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 AN ENRICHMENT FOR TEMPERATURE-SENSITIVE MUTANTS IN TETRAHYMENA THERMOPHILA

Genetics ◽  
1979 ◽  
Vol 92 (4) ◽  
pp. 1079-1092
Author(s):  
Duane W Martindale ◽  
Ronald E Pearlman
Keyword(s):  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Macromolecular Synthesis ◽  
Treatment Results ◽  
Uv Treatment ◽  
Temperature Sensitive Mutants ◽  
Near Ultraviolet ◽  
Treatment Data ◽  
Resistant Mutants

ABSTRACT The parameters for the killing of Tetrahymena by 5-bromodeoxyuridine (BUdR) and near-ultraviolet light have been determined. Significant preferential killing by UV* of cells that have incorporated BUdR was obtained when the cells were irradiated in a nonnutrient buffer. UV alone was found to be toxic to cells irradiated in growth medium. Mutants defective in division at a restrictive temperature were isolated from mutagenized cultures that had been treated with BUdR and UV and from mutagenized cultures that had no such treatment. Results indicate that the number of temperature sensitive (ts) growth mutants can be increased five to six times using the BUdR/UV treatment. Data are presented that indicate differences in the frequency of occurrence of various types of ts mutants, with and without enrichment. A mutant that immediately stopped macromolecular synthesis and cell division upon being placed at the restrictive temperature was more resistant to BUdR/UV treatment than wild type by 1000-fold. Using the above techniques, BUdR-resistant mutants altered in the phosphorylation of thymidine have been isolated.

Temperature-sensitive mutants in rfaI and rfaJ, genes for galactosyltransferase I and glucosyltransferase II, for synthesis of lipopolysaccharide in Salmonella typhimurium

1985 ◽  
Vol 31 (9) ◽  
pp. 861-869 ◽  
Author(s):  
Sunil K. Kadam ◽  
Mark S. Peppler ◽  
Kenneth E. Sanderson
Keyword(s):  
Salmonella Typhimurium ◽  
Temperature Sensitive ◽  
Side Chains ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Temperature Sensitive Mutants ◽  
Specific Phage ◽  
Phage P22 ◽  
Phage Sensitivity ◽  
Sensitivity Data

Certain rough mutants of Salmonella typhimurium LT2 were shown to be temperature sensitive for the production of lipopolysaccharide (LPS). When grown at the restrictive temperature (42 or 45 °C), the cells contained LPS deficient in O (somatic) side chains, based on phage-sensitivity data and gel electrophoresis of the LPS. Cells grown at the permissive temperature, 30 °C, made LPS resembling that of smooth cells. The mobility of the LPS in gels, the phage sensitivity patterns, and gas chromatographic analysis indicate that LPS of 45 °C-grown cells of SA126 (rfaJ3012) is of chemotype Rb2, with one glucose and two galactose units (and thus inferred to be due to a mutation in rfaJ), and LPS of 45 °C-grown cells of SA134 (rfaI3020) is of chemotype Rb3, with one glucose and one galactose unit (inferred to be rfaI). These inferences were confirmed, for pKZ26 (pBR322-rfaGBIJ) and pKZ27 (pBR322-rfaGBI) both complement rfaI3020, but only pKZ26 complemented rfaJ3012. In addition, pKZ26 carrying a Tn5 insertion resulting in loss of complementation of a known rfaJ mutation, but not of rfaG, B, or I, also resulted in loss of rfaJ3012 complementation. Based on gel analysis, there is a small amount of the LPS containing smooth side chains in cells of SA126 grown at 45 °C; following a switch to 30 °C, the amount of LPS with O side chains gradually increased, and the amount of core LPS was reduced, though even after 3 h the LPS does not fully resemble that of smooth strains. Cells grown at 42 °C show limited capacity to adsorb a smooth-specific phage, P22, but the capacity to adsorb the phage increased fourfold after 5 min growth at 30 °C, and by 60 min at 30 °C adsorption resembled that seen by smooth cells. These data indicate that synthesis of the smooth LPS begins less than 5 min after shift to the permissive temperature, and that phage adsorption is a more sensitive test for the appearance of smooth side chains than the use of polyacrylamide gels with silver stains.

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 Intragenic suppressors of folding defects in the P22 tailspike protein.

Genetics ◽  
1991 ◽  
Vol 127 (2) ◽  
pp. 263-277 ◽  
Author(s):  
B Fane ◽  
J King
Keyword(s):  
Amino Acid Sequences ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Chain Folding ◽  
Phage P22 ◽  
Nonsense Codons ◽  
P22 Tailspike ◽  
Polypeptide Chains ◽  
Tailspike Protein

Abstract Within the amino acid sequences of polypeptide chains little is known of the distribution of sites and sequences critical for directing chain folding and assembly. Temperature-sensitive folding (tsf) mutations identifying such sites have been previously isolated and characterized in gene 9 of phage P22 encoding the tailspike endorhamnosidase. We report here the isolation of a set of second-site conformational suppressors which alleviate the defect in such folding mutants. The suppressors were selected for their ability to correct the defects of missense tailspike polypeptide chains, generated by growth of gene 9 amber mutants on Salmonella host strains inserting either tyrosine, serine, glutamine or leucine at the nonsense codons. Second-site suppressors were recovered for 13 of 22 starting sites. The suppressors of defects at six sites mapped within gene 9. (Suppressors for seven other sites were extragenic and distant from gene 9.) The missense polypeptide chains generated from all six suppressible sites displayed ts phenotypes. Temperature-sensitive alleles were isolated at these amber sites by pseudoreversion. The intragenic suppressors restored growth at the restrictive temperature of these presumptive tsf alleles. Characterization of protein maturation in cells infected with mutant phages carrying the intragenic suppressors indicates that the suppression is acting at the level of polypeptide chain folding and assembly.

scholarly journals Role of SpoVA Proteins in Release of Dipicolinic Acid during Germination of Bacillus subtilis Spores Triggered by Dodecylamine or Lysozyme

2006 ◽  
Vol 189 (5) ◽  
pp. 1565-1572 ◽  
Author(s):  
Venkata Ramana Vepachedu ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Small Molecules ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Ph Optimum ◽  
Inner Membrane ◽  
Temperature Sensitive Mutants

ABSTRACT The release of dipicolinic acid (DPA) during the germination of Bacillus subtilis spores by the cationic surfactant dodecylamine exhibited a pH optimum of ∼9 and a temperature optimum of 60°C. DPA release during dodecylamine germination of B. subtilis spores with fourfold-elevated levels of the SpoVA proteins that have been suggested to be involved in the release of DPA during nutrient germination was about fourfold faster than DPA release during dodecylamine germination of wild-type spores and was inhibited by HgCl2. Spores carrying temperature-sensitive mutants in the spoVA operon were also temperature sensitive in DPA release during dodecylamine germination as well as in lysozyme germination of decoated spores. In addition to DPA, dodecylamine triggered the release of amounts of Ca2+ almost equivalent to those of DPA, and at least one other abundant spore small molecule, glutamic acid, was released in parallel with Ca2+ and DPA. These data indicate that (i) dodecylamine triggers spore germination by opening a channel in the inner membrane for Ca2+-DPA and other small molecules, (ii) this channel is composed at least in part of proteins, and (iii) SpoVA proteins are involved in the release of Ca2+-DPA and other small molecules during spore germination, perhaps by being a part of a channel in the spore's inner membrane.

Genetic Analysis of Yeast RPA1 Reveals Its Multiple Functions in DNA Metabolism

Genetics ◽  
1998 ◽  
Vol 148 (3) ◽  
pp. 989-1005 ◽  
Author(s):  
Keiko Umezu ◽  
Neal Sugawara ◽  
Clark Chen ◽  
James E Haber ◽  
Richard D Kolodner
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Protein A ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Physical Analysis ◽  
Single Stranded Dna ◽  
Temperature Sensitive Mutants

Abstract Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 104 to 105 times increased sensitivity to these agents. Some of the UV- and MMS-sensitive mutants were killed by an HO-induced double-strand break at MAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages.

Mutational blockage of DNA synthesis in Paramecium tetraurelia

1976 ◽  
Vol 54 (12) ◽  
pp. 2089-2097 ◽  
Author(s):  
E. L. Peterson ◽  
J. D. Berger
Keyword(s):  
Dna Synthesis ◽  
Deoxyribonucleic Acid ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Paramecium Tetraurelia ◽  
Selection System ◽  
Replication Process ◽  
Temperature Sensitive Mutants ◽  
Fold Enrichment ◽  
Comparison Of Experiments

One hundred and ninety-eight temperature-sensitive mutants of Paramecium tetraurelia were isolated after nitrosoguanidine mutagenesis. In some experiments, mutants were recovered with the aid of a bromouracil (BU) selection system. Fifty-six mutants showed cessation of cell division within one cell cycle at the restrictive temperature and were designated ts-0. Fourteen of the ts-0's showed a greater than 90% reduction in rnacronuclear deoxyribonucleic acid (DNA) synthesis at the restrictive temperature. Two ts-0. DNA-defective lines continued protein synthesis at greater than 50% the normal rate after arrest of DNA synthesis. Hence, these two mutants may be directly affected in the replication process itself. The two mutants are allelic and, in addition, a third 'leaky' allele was recovered. Comparison of experiments in which either BU selection or no selection was employed shows that a greater than 10-fold enrichment for ts mutants resulted from BU selection.

Correction of the defect in initiation of DNA replication in a temperature-sensitive mutant hamster cell line by in vitro addition of extracts from normal cells

1985 ◽  
Vol 5 (4) ◽  
pp. 902-905
Author(s):  
M Narkhammar ◽  
R Hand
Keyword(s):  
Cell Line ◽  
Nuclear Extract ◽  
Cell Extract ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Whole Cell ◽  
Mutant Cells

ts BN-2 is a temperature-sensitive hamster cell line that is defective in DNA synthesis at the restrictive temperature. The mutant expresses its defect during in vitro replication in whole-cell lysates. Addition of a high-salt-concentration extract from wild-type BHK-21, revertant RBN-2, or CHO cells to mutant cells lysed with 0.01% Brij 58 increased the activity in the mutant three- to fourfold, so that it reached 85% of the control value, and restored replicative synthesis. The presence of extract had an insignificant effect on wild-type and revertant replication and on mutant replication at the permissive temperature. Extract prepared from mutant cells was less effective than the wild-type cell extract was. Also, the stimulatory activity was more heat labile in the mutant than in the wild-type extract. Nuclear extract was as active as whole-cell extract.

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