scholarly journals Identification of two proteins encoded by the Saccharomyces cerevisiae GAL4 gene.

1984 ◽  
Vol 4 (2) ◽  
pp. 268-275 ◽  
Author(s):  
A Laughon ◽  
R Driscoll ◽  
N Wills ◽  
R F Gesteland
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Regulatory System ◽  
Wild Type ◽  
High Level Expression ◽  
Gal1 Promoter ◽  
Encoded Proteins ◽  
High Level

We placed the Saccharomyces cerevisiae GAL4 gene under control of the galactose regulatory system by fusing it to the S. cerevisiae GAL1 promoter. After induction with galactose, GAL4 is now transcribed at about 1,000-fold higher levels than in wild-type S. cerevisiae. This regulated high-level expression has enabled us to tentatively identify two GAL4-encoded proteins.

Identification of two proteins encoded by the Saccharomyces cerevisiae GAL4 gene

1984 ◽  
Vol 4 (2) ◽  
pp. 268-275
Author(s):  
A Laughon ◽  
R Driscoll ◽  
N Wills ◽  
R F Gesteland
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Regulatory System ◽  
Wild Type ◽  
High Level Expression ◽  
Gal1 Promoter ◽  
Encoded Proteins ◽  
High Level

We placed the Saccharomyces cerevisiae GAL4 gene under control of the galactose regulatory system by fusing it to the S. cerevisiae GAL1 promoter. After induction with galactose, GAL4 is now transcribed at about 1,000-fold higher levels than in wild-type S. cerevisiae. This regulated high-level expression has enabled us to tentatively identify two GAL4-encoded proteins.

Translation in Saccharomyces cerevisiae: initiation factor 4E-dependent cell-free system

1989 ◽  
Vol 9 (10) ◽  
pp. 4467-4472
Author(s):  
M Altmann ◽  
N Sonenberg ◽  
H Trachsel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Point Mutations ◽  
Translation Initiation Factor ◽  
Apparent Temperature ◽  
Initiation Factor ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Free System ◽  
Gal1 Promoter

The gene encoding translation initiation factor 4E (eIF-4E) from Saccharomyces cerevisiae was randomly mutagenized in vitro. The mutagenized gene was reintroduced on a plasmid into S. cerevisiae cells having their only wild-type eIF-4E gene on a plasmid under the control of the regulatable GAL1 promoter. Transcription from the GAL1 promoter (and consequently the production of wild-type eIF-4E) was then shut off by plating these cells on glucose-containing medium. Under these conditions, the phenotype conferred upon the cells by the mutated eIF-4E gene became apparent. Temperature-sensitive S. cerevisiae strains were identified by replica plating. The properties of one strain, 4-2, were further analyzed. Strain 4-2 has two point mutations in the eIF-4E gene. Upon incubation at 37 degrees C, incorporation of [35S]methionine was reduced to 15% of the wild-type level. Cell-free translation systems derived from strain 4-2 were dependent on exogenous eIF-4E for efficient translation of certain mRNAs, and this dependence was enhanced by preincubation of the extract at 37 degrees C. Not all mRNAs tested required exogenous eIF-4E for translation.

Characterization of cDNA encoding mouse DNA repair protein O6-methylguanine-DNA methyltransferase and high-level expression of the wild-type and mutant proteins in E. coli

Biochemistry ◽  
1992 ◽  
Vol 31 (7) ◽  
pp. 1897-1903 ◽  
Author(s):  
Susumu Shiota ◽  
Mathew A. Von Wronski ◽  
Keizo Tano ◽  
Darell D. Bigner ◽  
Thomas P. Brent ◽  
...  
Keyword(s):  
Dna Methyltransferase ◽  
Wild Type ◽  
High Level Expression ◽  
Mutant Proteins ◽  
E Coli ◽  
Methylguanine Dna Methyltransferase ◽  
Repair Protein ◽  
Dna Repair Protein ◽  
High Level

Stoichiometry of G protein subunits affects the Saccharomyces cerevisiae mating pheromone signal transduction pathway

1990 ◽  
Vol 10 (2) ◽  
pp. 510-517
Author(s):  
G M Cole ◽  
D E Stone ◽  
S I Reed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Signal Transduction ◽  
G Protein ◽  
Signal Transduction Pathway ◽  
Mating Pheromone ◽  
Gal1 Promoter ◽  
Gamma Subunits ◽  
Adaptation Response ◽  
High Level

The Saccharomyces cerevisiae GPA1, STE4, and STE18 genes encode products homologous to mammalian G-protein alpha, beta, and gamma subunits, respectively. All three genes function in the transduction of the signal generated by mating pheromone in haploid cells. To characterize more completely the role of these genes in mating, we have conditionally overexpressed GPA1, STE4, and STE18, using the galactose-inducible GAL1 promoter. Overexpression of STE4 alone, or STE4 together with STE18, generated a response in haploid cells suggestive of pheromone signal transduction: arrest in G1 of the cell cycle, formation of cellular projections, and induction of the pheromone-inducible transcript FUS1 25- to 70-fold. High-level STE18 expression alone had none of these effects, nor did overexpression of STE4 in a MATa/alpha diploid. However, STE18 was essential for the response, since overexpression of STE4 was unable to activate a response in a ste18 null strain. GPA1 hyperexpression suppressed the phenotype of STE4 overexpression. In addition, cells that overexpressed GPA1 were more resistant to pheromone and recovered more quickly from pheromone than did wild-type cells, which suggests that GPA1 may function in an adaptation response to pheromone.

scholarly journals Effects of controlled RAD52 expression on repair and recombination in Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (4) ◽  
pp. 2013-2017 ◽  
Author(s):  
K J Dornfeld ◽  
D M Livingston
Keyword(s):  
Fold Increase ◽  
Mitotic Recombination ◽  
Protein Product ◽  
Wild Type ◽  
Recombination Rates ◽  
Plasmid Recombination ◽  
Gal1 Promoter ◽  
High Level ◽  
Rate Limiting ◽  
Mutant Cells

We have examined the effects of RAD52 overexpression on methyl methanesulfonate (MMS) sensitivity and spontaneous mitotic recombination rates. Cells expressing a 10-fold excess of RAD52 mRNA from the ENO1 promoter are no more resistant to MMS than are wild-type cells. Similarly, under the same conditions, the rate of mitotic recombination within a reporter plasmid does not exceed that measured in wild-type cells. This high level of expression is capable of correcting the defects of rad52 mutant cells in carrying out repair and recombination. From these observations, we conclude that wild-type amounts of Rad52 are not rate limiting for repair of MMS-induced lesions or plasmid recombination. By placing RAD52 under the control of the inducible GAL1 promoter, we find that induction results in a 12-fold increase in the fraction of recombinants within 4 h. After this time, the fraction increases less rapidly. When RAD52 expression is quickly repressed during induction, the amount of RAD52 mRNA decreases rapidly and no nascent recombinants are formed. This result suggests a short active half-life for the protein product. Induction of RAD52 in G1-arrested mutant cells also causes a rapid increase in recombinants, suggesting that replication is not necessary for plasmid recombination.

scholarly journals Posttranslational control of Ty1 retrotransposition occurs at the level of protein processing.

1992 ◽  
Vol 12 (6) ◽  
pp. 2813-2825 ◽  
Author(s):  
M J Curcio ◽  
D J Garfinkel
Keyword(s):  
Protein Processing ◽  
Yeast Cells ◽  
Translational Efficiency ◽  
Gene Products ◽  
Protease Domain ◽  
High Level Expression ◽  
Gal1 Promoter ◽  
Low Levels ◽  
Ty1 Retrotransposition ◽  
High Level

High-level expression of a transpositionally competent Ty1 element fused to the inducible GAL1 promoter on a 2 microns plasmid (pGTy1) overcomes transpositional dormancy in Saccharomyces cerevisiae. To investigate the mechanisms controlling the rate of Ty1 retrotransposition, we quantitated transposition and Ty1 gene products in cells induced and uninduced for expression of pGTy1. The increase in Ty1 transposition was 45- to 125-fold greater than the increase in Ty1 RNA effected by pGTy1 induction. Translational efficiency of Ty1 RNA was not altered in transposition-induced cells, since p190TYA1-TYB1 protein synthesis increased in proportion to steady-state Ty1 RNA levels. Therefore, expression of a pGTy1 element increases the efficiency of Ty1 transposition at a posttranslational level. Galactose induction of pGTy1 enhanced TYA1 protein processing and allowed detection of processed TYB1 proteins, which are normally present at very low levels in uninduced cells. When the ability of genomic Ty1 elements to complement defined mutations in HIS3-marked pGTy1 elements was examined, mutations in the protease domain or certain mutations in the integrase domain failed to be complemented, but mutations in the reverse transcriptase domain were partially complemented by genomic Ty1 elements. Therefore, the activity of Ty1 elements in yeast cells may be limited by the availability of Ty1 protease and possibly integrase. These results suggest that Ty1 transposition is regulated at the level of protein processing and that this regulation is overcome by expression of a pGTy1 element.

scholarly journals Structural Insights into the Azole Resistance of the Candida albicans Darlington Strain Using Saccharomyces cerevisiae Lanosterol 14α-Demethylase as a Surrogate

2021 ◽  
Vol 7 (11) ◽  
pp. 897
Author(s):  
Danyon O. Graham ◽  
Rajni K. Wilson ◽  
Yasmeen N. Ruma ◽  
Mikhail V. Keniya ◽  
Joel D. A. Tyndall ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
Crystal Structures ◽  
Differential Susceptibility ◽  
Azole Resistance ◽  
Wild Type ◽  
Gene Encoding ◽  
High Level ◽  
Structural Insights ◽  
Level Resistance

Target-based azole resistance in Candida albicans involves overexpression of the ERG11 gene encoding lanosterol 14α-demethylase (LDM), and/or the presence of single or multiple mutations in this enzyme. Overexpression of Candida albicans LDM (CaLDM) Y132H I471T by the Darlington strain strongly increased resistance to the short-tailed azoles fluconazole and voriconazole, and weakly increased resistance to the longer-tailed azoles VT-1161, itraconazole and posaconazole. We have used, as surrogates, structurally aligned mutations in recombinant hexahistidine-tagged full-length Saccharomyces cerevisiae LDM6×His (ScLDM6×His) to elucidate how differential susceptibility to azole drugs is conferred by LDM of the C. albicans Darlington strain. The mutations Y140H and I471T were introduced, either alone or in combination, into ScLDM6×His via overexpression of the recombinant enzyme from the PDR5 locus of an azole hypersensitive strain of S. cerevisiae. Phenotypes and high-resolution X-ray crystal structures were determined for the surrogate enzymes in complex with representative short-tailed (voriconazole) and long-tailed (itraconazole) triazoles. The preferential high-level resistance to short-tailed azoles conferred by the ScLDM Y140H I471T mutant required both mutations, despite the I471T mutation conferring only a slight increase in resistance. Crystal structures did not detect changes in the position/tilt of the heme co-factor of wild-type ScLDM, I471T and Y140H single mutants, or the Y140H I471T double-mutant. The mutant threonine sidechain in the Darlington strain CaLDM perturbs the environment of the neighboring C-helix, affects the electronic environment of the heme, and may, via differences in closure of the neck of the substrate entry channel, increase preferential competition between lanosterol and short-tailed azole drugs.

High-Level Expression and In Vivo Processing of Chimeric Ubiquitin Fusion Proteins in Saccharomyces Cerevisiae

1989 ◽  
Vol 7 (7) ◽  
pp. 705-709 ◽  
Author(s):  
Elizabeth A. Sabin ◽  
Chun Ting Lee-Ng ◽  
Jeffrey R. Shuster ◽  
Philip J. Barr
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fusion Proteins ◽  
High Level Expression ◽  
High Level
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