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 CHARACTERIZATION OF TEMPERATURE-SENSITIVE, FERTILIZATION-DEFECTIVE MUTANTS OF THE NEMATODE CAENORHABDITIS ELEGANS

Genetics ◽  
1978 ◽  
Vol 88 (2) ◽  
pp. 285-303 ◽  
Author(s):  
Samuel Ward ◽  
Johji Miwa
Keyword(s):  
Caenorhabditis Elegans ◽  
Sensitive Period ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Normal Numbers ◽  
Primary Defect ◽  
Isolation And Characterization ◽  
Pass Through

ABSTRACT The isolation and characterization of three Caenorhabditis elegans temperature-sensitive mutants that are defective at fertilization are described. All three are alleles of the gene fer-1. At the restrictive temperature of 25°, mutant hermaphrodites make sperm and oocytes in normal numbers. No oocytes are fertilized, although they pass through the spermatheca and uterus normally. The oocytes can be fertilized by sperm transferred by wild-type males, indicating that the mutant defect is in the sperm. The temperature-sensitive period for the mutants coincides with spermatogenesis. Sperm made by mutants at 25° cannot be distinguished from wild-type sperm by light microscopy. The sperm do contact oocytes in mutant hermaphrodites, but do not fertilize. Mutant sperm appear to be nonmotile. Mutant males are also sterile when grown at 25°. They transfer normal numbers of sperm to hermaphrodites at mating, but these sperm fail to migrate to the spermatheca and are infertile. The phenotype of these mutants is consistent with a primary defect in sperm motility, but the cause of this defect is not known.

Identification of seven new cut genes involved in Schizosaccharomyces pombe mitosis

1993 ◽  
Vol 105 (1) ◽  
pp. 135-143 ◽  
Author(s):  
I. Samejima ◽  
T. Matsumoto ◽  
Y. Nakaseko ◽  
D. Beach ◽  
M. Yanagida
Keyword(s):  
Topoisomerase Ii ◽  
Nuclear Division ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Dna Topoisomerase ◽  
Isolation And Characterization ◽  
Genes Encoding ◽  
Mixed Phenotype

Fission yeast cut mutants cause cytokinesis in the absence of normal nuclear division. These mutants show abnormal uncoupled mitosis and are known to be the result of mutations in the genes encoding DNA topoisomerase II, proteins related to spindle pole duplication, and a kinesin-related mitotic motor. We have screened 717 temperature-sensitive (ts) mutants by individually observing their cytological phenotypes at the restrictive temperature, and have newly isolated 25 cut mutants. Genetic analyses indicate that 14 of them fall into five previously identified loci, namely, top2, cut1, cut5, cut7 and cut9, whereas nine have been mapped onto seven new loci, designated cut13 to cut19. The cytological phenotypes of the newly identified cut mutants can be classified into three groups. One group consists of mutants in which a portion of the nuclear chromatin is stretched by the elongated spindle but the entire nucleus is not separated, reminiscent of, but not identical to, the phenotypes of top2 and cut1; mutants cut14-208, cut15-85, cut16-267 and cut17-275 display such a phenotype. Another group exhibits non-disjunctioned and condensed chromosomes in the presence of the spindle; cut13-131 belongs to this group. The cut19-708 mutant has also been found to have condensed chromosomes. The remaining group has a mixed phenotype of the above two groups; namely, stretched chromatin and condensed chromosomes; cut18-447 exhibits such a phenotype. The isolation and characterization of the mutated genes will be the subjects of future investigations.

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 Temperature-Sensitive Salmonella enterica Serovar Enteritidis PT13a Expressing Essential Proteins of Psychrophilic Bacteria

2015 ◽  
Vol 81 (19) ◽  
pp. 6757-6766 ◽  
Author(s):  
Barry N. Duplantis ◽  
Stephanie M. Puckett ◽  
Everett L. Rosey ◽  
Keith A. Ameiss ◽  
Angela D. Hartman ◽  
...  
Keyword(s):  
Salmonella Enterica ◽  
Type Strain ◽  
Wild Type Strain ◽  
Phage Type ◽  
The Body ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Psychrophilic Bacteria ◽  
Content Type

ABSTRACTSynthetic genes based on deduced amino acid sequences of the NAD-dependent DNA ligase (ligA) and CTP synthetase (pyrG) of psychrophilic bacteria were substituted for their native homologues in the genome ofSalmonella entericaserovar Enteritidis phage type 13a (PT13a). The resulting strains were rendered temperature sensitive (TS) and did not revert to temperature resistance at a detectable level. At permissive temperatures, TS strains grew like the parental strain in broth medium and in macrophage-like cells, but their growth was slowed or stopped when they were shifted to a restrictive temperature. When injected into BALB/c mice at the base of the tail, representing a cool site of the body, the strains with restrictive temperatures of 37, 38.5, and 39°C persisted for less than 1 day, 4 to 7 days, and 20 to 28 days, respectively. The wild-type strain persisted at the site of inoculation for at least 28 days. The wild-type strain, but not the TS strains, was also found in spleen-plus-liver homogenates within 1 day of inoculation of the tail and was detectable in these organs for at least 28 days. Intramuscular vaccination of White Leghorn chickens with the PT13a strain carrying the psychrophilicpyrGgene provided some protection against colonization of the reproductive tract and induced an anti-S. entericaantibody response.

scholarly journals Isolation and Characterization of Burkholderia cenocepacia Mutants Deficient in Pyochelin Production: Pyochelin Biosynthesis Is Sensitive to Sulfur Availability

2004 ◽  
Vol 186 (2) ◽  
pp. 270-277 ◽  
Author(s):  
Kate L. Farmer ◽  
Mark S. Thomas
Keyword(s):  
Wild Type Strain ◽  
Opportunistic Pathogen ◽  
Burkholderia Cenocepacia ◽  
Wild Type ◽  
Iron Starvation ◽  
Gene Encoding ◽  
Isolation And Characterization ◽  
Putative Operon ◽  
Green Fluorescent

ABSTRACT The opportunistic pathogen Burkholderia cenocepacia produces the yellow-green fluorescent siderophore, pyochelin. To isolate mutants which do not produce this siderophore, we mutagenized B. cenocepacia with the transposon mini-Tn5Tp. Two nonfluorescent mutants were identified which were unable to produce pyochelin. In both mutants, the transposon had integrated into a gene encoding an orthologue of CysW, a component of the sulfate/thiosulfate transporter. The cysW gene was located within a putative operon encoding other components of the transporter and a polypeptide exhibiting high homology to the LysR-type regulators CysB and Cbl. Sulfate uptake assays confirmed that both mutants were defective in sulfate transport. Growth in the presence of cysteine, but not methionine, restored the ability of the mutants to produce pyochelin, suggesting that the failure to produce the siderophore was the result of a depleted intracellular pool of cysteine, a biosynthetic precursor of pyochelin. Consistent with this, the wild-type strain did not produce pyochelin when grown in the presence of lower concentrations of sulfate that still supported efficient growth. We also showed that whereas methionine and certain organosulfonates can serve as sole sulfur sources for this bacterium, they do not facilitate pyochelin biosynthesis. These observations suggest that, under conditions of sulfur depletion, cysteine cannot be spared for production of pyochelin even under iron starvation conditions.

scholarly journals Genetic properties of temperature-sensitive folding mutants of the coat protein of phage P22.

Genetics ◽  
1994 ◽  
Vol 136 (2) ◽  
pp. 427-438 ◽  
Author(s):  
C L Gordon ◽  
J King
Keyword(s):  
Coat Protein ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Temperature Sensitive Mutants ◽  
Intragenic Complementation ◽  
Genes Encoding ◽  
Phage P22 ◽  
Polypeptide Chains ◽  
Synthetic Lethals

Abstract Temperature-sensitive mutations fall into two general classes: those generating thermolabile proteins; and those generating defects in protein synthesis, folding or assembly. Temperature-sensitive mutations at 17 sites in the gene for the coat protein of Phage P22 are of the latter class, preventing the productive folding of the polypeptide chain at restrictive temperature. We show here that, though the coat subunits interact intimately to form the viral shell, these temperature-sensitive folding (TSF) mutations were all recessive to wild type. The mutant polypeptide chains were not rescued by the presence of wild-type polypeptide chains. Missense substitutions in multimeric proteins frequently exhibit intragenic complementation; however, all pairs of coat protein TSF mutants tested failed to complement. The recessive phenotypes, absence of rescue and absence of intragenic complementation are all accounted for by the TSF defect, in which destabilization of a folding intermediate at restrictive temperature prevents the mutant chain from reaching the conformation required for subunit/subunit recognition. We suggest that absence of intragenic complementation should be a general property of TSF mutations in genes encoding multimeric proteins. The spectra of new loci identified by isolating second-site suppressors and synthetic lethals of temperature sensitive mutants will also differ depending on the nature of the defect. In the case of TSF mutations, where folding intermediates are defective rather than the native molecule, the spectra of other genes identified should shift from those whose products interact with the native molecule to those whose products influence the folding process.

scholarly journals Biochemical genetics of Neurospora nuclease I: Isolation and characterization of nuclease (nuc) mutants

1983 ◽  
Vol 41 (3) ◽  
pp. 271-286 ◽  
Author(s):  
A. M. Forsthoefel ◽  
N. C. Mishra
Keyword(s):  
Nucleic Acids ◽  
Type Strain ◽  
Wild Type Strain ◽  
Extracellular Enzymes ◽  
Spontaneous Mutation ◽  
Subsequent Paper ◽  
Wild Type ◽  
Isolation And Characterization ◽  
Phosphorus Source

SUMMARYIsolation and characterization of five new nuclease (nuc) deficient mutants ofNeurosporahave been described. The new mutants are unable to utilize nucleic acids as the sole phosphorus source and possess growth characteristics similar to thosenuc(nuc-1andnuc-2) mutants described previously. Two new mutants (nuc-4andnuc-5) were able to use RNA or predigested DNA (but not intact DNA) as phosphorus source and showed temperature sensitive growth at 37 °C. Based on the data from complementation and genetic analyses the five new nuc mutants (nuc-3, nuc-4, nuc-5, nuc-6andnuc-7) were found nonallelic to each other and to previously describednuc(nuc-1andnuc-2) mutants; the newnucmutants mapped to the right ofarg-12on linkage group II. On biochemical analyses, thesenucmutants were found to possess a lower level of extracellular nucleases and alkaline phosphatase as compared to the wild type strain. The ds DNase activity of the new mutants was only about 2–12% of that of the wild type strain; thus, the low level of these extracellular enzymes in thenucmutants causes their inability to utilize nucleic acids as the sole phosphorus source. Wild type levels of these enzymes were restored in the complementing heterokaryons capable of full growth on the DNA medium. Data from intercrosses, mutagen sensitivity and spontaneous mutation-frequency studies (as discussed in a subsequent paper) indicated the involvement of thenucgenes in DNA repair and recombination.

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.

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