scholarly journals ACE1 regulates expression of the Saccharomyces cerevisiae metallothionein gene.

1988 ◽  
Vol 8 (7) ◽  
pp. 2745-2752 ◽  
Author(s):  
D J Thiele
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metal Binding ◽  
Gene Deletion ◽  
Copper Resistance ◽  
Low Molecular Weight ◽  
Wild Type ◽  
Positive Role ◽  
Cup1 Gene ◽  
Transcriptional Induction

Copper resistance in Saccharomyces cerevisiae is mediated, in large part, by the CUP1 locus, which encodes a low-molecular-weight, cysteine-rich metal-binding protein. Expression of the CUP1 gene is regulated at the level of transcriptional induction in response to high environmental copper levels. This report describes the isolation of a yeast mutant, ace1-1, which is defective in the activation of CUP1 expression upon exposure to exogenous copper. The ace1-1 mutation is recessive and lies in a genetic element that encodes a trans-acting CUP1 regulatory factor. The wild-type ACE1 gene was isolated by in vivo complementation and restores copper inducibility of CUP1 expression and copper resistance to the otherwise copper-sensitive ace1-1 mutant. Linkage analysis and gene deletion experiments verified that this gene represents the authentic ACE1 locus. ACE1 maps to the left arm of chromosome VII, 9 centimorgans centromere distal to lys5. The ACE1 gene appears to play a direct or indirect positive role in activation of CUP1 expression in response to elevated copper concentrations.

ACE1 regulates expression of the Saccharomyces cerevisiae metallothionein gene

1988 ◽  
Vol 8 (7) ◽  
pp. 2745-2752
Author(s):  
D J Thiele
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Metal Binding ◽  
Gene Deletion ◽  
Copper Resistance ◽  
Low Molecular Weight ◽  
Wild Type ◽  
Positive Role ◽  
Cup1 Gene ◽  
Transcriptional Induction

Copper resistance in Saccharomyces cerevisiae is mediated, in large part, by the CUP1 locus, which encodes a low-molecular-weight, cysteine-rich metal-binding protein. Expression of the CUP1 gene is regulated at the level of transcriptional induction in response to high environmental copper levels. This report describes the isolation of a yeast mutant, ace1-1, which is defective in the activation of CUP1 expression upon exposure to exogenous copper. The ace1-1 mutation is recessive and lies in a genetic element that encodes a trans-acting CUP1 regulatory factor. The wild-type ACE1 gene was isolated by in vivo complementation and restores copper inducibility of CUP1 expression and copper resistance to the otherwise copper-sensitive ace1-1 mutant. Linkage analysis and gene deletion experiments verified that this gene represents the authentic ACE1 locus. ACE1 maps to the left arm of chromosome VII, 9 centimorgans centromere distal to lys5. The ACE1 gene appears to play a direct or indirect positive role in activation of CUP1 expression in response to elevated copper concentrations.

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (9) ◽  
pp. 4084-4092
Author(s):  
P C McCabe ◽  
H Haubruck ◽  
P Polakis ◽  
F McCormick ◽  
M A Innis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Bound States ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Amino Terminal ◽  
Loop Region

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

scholarly journals Topoisomerase I involvement in illegitimate recombination in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (4) ◽  
pp. 1805-1812 ◽  
Author(s):  
J Zhu ◽  
R H Schiestl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Aberrations ◽  
Topoisomerase I ◽  
Wild Type Strain ◽  
Hot Spot ◽  
Illegitimate Recombination ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae

Chromosome aberrations may cause cancer and many heritable diseases. Topoisomerase I has been suspected of causing chromosome aberrations by mediating illegitimate recombination. The effects of deletion and of overexpression of the topoisomerase I gene on illegitimate recombination in the yeast Saccharomyces cerevisiae have been studied. Yeast transformations were carried out with DNA fragments that did not have any homology to the genomic DNA. The frequency of illegitimate integration was 6- to 12-fold increased in a strain overexpressing topoisomerase I compared with that in isogenic control strains. Hot spot sequences [(G/C)(A/T)T] for illegitimate integration target sites accounted for the majority of the additional events after overexpression of topoisomerase I. These hot spot sequences correspond to sequences previously identified in vitro as topoisomerase I preferred cleavage sequences in other organisms. Furthermore, such hot spot sequences were found in 44% of the integration events present in the TOP1 wild-type strain and at a significantly lower frequency in the top1delta strain. Our results provide in vivo evidence that a general eukaryotic topoisomerase I enzyme nicks DNA and ligates nonhomologous ends, leading to illegitimate recombination.

scholarly journals Domain structure and functional analysis of the carboxyl-terminal polyacidic sequence of the RAD6 protein of Saccharomyces cerevisiae.

1988 ◽  
Vol 8 (3) ◽  
pp. 1179-1185 ◽  
Author(s):  
A Morrison ◽  
E J Miller ◽  
L Prakash
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Proteinase K ◽  
Intrinsic Activity ◽  
Structural Domain ◽  
Globular Domain ◽  
Wild Type ◽  
Calculated Mass ◽  
Stokes Radius ◽  
Carboxyl Terminal

The RAD6 gene of Saccharomyces cerevisiae, which is required for normal tolerance of DNA damage and for sporulation, encodes a 172-residue protein whose 23 carboxyl-terminal residues are almost all acidic. We show that this polyacidic sequence appends to RAD6 protein as a polyanionic tail and that its function in vivo does not require stoichiometry of length. RAD6 protein was purified to near homogeneity from a yeast strain carrying a RAD6 overproducing plasmid. Approximately the first 150 residues of RAD6 protein composed a structural domain that was resistant to proteinase K and had a Stokes radius typical of a globular protein of its calculated mass. The carboxyl-terminal polyacidic sequence was sensitive to proteinase K, and it endowed RAD6 protein with an aberrantly large Stokes radius that indicates an asymmetric shape. We deduce that RAD6 protein is monomeric and comprises a globular domain with a freely extending polyacidic tail. We tested the phenotypic effects of partial or complete deletion of the polyacidic sequence, demonstrating the presence of the shortened proteins in the cell by using antibody to RAD6 protein. Removal of the entire polyacidic sequence severely reduced sporulation but only slightly affected survival after UV irradiation or UV-induced mutagenesis. Strains with deletions of all but the first 4 or 15 residues of the polyacidic sequence were phenotypically almost wild type or wild type, respectively. We conclude that the intrinsic activity of RAD6 protein resides in the globular domain, that the polyacidic sequence has a stimulatory or modifying role evident primarily in sporulation, and that only a short section apparently functions as effectively as the entire polyacidic sequence.

scholarly journals Impact of Pus1 Pseudouridine Synthase on Specific Decoding Events in Saccharomyces cerevisiae

Biomolecules ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 729
Author(s):  
Bahar Khonsari ◽  
Roland Klassen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Elevated Temperature ◽  
Synthetic Lethality ◽  
Positive Role ◽  
Functional Impact ◽  
Pseudouridine Synthase ◽  
Decoding Efficiency ◽  
Cognate Trna

Pus1-dependent pseudouridylation occurs in many tRNAs and at multiple positions, yet the functional impact of this modification is incompletely understood. We analyzed the consequences of PUS1 deletion on the essential decoding of CAG (Gln) codons by tRNAGlnCUG in yeast. Synthetic lethality was observed upon combining the modification defect with destabilized variants of tRNAGlnCUG, pointing to a severe CAG-decoding defect of the hypomodified tRNA. In addition, we demonstrated that misreading of UAG stop codons by a tRNAGlnCUG variant is positively affected by Pus1. Genetic approaches further indicated that mildly elevated temperature decreases the decoding efficiency of CAG and UAG via destabilized tRNAGlnCAG variants. We also determined the misreading of CGC (Arg) codons by tRNAHisGUG, where the CGC decoder tRNAArgICG contains Pus1-dependent pseudouridine, but not the mistranslating tRNAHis. We found that the absence of Pus1 increased CGC misreading by tRNAHis, demonstrating a positive role of the modification in the competition against non-synonymous near-cognate tRNA. Part of the in vivo decoding defects and phenotypes in pus1 mutants and strains carrying destabilized tRNAGlnCAG were suppressible by additional deletion of the rapid tRNA decay (RTD)-relevant MET22, suggesting the involvement of RTD-mediated tRNA destabilization.

scholarly journals Knock out of sHSP genes determines some modifications in the probiotic attitude of Lactiplantibacillus plantarum

2020 ◽  
Author(s):  
Angela Longo ◽  
Pasquale Russo ◽  
Vittorio Capozzi ◽  
Giuseppe Spano ◽  
Daniela Fiocco
Keyword(s):  
Small Heat Shock Protein ◽  
Cellular Adhesion ◽  
Probiotic Properties ◽  
Wild Type ◽  
Knock Out ◽  
Human Enterocyte ◽  
Transcriptional Induction ◽  
Pro Inflammatory Cytokines

Abstract Objective We investigated whether the knock out of small heat shock protein (sHSP) genes (hsp1, hsp2 and hsp3) impact on probiotic features of Lactiplantibacillus plantarum WCFS1, aiming to find specific microbial effectors involved in microbe-host interplay. Results The probiotic properties of L. plantarum WCFS1 wild type, hsp1, hsp2 and hsp3 mutant clones were evaluated and compared through in vitro trials. Oro-gastro-intestinal assays pointed to significantly lower survival for hsp1 and hsp2 mutants under stomach-like conditions, and for hsp3 mutant under intestinal stress. Adhesion to human enterocyte-like cells was similar for all clones, though the hsp2 mutant exhibited higher adhesiveness. L. plantarum cells attenuated the transcriptional induction of pro-inflammatory cytokines on lipopolysaccharide-treated human macrophages, with some exception for the hsp1 mutant. Intriguingly, this clone also induced a higher IL10/IL12 ratio, which is assumed to indicate the anti-inflammatory potential of probiotics. Conclusions sHSP genes deletion determined some differences in gut stress resistance, cellular adhesion and immuno-modulation, also implying effects on in vivo interaction with the host. HSP1 might contribute to immunomodulatory mechanisms, though additional experiments are necessary to test this feature.

scholarly journals Soluble epoxide hydrolase inhibition and gene deletion are protective against myocardial ischemia-reperfusion injury in vivo

2008 ◽  
Vol 295 (5) ◽  
pp. H2128-H2134 ◽  
Author(s):  
Atsuko Motoki ◽  
Matthias J. Merkel ◽  
William H. Packwood ◽  
Zhiping Cao ◽  
Lijuan Liu ◽  
...  
Keyword(s):  
Myocardial Ischemia ◽  
Reperfusion Injury ◽  
Epoxide Hydrolase ◽  
Gene Deletion ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Wild Type ◽  
Myocardial Ischemia Reperfusion Injury ◽  
Myocardial Ischemia Reperfusion

Soluble epoxide hydrolase (sEH) metabolizes epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids. EETs are formed from arachidonic acid during myocardial ischemia and play a protective role against ischemic cell death. Deletion of sEH has been shown to be protective against myocardial ischemia in the isolated heart preparation. We tested the hypothesis that sEH inactivation by targeted gene deletion or pharmacological inhibition reduces infarct size (I) after regional myocardial ischemia-reperfusion injury in vivo. Male C57BL\6J wild-type or sEH knockout mice were subjected to 40 min of left coronary artery (LCA) occlusion and 2 h of reperfusion. Wild-type mice were injected intraperitoneally with 12-(3-adamantan-1-yl-ureido)-dodecanoic acid butyl ester (AUDA-BE), a sEH inhibitor, 30 min before LCA occlusion or during ischemia 10 min before reperfusion. 14,15-EET, the main substrate for sEH, was administered intravenously 15 min before LCA occlusion or during ischemia 5 min before reperfusion. The EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (EEZE) was given intravenously 15 min before reperfusion. Area at risk (AAR) and I were assessed using fluorescent microspheres and triphenyltetrazolium chloride, and I was expressed as I/AAR. I was significantly reduced in animals treated with AUDA-BE or 14,15-EET, independent of the time of administration. The cardioprotective effect of AUDA-BE was abolished by the EET antagonist 14,15-EEZE. Immunohistochemistry revealed abundant sEH protein expression in left ventricular tissue. Strategies to increase 14,15-EET, including sEH inactivation, may represent a novel therapeutic approach for cardioprotection against myocardial ischemia-reperfusion injury.

scholarly journals Effect of Targeted Deletion of the Heme Oxygenase-2 Gene on Hemoglobin Toxicity in the Striatum

2005 ◽  
Vol 25 (11) ◽  
pp. 1466-1475 ◽  
Author(s):  
Yan Qu ◽  
Jing Chen ◽  
Luna Benvenisti-Zarom ◽  
Xin Ma ◽  
Raymond F Regan
Keyword(s):  
Heme Oxygenase ◽  
Knockout Mice ◽  
Protein Oxidation ◽  
Gene Deletion ◽  
Wild Type ◽  
Cell Culture Study ◽  
Targeted Deletion ◽  
Rate Limiting Step ◽  
Diphenyltetrazolium Bromide

The heme oxygenase (HO) enzymes catalyze the rate-limiting step in the breakdown of heme to iron, carbon monoxide, and biliverdin. A prior cell culture study demonstrated that deletion of HO-2, the isoform constitutively expressed in neurons, attenuated hemoglobin (Hb) neurotoxicity. The present study tested the hypothesis that HO-2 gene deletion is cytoprotective in a model of Hb toxicity in vivo. Stereotactic injection of 6 μL stroma-free Hb (SFHb) into the striatum significantly increased protein oxidation in wild-type mice at 24 to 72 h, as detected by an assay for carbonyl groups. At 72 h, carbonylation was increased 2.5-fold compared with that in the contralateral striatum. In HO-2 knockout mice, protein oxidation was not increased at 24 h, and was increased by only 1.7-fold at 72 h. Similarly, striatal lipid peroxidation, as detected by the malondialdehyde assay, was significantly greater in the SFHb-injected striata of wild-type mice than in knockout mice. Striatal cell viability, determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, was 45.0%±6.3% of that in contralateral striata in wild-type mice at 72 h; it was increased to 85%±8% in knockouts. Heme oxygenase-2 gene deletion did not alter weight loss or mortality after SFHb injection. Baseline striatal HO-1 expression was similar in knockout and wild-type mice; induction after SFHb injection occurred more rapidly in the latter. These results suggest that HO-2 gene deletion protects striatal cells from the oxidative toxicity of Hb in vivo. Pharmacologic or genetic strategies that target HO-2 may be beneficial after central nervous system hemorrhage, and warrant further investigation.

scholarly journals Yeast Est2p Affects Telomere Length by Influencing Association of Rap1p with Telomeric Chromatin

2008 ◽  
Vol 28 (7) ◽  
pp. 2380-2390 ◽  
Author(s):  
Hong Ji ◽  
Christopher J. Adkins ◽  
Bethany R. Cartwright ◽  
Katherine L. Friedman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Telomere Length ◽  
Specific Binding ◽  
Telomerase Activity ◽  
Negative Regulator ◽  
Wild Type ◽  
Telomeric Dna ◽  
Telomere Length Homeostasis

ABSTRACT In Saccharomyces cerevisiae, the sequence-specific binding of the negative regulator Rap1p provides a mechanism to measure telomere length: as the telomere length increases, the binding of additional Rap1p inhibits telomerase activity in cis. We provide evidence that the association of Rap1p with telomeric DNA in vivo occurs in part by sequence-independent mechanisms. Specific mutations in EST2 (est2-LT) reduce the association of Rap1p with telomeric DNA in vivo. As a result, telomeres are abnormally long yet bind an amount of Rap1p equivalent to that observed at wild-type telomeres. This behavior contrasts with that of a second mutation in EST2 (est2-up34) that increases bound Rap1p as expected for a strain with long telomeres. Telomere sequences are subtly altered in est2-LT strains, but similar changes in est2-up34 telomeres suggest that sequence abnormalities are a consequence, not a cause, of overelongation. Indeed, est2-LT telomeres bind Rap1p indistinguishably from the wild type in vitro. Taken together, these results suggest that Est2p can directly or indirectly influence the binding of Rap1p to telomeric DNA, implicating telomerase in roles both upstream and downstream of Rap1p in telomere length homeostasis.

scholarly journals Characterization of the DNA-binding activity of GCR1: in vivo evidence for two GCR1-binding sites in the upstream activating sequence of TPI of Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (6) ◽  
pp. 2690-2700 ◽  
Author(s):  
M A Huie ◽  
E W Scott ◽  
C M Drazinic ◽  
M C Lopez ◽  
I K Hornstra ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Protein ◽  
Binding Site ◽  
Gene Function ◽  
Binding Sites ◽  
Sequence Motif ◽  
Wild Type ◽  
Upstream Activating Sequence ◽  
Sequence Elements

GCR1 gene function is required for high-level glycolytic gene expression in Saccharomyces cerevisiae. Recently, we suggested that the CTTCC sequence motif found in front of many genes encoding glycolytic enzymes lay at the core of the GCR1-binding site. Here we mapped the DNA-binding domain of GCR1 to the carboxy-terminal 154 amino acids of the polypeptide. DNase I protection studies showed that a hybrid MBP-GCR1 fusion protein protected a region of the upstream activating sequence of TPI (UASTPI), which harbored the CTTCC sequence motif, and suggested that the fusion protein might also interact with a region of the UAS that contained the related sequence CATCC. A series of in vivo G methylation protection experiments of the native TPI promoter were carried out with wild-type and gcr1 deletion mutant strains. The G doublets that correspond to the C doublets in each site were protected in the wild-type strain but not in the gcr1 mutant strain. These data demonstrate that the UAS of TPI contains two GCR1-binding sites which are occupied in vivo. Furthermore, adjacent RAP1/GRF1/TUF- and REB1/GRF2/QBP/Y-binding sites in UASTPI were occupied in the backgrounds of both strains. In addition, DNA band-shift assays were used to show that the MBP-GCR1 fusion protein was able to form nucleoprotein complexes with oligonucleotides that contained CTTCC sequence elements found in front of other glycolytic genes, namely, PGK, ENO1, PYK, and ADH1, all of which are dependent on GCR1 gene function for full expression. However, we were unable to detect specific interactions with CTTCC sequence elements found in front of the translational component genes TEF1, TEF2, and CRY1. Taken together, these experiments have allowed us to propose a consensus GCR1-binding site which is 5'-(T/A)N(T/C)N(G/A)NC(T/A)TCC(T/A)N(T/A)(T/A)(T/G)-3'.

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