scholarly journals sir2 mutants of Kluyveromyces lactis are hypersensitive to DNA-targeting drugs.

1994 ◽  
Vol 14 (7) ◽  
pp. 4501-4508 ◽  
Author(s):  
X J Chen ◽  
G D Clark-Walker
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Function ◽  
Kluyveromyces Lactis ◽  
Functional Equivalence ◽  
Striking Difference ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Dna Targeting ◽  
Mating Efficiency ◽  
Type Gene

A Kluyveromyces lactis mutant, hypersensitive to the DNA-targeting drugs ethidium bromide (EtBr), berenil, and HOE15030, can be complemented by a wild-type gene with homology to SIR2 of Saccharomyces cerevisiae (ScSIR2). The deduced amino acid sequence of the K. lactis Sir2 protein has 53% identity with ScSir2 protein but is 108 residues longer. K. lactis sir2 mutants show decreased mating efficiency, deficiency in sporulation, an increase in recombination at the ribosomal DNA locus, and EtBr-induced death. Some functional equivalence between the Sir2 proteins of K. lactis and S. cerevisiae has been demonstrated by introduction of ScSIR2 into a sir2 mutant of K. lactis. Expression of ScSIR2 on a multicopy plasmid restores resistance to EtBr and complements sporulation deficiency. Similarly, mating efficiency of a sir2 mutant of S. cerevisiae is partially restored by K. lactis SIR2 on a multicopy plasmid. Although these observations suggest that there has been some conservation of Sir2 protein function, a striking difference is that sir2 mutants of S. cerevisiae, unlike their K. lactis counterparts, are not hypersensitive to DNA-targeting drugs.

sir2 mutants of Kluyveromyces lactis are hypersensitive to DNA-targeting drugs

1994 ◽  
Vol 14 (7) ◽  
pp. 4501-4508
Author(s):  
X J Chen ◽  
G D Clark-Walker
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Function ◽  
Kluyveromyces Lactis ◽  
Functional Equivalence ◽  
Striking Difference ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Dna Targeting ◽  
Mating Efficiency ◽  
Type Gene

A Kluyveromyces lactis mutant, hypersensitive to the DNA-targeting drugs ethidium bromide (EtBr), berenil, and HOE15030, can be complemented by a wild-type gene with homology to SIR2 of Saccharomyces cerevisiae (ScSIR2). The deduced amino acid sequence of the K. lactis Sir2 protein has 53% identity with ScSir2 protein but is 108 residues longer. K. lactis sir2 mutants show decreased mating efficiency, deficiency in sporulation, an increase in recombination at the ribosomal DNA locus, and EtBr-induced death. Some functional equivalence between the Sir2 proteins of K. lactis and S. cerevisiae has been demonstrated by introduction of ScSIR2 into a sir2 mutant of K. lactis. Expression of ScSIR2 on a multicopy plasmid restores resistance to EtBr and complements sporulation deficiency. Similarly, mating efficiency of a sir2 mutant of S. cerevisiae is partially restored by K. lactis SIR2 on a multicopy plasmid. Although these observations suggest that there has been some conservation of Sir2 protein function, a striking difference is that sir2 mutants of S. cerevisiae, unlike their K. lactis counterparts, are not hypersensitive to DNA-targeting drugs.

A deletion that includes the segment coding for the signal peptidase cleavage site delays release of Saccharomyces cerevisiae acid phosphatase from the endoplasmic reticulum

1986 ◽  
Vol 6 (2) ◽  
pp. 723-729
Author(s):  
R Haguenauer-Tsapis ◽  
M Nagy ◽  
A Ryter
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Acid Phosphatase ◽  
Cleavage Site ◽  
Ultrastructural Localization ◽  
Signal Peptidase ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Sugar Chains ◽  
Pho5 Gene

We studied ultrastructural localization of acid phosphatase in derepressed Saccharomyces cerevisiae cells transformed with a multicopy plasmid carrying either the wild-type PHO5 gene or a PHO5 gene deleted in the region overlapping the signal peptidase cleavage site. Wild-type enzyme was located in the cell wall, as was 50% of the modified protein, which carried high-mannose-sugar chains. The remaining 50% of the protein was active and core glycosylated, and it accumulated in the endoplasmic reticulum cisternae. The signal peptide remained uncleaved in both forms. Cells expressing the modified protein exhibited an exaggerated endoplasmic reticulum with dilated lumen.

scholarly journals The GTS1 gene, which contains a Gly-Thr repeat, affects the timing of budding and cell size of the yeast Saccharomyces cerevisiae.

1994 ◽  
Vol 14 (8) ◽  
pp. 5569-5578 ◽  
Author(s):  
K Mitsui ◽  
S Yaguchi ◽  
K Tsurugi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Cell Size ◽  
Single Cells ◽  
Exponential Growth Phase ◽  
Restrictive Temperature ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mean Cell Volume

A gene with an open reading frame encoding a protein of 417 amino acid residues with a Gly-Thr repeat was isolated from the yeast Saccharomyces cerevisiae by using synthetic oligonucleotides encoding three Gly-Thr dimers as probes. The deduced amino acid sequence showed partial homology to the clock-affecting gene, per, of Drosophila melanogaster in the regions including the GT repeat. The function of the gene, named GTS1, was examined by characterizing the phenotypes of transformants with different copy numbers of the GTS1 gene produced either by inactivating the GTS1 gene by gene disruption (TM delta gts1) or by transformation with multicopy plasmid pPER119 (TMpGTS1). They grew at similar rates during the exponential growth phase, but the lag phases were shorter for TM delta gts1 and longer for TMpGTS1 cells than that for the wild type. Analyses of their cell cycle parameters using synchronized cells revealed that the unbudding period changed as a function of gene dosage; that is, the periods of TM delta gts1 and TMpGTS1 were about 20% shorter and longer, respectively, than that of the wild-type. Another significant change in the transformants was detected in the distribution of the cell size. The mean cell volume of the TM delta gts1 cells in the unbudded period (single cells) was 27% smaller than that of single wild-type cells, whereas that of single TMpGTS1 cells was 48% larger. Furthermore, in the temperature-sensitive cdc4 mutant, the GTS1 gene affected the timing of budding at the restrictive temperature. Thus, the GTS1 gene product appears to modulate the timing of budding to obtain an appropriate cell size independent of the DNA replication cycle.

Two related regulatory sequences are required for maximal induction of Saccharomyces cerevisiae his3 transcription

1987 ◽  
Vol 7 (1) ◽  
pp. 104-110
Author(s):  
K Struhl ◽  
D E Hill
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Regulatory Region ◽  
Constitutive Expression ◽  
Initiation Site ◽  
Regulatory Sequence ◽  
Regulatory Sequences ◽  
Wild Type ◽  
Base Pair Deletion ◽  
Type Gene ◽  
Maximal Induction

In Saccharomyces cerevisiae, the coordinate induction of his3 and other amino acid biosynthesis genes is mediated by the binding of GCN4 activator protein to specific promoter sequences. The his3 regulatory region contains the sequence TGACTC, which with some variation is repeated six times upstream of the mRNA initiation site. The requirements for maximal his3 induction were examined with a series of sequential 5' deletion mutations as well as a set of small internal deletions. Deletions encroaching as far downstream as position -142 behave indistinguishably from the wild-type gene, thus indicating that the two proximal copies of the regulatory sequence are sufficient for maximal induction. Deletions with breakpoints between -137 and -99 confer inducibility, but not to the normal wild-type level. A deletion ending immediately upstream of the proximal TGACTC sequence (position -99) shows some constitutive expression that is independent of the gcn4 gene product. Deletions extending to -94 or beyond do not produce detectable levels of his3 mRNA. Small internal deletions that only remove the proximal regulatory sequence and a 1-base-pair deletion of the thymine residue at -99 abolish induction, but do not affect the basal level of transcription. These results indicate that the proximal copy between -99 and -94 is absolutely required for his3 induction, whereas the copy between -142 and -137 is required only for the maximal level of induction and is inactive by itself. From these and other observations, we suggest the possibility that these related regulatory sequences may be targets for two distinct proteins.

Sequence analysis of temperature-sensitive mutations in the Saccharomyces cerevisiae gene CDC28

1986 ◽  
Vol 6 (11) ◽  
pp. 4099-4103
Author(s):  
A T Lörincz ◽  
S I Reed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Structural Analysis ◽  
Sequence Analysis ◽  
Structural Transition ◽  
Point Mutations ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Type Gene

Eleven independently isolated temperature-sensitive mutations in the cell division cycle gene CDC28 were mapped with respect to the DNA sequence of the wild-type gene and then sequenced to determine the precise nature of each mutation. The set yielded six different point mutations, each of which predicts a single amino acid substitution in the CDC28 product. The positions of the mutations did not correlate in any obvious way with observable biological characteristics of the mutant alleles. When the positions of substitutions were collated with a predicted secondary structural analysis of the CDC28 protein kinase, they were found to correlate strongly with probable regions of structural transition.

scholarly journals Assessment of the Toxicity of CuO Nanoparticles by Using Saccharomyces cerevisiae Mutants with Multiple Genes Deleted

2015 ◽  
Vol 81 (23) ◽  
pp. 8098-8107 ◽  
Author(s):  
Shaopan Bao ◽  
Qicong Lu ◽  
Tao Fang ◽  
Heping Dai ◽  
Chao Zhang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Single Gene ◽  
Cuo Nanoparticles ◽  
Copper Salt ◽  
Cell Permeability ◽  
Striking Difference ◽  
Wild Type ◽  
Multiple Gene ◽  
Content Type ◽  
Cuo Nps

ABSTRACTTo develop applicable and susceptible models to evaluate the toxicity of nanoparticles, the antimicrobial effects of CuO nanoparticles (CuO-NPs) on variousSaccharomyces cerevisiae(S. cerevisiae) strains (wild type, single-gene-deleted mutants, and multiple-gene-deleted mutants) were determined and compared. Further experiments were also conducted to analyze the mechanisms associated with toxicity using copper salt, bulk CuO (bCuO), carbon-shelled copper nanoparticles (C/Cu-NPs), and carbon nanoparticles (C-NPs) for comparisons. The results indicated that the growth inhibition rates of CuO-NPs for the wild-type and the single-gene-deleted strains were comparable, while for the multiple-gene deletion mutant, significantly higher toxicity was observed (P< 0.05). When the toxicity of the CuO-NPs to yeast cells was compared with the toxicities of copper salt and bCuO, we concluded that the toxicity of CuO-NPs should be attributed to soluble copper rather than to the nanoparticles. The striking difference in adverse effects of C-NPs and C/Cu-NPs with equivalent surface areas also proved this. A toxicity assay revealed that the multiple-gene-deleted mutant was significantly more sensitive to CuO-NPs than the wild type. Specifically, compared with the wild-type strain, copper was readily taken up by mutant strains when cell permeability genes were knocked out, and the mutants with deletions of genes regulated under oxidative stress (OS) were likely producing more reactive oxygen species (ROS). Hence, as mechanism-based gene inactivation could increase the susceptibility of yeast, the multiple-gene-deleted mutants should be improved model organisms to investigate the toxicity of nanoparticles.

scholarly journals Genetic Analysis of the Hsm3 Protein Function in Yeast Saccharomyces cerevisiae NuB4 Complex

Genes ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 1083
Author(s):  
Tatiyana A. Evstyukhina ◽  
Elena A. Alekseeva ◽  
Dmitriy V. Fedorov ◽  
Vyacheslav T. Peshekhonov ◽  
Vladimir G. Korolev
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Function ◽  
Wild Type ◽  
Core Complex ◽  
Yeast Saccharomyces Cerevisiae ◽  
Nuclear Compartment ◽  
Spontaneous Mutagenesis ◽  
Chaperone Function ◽  
Induced Mutagenesis

In the nuclear compartment of yeast, NuB4 core complex consists of three proteins, Hat1, Hat2, and Hif1, and interacts with a number of other factors. In particular, it was shown that NuB4 complex physically interacts with Hsm3p. Early we demonstrated that the gene HSM3 participates in the control of replicative and reparative spontaneous mutagenesis, and that hsm3Δ mutants increase the frequency of mutations induced by different mutagens. It was previously believed that the HSM3 gene controlled only some minor repair processes in the cell, but later it was suggested that it had a chaperone function with its participation in proteasome assembly. In this work, we analyzed the properties of three hsm3Δ, hif1Δ, and hat1Δ mutants. The results obtained showed that the Hsm3 protein may be a functional subunit of NuB4 complex. It has been shown that hsm3- and hif1-dependent UV-induced mutagenesis is completely suppressed by inactivation of the Polη polymerase. We showed a significant role of Polη for hsm3-dependent mutagenesis at non-bipyrimidine sites (NBP sites). The efficiency of expression of RNR (RiboNucleotid Reducase) genes after UV irradiation in hsm3Δ and hif1Δ mutants was several times lower than in wild-type cells. Thus, we have presented evidence that significant increase in the dNTP levels suppress hsm3- and hif1-dependent mutagenesis and Polη is responsible for hsm3- and hif1-dependent mutagenesis.

scholarly journals Sequence analysis of temperature-sensitive mutations in the Saccharomyces cerevisiae gene CDC28.

1986 ◽  
Vol 6 (11) ◽  
pp. 4099-4103 ◽  
Author(s):  
A T Lörincz ◽  
S I Reed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Structural Analysis ◽  
Sequence Analysis ◽  
Structural Transition ◽  
Point Mutations ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Type Gene

Eleven independently isolated temperature-sensitive mutations in the cell division cycle gene CDC28 were mapped with respect to the DNA sequence of the wild-type gene and then sequenced to determine the precise nature of each mutation. The set yielded six different point mutations, each of which predicts a single amino acid substitution in the CDC28 product. The positions of the mutations did not correlate in any obvious way with observable biological characteristics of the mutant alleles. When the positions of substitutions were collated with a predicted secondary structural analysis of the CDC28 protein kinase, they were found to correlate strongly with probable regions of structural transition.

scholarly journals Bul1, a new protein that binds to the Rsp5 ubiquitin ligase in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (7) ◽  
pp. 3255-3263 ◽  
Author(s):  
H Yashiroda ◽  
T Oguchi ◽  
Y Yasuda ◽  
A Toh-E ◽  
Y Kikuchi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ubiquitin Ligase ◽  
Gradient Centrifugation ◽  
High Dose ◽  
Temperature Sensitive ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Protein Ubiquitination ◽  
C Terminus ◽  
In The Wild

We characterized a temperature-sensitive mutant of Saccharomyces cerevisiae in which a mini-chromosome was unstable at a high temperature and cloned a new gene which encodes a basic and hydrophilic protein (110 kDa). The disruption of this gene caused the same temperature-sensitive growth as the original mutation. By using the two-hybrid system, we further isolated RSP5 (reverses Spt- phenotype), which encodes a hect (homologous to E6-AP C terminus) domain, as a gene encoding a ubiquitin ligase. Thus, we named our gene BUL1 (for a protein that binds to the ubiquitin ligase). BUL1 seems to be involved in the ubiquitination pathway, since a high dose of UBI1, encoding a ubiquitin, partially suppressed the temperature sensitivity of the bul1 disruptant as well as that of a rsp5 mutant. Coexpression of RSP5 and BUL1 on a multicopy plasmid was toxic for mitotic growth of the wild-type cells. Pulse-chase experiments revealed that Bul1 in the wild-type cells remained stable, while the bands of Bul1 in the rsp5 cells were hardly detected. Since the steady-state levels of the protein were the same in the two strains as determined by immunoblotting analysis, Bul1 might be easily degraded during immunoprecipitation in the absence of intact Rsp5. Furthermore, both Bul1 and Rsp5 appeared to be associated with large complexes which were separated through a sucrose gradient centrifugation, and Rsp5 was coimmunoprecipitated with Bul1. We discuss the possibility that Bul1 functions together with Rsp5 in protein ubiquitination.

scholarly journals Heterogeneous functional Ty1 elements are abundant in the Saccharomyces cerevisiae genome.

Genetics ◽  
1994 ◽  
Vol 136 (4) ◽  
pp. 1245-1259 ◽  
Author(s):  
M J Curcio ◽  
D J Garfinkel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rare Event ◽  
Yeast Genome ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Negative Effect ◽  
In Trans ◽  
Ty1 Retrotransposition ◽  
In Cis ◽  
Restriction Site Polymorphisms

Abstract Despite the abundance of Ty1 RNA in Saccharomyces cerevisiae, Ty1 retrotransposition is a rare event. To determine whether transpositional dormancy is the result of defective Ty1 elements, functional and defective alleles of the retrotransposon in the yeast genome were quantitated. Genomic Ty1 elements were isolated by gap repair-mediated recombination of pGTy1-H3(delta 475-3944) HIS3, a multicopy plasmid containing a GAL1/Ty1-H3 fusion element lacking most of the gag domain (TYA) and the protease (PR) and integrase (IN) domains. Of 39 independent gap repaired pGTyHIS3 elements isolated, 29 (74%) transposed at high levels following galactose induction. The presence of restriction site polymorphisms within the gap repaired region of the 29 functional pGTyHIS3 elements indicated that they were derived from at least eight different genomic Ty1 elements and one Ty2 element. Of the 10 defective pGTyHIS3 elements, one was a partial gap repair event while the other nine were derived from at least six different genomic Ty1 elements. These results suggest that most genomic Ty1 elements encode functional TYA, PR and IN proteins. To understand how functional Ty1 elements are regulated, we tested the hypothesis that a TYB protein associates preferentially in cis with the RNA template that encodes it, thereby promoting transposition of its own element. A genomic Ty1 mhis3AI element containing either an in-frame insertion in PR or a deletion in TYB transposed at the same rate as a wild-type Ty1mhis3AI allele, indicating that TYB proteins act efficiently in trans. This result suggests in principle that defective genomic Ty1 elements could encode trans-acting repressors of transposition; however, expression of only one of the nine defective pGTy1 isolates had a negative effect on genomic Ty1 mhis3AI element transposition in trans, and this effect was modest. Therefore, the few defective Ty1 elements in the genome are not responsible for transpositional dormancy.

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