential gene, and no increased transcription of this gene is observed in cells exposed to the DNA-damaging agent 4-nitroquinoline-1-oxide. The site of inactivation of RAD4 in a particular plasmid propagated in E. coli was localized to a 100-base-pair region by gene disruption and gap repair experiments. In addition, we have identified the approximate locations of the chromosomal rad4-2, rad4-3, and rad4-4 mutations.

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