Characterization of a temperature-sensitive mutant of Saccharomyces cerevisiae that undergoes uncontrolled protein synthesis

1976 ◽  
Vol 22 (6) ◽  
pp. 873-883 ◽  
Author(s):  
James M. Gentile ◽  
Mathew J. Nadakavukaren ◽  
Arlan Richardson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Synthesis ◽  
Wild Type Strain ◽  
Nuclear Gene ◽  
High Rate ◽  
Recessive Mutation ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Inhibitor Of Protein Synthesis

A mutant of Saccharomyces cerevisiae, DW137, was isolated after treatment of a wild-type strain with ICR-170. The mutant was respiration-deficient and showed abnormal cell division when grown at 30 °C. In addition, the mutant was temperature-sensitive and underwent lysis when grown at 37 °C. Random spore analysis, induced reversion profiles, and complementation analysis indicated that the abnormal phenotypes were under the control of a single recessive mutation caused by a base-pair substitution in a nuclear gene. Macromolecular analysis of the mutant at permissive and restrictive temperatures showed that at restrictive temperatures the mutant cannot synthesize DNA. Surprisingly, at restrictive temperatures, protein synthesis in the mutant continued at a rate greater than that observed at permissive temperatures. Cell death and lysis of the mutant could be prevented by treatment of cultures with cycloheximide, an inhibitor of protein synthesis. The data suggest that the abnormally high rate of protein synthesis and the inability to synthesize DNA are jointly responsible for death of the cells, and most probably play an integrating role in the incipient cell lysis.

Isolation of the thymidylate synthetase gene (TMP1) by complementation in Saccharomyces cerevisiae

1982 ◽  
Vol 2 (4) ◽  
pp. 437-442
Author(s):  
G R Taylor ◽  
B J Barclay ◽  
R K Storms ◽  
J D Friesen ◽  
R H Haynes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Frequency ◽  
Wild Type Strain ◽  
Biologically Active ◽  
Thymidylate Synthetase ◽  
Temperature Sensitive ◽  
Synthetase Activity ◽  
Enzymatic Conversion ◽  
Wild Type ◽  
Sensitive Mutant

The structural gene (TMP1) for yeast thymidylate synthetase (thymidylate synthase; EC 2.1.1.45) was isolated from a chimeric plasmid bank by genetic complementation in Saccharomyces cerevisiae. Retransformation of the dTMP auxotroph GY712 and a temperature-sensitive mutant (cdc21) with purified plasmid (pTL1) yielded Tmp+ transformants at high frequency. In addition, the plasmid was tested for the ability to complement a bacterial thyA mutant that lacks functional thymidylate synthetase. Although it was not possible to select Thy+ transformants directly, it was found that all pTL1 transformants were phenotypically Thy+ after several generations of growth in nonselective conditions. Thus, yeast thymidylate synthetase is biologically active in Escherichia coli. Thymidylate synthetase was assayed in yeast cell lysates by high-pressure liquid chromatography to monitor the conversion of [6-3H]dUMP to [6-3H]dTMP. In protein extracts from the thymidylate auxotroph (tmp1-6) enzymatic conversion of dUMP to dTMP was barely detectable. Lysates of pTL1 transformants of this strain, however, had thymidylate synthetase activity that was comparable to that of the wild-type strain.

scholarly journals Isolation of the thymidylate synthetase gene (TMP1) by complementation in Saccharomyces cerevisiae.

1982 ◽  
Vol 2 (4) ◽  
pp. 437-442 ◽  
Author(s):  
G R Taylor ◽  
B J Barclay ◽  
R K Storms ◽  
J D Friesen ◽  
R H Haynes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Frequency ◽  
Wild Type Strain ◽  
Biologically Active ◽  
Thymidylate Synthetase ◽  
Temperature Sensitive ◽  
Synthetase Activity ◽  
Enzymatic Conversion ◽  
Wild Type ◽  
Sensitive Mutant

The structural gene (TMP1) for yeast thymidylate synthetase (thymidylate synthase; EC 2.1.1.45) was isolated from a chimeric plasmid bank by genetic complementation in Saccharomyces cerevisiae. Retransformation of the dTMP auxotroph GY712 and a temperature-sensitive mutant (cdc21) with purified plasmid (pTL1) yielded Tmp+ transformants at high frequency. In addition, the plasmid was tested for the ability to complement a bacterial thyA mutant that lacks functional thymidylate synthetase. Although it was not possible to select Thy+ transformants directly, it was found that all pTL1 transformants were phenotypically Thy+ after several generations of growth in nonselective conditions. Thus, yeast thymidylate synthetase is biologically active in Escherichia coli. Thymidylate synthetase was assayed in yeast cell lysates by high-pressure liquid chromatography to monitor the conversion of [6-3H]dUMP to [6-3H]dTMP. In protein extracts from the thymidylate auxotroph (tmp1-6) enzymatic conversion of dUMP to dTMP was barely detectable. Lysates of pTL1 transformants of this strain, however, had thymidylate synthetase activity that was comparable to that of the wild-type strain.

scholarly journals The Ski7 Antiviral Protein Is an EF1-α Homolog That Blocks Expression of Non-Poly(A) mRNA in Saccharomyces cerevisiae

1999 ◽  
Vol 73 (4) ◽  
pp. 2893-2900 ◽  
Author(s):  
Lionel Benard ◽  
Kathleen Carroll ◽  
Rosaura C. P. Valle ◽  
Daniel C. Masison ◽  
Reed B. Wickner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Wild Type Strain ◽  
Untranslated Regions ◽  
Luciferase Expression ◽  
Temperature Sensitive ◽  
Rna Structures ◽  
Wild Type ◽  
Antiviral Protein ◽  
Double Stranded Rna ◽  
Translation Factors

ABSTRACT We mapped and cloned SKI7, a gene that negatively controls the copy number of L-A and M double-stranded RNA viruses inSaccharomyces cerevisiae. We found that it encodes a nonessential 747-residue protein with similarities to two translation factors, Hbs1p and EF1-α. The ski7 mutant was hypersensitive to hygromycin B, a result also suggesting a role in translation. The SKI7 product repressed the expression of nonpolyadenylated [non-poly(A)] mRNAs, whether capped or uncapped, thus explaining why Ski7p inhibits the propagation of the yeast viruses, whose mRNAs lack poly(A). The dependence of the Ski7p effect on 3′ RNA structures motivated a study of the expression of capped non-poly(A) luciferase mRNAs containing 3′ untranslated regions (3′UTRs) differing in length. In a wild-type strain, increasing the length of the 3′UTR increased luciferase expression due to both increased rates and duration of translation. Overexpression of Ski7p efficiently cured the satellite virus M2 due to a twofold-increased repression of non-poly(A) mRNA expression. Our experiments showed that Ski7p is part of the Ski2p-Ski3p-Ski8p antiviral system because a single ski7 mutation derepresses the expression of non-poly(A) mRNA as much as a quadruple ski2 ski3 ski7 ski8 mutation, and the effect of the overexpression of Ski7p is not obtained unless other SKI genes are functional. ski1/xrn1Δ ski2Δ and ski1/xrn1Δ ski7Δ mutants were viable but temperature sensitive for growth.

scholarly journals CDC37 is required for p60v-src activity in yeast.

1996 ◽  
Vol 7 (9) ◽  
pp. 1405-1417 ◽  
Author(s):  
B Dey ◽  
J J Lightbody ◽  
F Boschelli
Keyword(s):  
Protein Tyrosine Kinase ◽  
Wild Type Strain ◽  
Active Form ◽  
Biologically Active ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Isolation And Characterization ◽  
Genes Encoding

Mutations in genes encoding the molecular chaperones Hsp90 and Ydj1p suppress the toxicity of the protein tyrosine kinase p60v-src in yeast by reducing its levels or its kinase activity. We describe isolation and characterization of novel p60v-src-resistant, temperature-sensitive cdc37 mutants, cdc37-34 and cdc37-17, which produce less p60v-src than the parental wild-type strain at 23 degrees C. However, p60v-src levels are not low enough to account for the resistance of these strains. Asynchronously growing cdc37-34 and cdc37-17 mutants arrest in G1 and G2/M when shifted from permissive temperatures (23 degrees C) to the restrictive temperature (37 degrees C), but hydroxyurea-synchronized cdc37-34 and cdc37-17 mutants arrest in G2/M when released from the hydroxyurea block and shifted from 23 to 37 degrees C. The previously described temperature-sensitive cdc37-1 mutant is p60v-src-sensitive and produces wild-type amounts of p60v-src at permissive temperatures but becomes p60v-src-resistant at its restrictive temperature, 38 degrees C. In all three cdc37 mutants, inactivation of Cdc37p by incubation at 38 degrees C reduces p60v-src-dependent tyrosine phosphorylation of yeast proteins to low or undetectable levels. Also, p60v-src levels are enriched in urea-solubilized extracts and depleted in detergent-solubilized extracts of all three cdc37 mutants prepared from cells incubated at the restrictive temperature. These results suggest that Cdc37p is required for maintenance of p60v-src in a soluble, biologically active form.

scholarly journals Hierarchy of elements regulating synthesis of ribosomal proteins in Saccharomyces cerevisiae.

1981 ◽  
Vol 1 (11) ◽  
pp. 1016-1023 ◽  
Author(s):  
D R Kief ◽  
J R Warner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Synthesis ◽  
Ribosomal Protein ◽  
Ribonucleic Acid ◽  
Ribosomal Proteins ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Messenger Ribonucleic Acid ◽  
Ribosomal Protein Synthesis ◽  
Stimulation Of

Saccharomyces cerevisiae cells respond to a heat shock by temporarily slowing the synthesis of ribosomal proteins (C. Gorenstein and J. R. Warner, Proc. Natl. Acad. Sci. U.S.A. 73:1574-1551, 1976). When cultures growing oxidatively on ethanol as the sole carbon source were shifted from 23 to 36 degrees C, the synthesis of ribosomal proteins was coordinately inhibited twice as rapidly and 45% more severely than in comparable cultures growing fermentatively on glucose. Within 15 min, the relative rates of synthesis of at least 30 ribosomal proteins declined to less than one-sixth their initial values, whereas the overall rate of protein synthesis increased at least threefold. We suggest that this is due primarily to controls at the level of synthesis of messenger ribonucleic acid for ribosomal proteins but may also involve changes in messenger ribonucleic acid stability. In contrast, a nutritional shift-up causes a stimulation of the synthesis of ribosomal proteins. Experiments designed to determine the hierarchy of stimuli affecting the synthesis of these proteins demonstrated that temperature shock was dominant to glucose stimulation. When a culture growing on ethanol was shifted from 23 to 36 degrees C and glucose was added shortly afterward, the decline in ribosomal protein synthesis continued unabated. However, in wild-type cells ribosomal protein synthesis began to recover within 15 min. In mutants temperature sensitive for ribosome synthesis, e.g., rna2, there was no recovery in the synthesis of most ribosomal proteins, suggesting that the product of rna2 is essential for the production of these proteins under all vegetative conditions.

scholarly journals L-Phenylalanine Transport in Saccharomyces cerevisiae: Participation of GAP1, BAP2, and AGP1

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Daniel A. Sáenz ◽  
Mónica S. Chianelli ◽  
Carlos A. Stella
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Substrate Specificity ◽  
Nitrogen Source ◽  
Type Strain ◽  
Wild Type Strain ◽  
Ammonium Ion ◽  
Wild Type ◽  
Synthetic Media ◽  
Phenylalanine Transport

We focused on the participation of GAP1, BAP2, and AGP1 in L-phenylalanine transport in yeast. In order to study the physiological functions of GAP1, BAP2, and AGP1 in L-phenylalanine transport, we examined the kinetics, substrate specificity, and regulation of these systems, employing isogenic haploid strains with the respective genes disrupted individually and in combination. During the characterization of phenylalanine transport, we noted important regulatory phenomena associated with these systems. Our results show that Agp1p is the major transporter of the phenylalanine in a gap1 strain growing in synthetic media with leucine present as an inducer. In a wild type strain grown in the presence of leucine, when ammonium ion was the nitrogen source, Bap2p is the principal phenylalanine carrier.

Hierarchy of elements regulating synthesis of ribosomal proteins in Saccharomyces cerevisiae

1981 ◽  
Vol 1 (11) ◽  
pp. 1016-1023
Author(s):  
D R Kief ◽  
J R Warner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Synthesis ◽  
Ribosomal Protein ◽  
Ribonucleic Acid ◽  
Ribosomal Proteins ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Messenger Ribonucleic Acid ◽  
Ribosomal Protein Synthesis ◽  
Stimulation Of

Saccharomyces cerevisiae cells respond to a heat shock by temporarily slowing the synthesis of ribosomal proteins (C. Gorenstein and J. R. Warner, Proc. Natl. Acad. Sci. U.S.A. 73:1574-1551, 1976). When cultures growing oxidatively on ethanol as the sole carbon source were shifted from 23 to 36 degrees C, the synthesis of ribosomal proteins was coordinately inhibited twice as rapidly and 45% more severely than in comparable cultures growing fermentatively on glucose. Within 15 min, the relative rates of synthesis of at least 30 ribosomal proteins declined to less than one-sixth their initial values, whereas the overall rate of protein synthesis increased at least threefold. We suggest that this is due primarily to controls at the level of synthesis of messenger ribonucleic acid for ribosomal proteins but may also involve changes in messenger ribonucleic acid stability. In contrast, a nutritional shift-up causes a stimulation of the synthesis of ribosomal proteins. Experiments designed to determine the hierarchy of stimuli affecting the synthesis of these proteins demonstrated that temperature shock was dominant to glucose stimulation. When a culture growing on ethanol was shifted from 23 to 36 degrees C and glucose was added shortly afterward, the decline in ribosomal protein synthesis continued unabated. However, in wild-type cells ribosomal protein synthesis began to recover within 15 min. In mutants temperature sensitive for ribosome synthesis, e.g., rna2, there was no recovery in the synthesis of most ribosomal proteins, suggesting that the product of rna2 is essential for the production of these proteins under all vegetative conditions.

scholarly journals Genetic Analysis of Mutant Strains of Saccharomyces cerevisiae with Defects in Mannoprotein Synthesis

Proceedings ◽  
2020 ◽  
Vol 70 (1) ◽  
pp. 18
Author(s):  
Patricia Gil ◽  
Alberto Martínez ◽  
Elena Palencia ◽  
Rocío Velázquez ◽  
Manuel Ramírez ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Type Strain ◽  
Wild Type Strain ◽  
Point Mutations ◽  
Random Mutagenesis ◽  
Selection Method ◽  
Wild Type ◽  
Truncated Protein ◽  
Mutant Strains

Mannan defective (mnn) mutants have constituted a fundamental tool in the study of the structure and biosynthesis of mannoproteins in Saccharomyces cerevisiae. They were isolated by the group of Dr. C.E. Ballou by random mutagenesis, and a selection method using specific antibodies obtained against the wild-type strain. Initially, the mutants were characterized biochemically, and in subsequent years the genes in which they were mutated were identified. All of them encode membrane proteins that catalyze the transfer of mannoses to N-oligosaccharides, sometimes isolated or as part of complexes made up of several proteins. However, the specific mutation of each of these mutants has only been identified in the case of mnn3. In this work, we have completed the characterization of the mutants by sequencing the mutated genes in each of them. As expected, they are point mutations that involve the change of one amino acid for another in the mutated protein, or for a stop signal, resulting in a truncated protein.

scholarly journals Effects of Mutations of RAD50, RAD51, RAD52, and Related Genes on Illegitimate Recombination in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 383-391 ◽  
Author(s):  
Yasumasa Tsukamoto ◽  
Jun-ichi Kato ◽  
Hideo Ikeda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quantitative Analysis ◽  
Structural Analysis ◽  
Recombination Rate ◽  
Type Strain ◽  
Negative Selection ◽  
Wild Type Strain ◽  
Illegitimate Recombination ◽  
Wild Type ◽  
In The Wild

Abstract To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rud51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.

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