Repair deficiency, mutator activity, and thermal prophage inducibility in dna-8132 strains of Bacillus subtilis.

A ts mutation, dna-8132 (Hara and Yoshikawa, 1973), in the region of chromosome replication origin of Bacillus subtilis was found to cause pleiotropic effects at a permissive temperature (30 C). Strains carrying this mutation were lethan at 48 C but exhibited higher spontaneous mutation frequency and a lower capacity for repairing radiation damages at 30C. Introduction of the polA59 (Gass et al., 1971) mutation further enhanced the repair deficiency and the mutator activity. These results suggest that the dna-8132 gene product may be directly involved in chromosome replication and repair. SPO2 lysogens carrying this mutation produced mature phages upon a temperature shift from 30 to 48 C. Phage production at nonpermissive temperature suggests that there are few defects in the precursors of deoxyribonucleic acid synthesis in the mutant.     

Bacillus subtilis mutant with temperature-sensitive net synthesis of phosphatidylethanolamine.

Bacillus subtilis mutants with temperature-sensitive growth on complex media were screened for defects in phospholipid metabolism. One mutant was isolated that showed temperature-sensitive net synthesis of phosphatidylethanolamine. The mutant did not accumulate phosphatidylserine at the nonpermissive temperature. In the presence of hydroxylamine, wild-type B. subtilis accumulated phosphatidylserine at both 32 and 45 degrees C, whereas the mutant did only at 32 degrees C. In vitro phosphatidylethanolamine synthesis with bacterial membranes is no more temperature sensitive with mutant membranes than with wild-type membranes. The mutation probably affects the synthesis indirectly, possibly by altering a membrane protein. The mutant bacteria grew at the nonpermissive temperature, 45 degrees C, in a phosphate buffer-based minimal medium, although net synthesis of phosphatidylethanolamine was also temperature sensitive in this medium. One mutation caused both temperature-sensitive growth on complex media and temperature-sensitive net synthesis of phosphatidylethanolamine. The mutation is linked to aroD by transformation.     

Escherichia coli rpoC397 encodes a temperature-sensitive C-terminal frameshift in the beta' subunit of RNA polymerase that blocks growth of bacteriophage P2.

Escherichia coli 397c is temperature sensitive for growth at 43.5 degrees C and unable to plate bacteriophage P2 at 33 degrees C. The mutation conferring these phenotypes was mapped to the rpoC gene. RNA synthesis is temperature sensitive in the mutant strain, and the beta' subunit of RNA polymerase isolated from this strain exhibits increased electrophoretic mobility. DNA sequence analysis revealed that the mutation is a deletion of 16 bp, resulting in a frameshift that leads to truncation of the beta' subunit at the carboxy terminus.     

Cold-Sensitive Mutation of Pseudomonas putida Affecting Enzyme Synthesis at Low Temperature

A cold-sensitive mutant of Pseudomonas putida has been isolated which grows normally at 30 C but is unable to grow on mandelate as a source of carbon at 15 C. The mutation results in the inability of the strain to carry out the reaction catalyzed by cis,cis-muconate lactonizing enzyme at low temperature and must lie in the structural gene for that enzyme, because the mutant enzyme produced at 30 C shows altered thermal stability. The mutant enzyme is not intrinsically cold-labile, nor is it cold-labile at the moment of synthesis. The activity of the mutant enzyme is not inhibited at low temperature. Evidence is presented to establish that this mutation in the structural gene coding for cis,cis-muconate lactonizing enzyme results in the lack of expression of that gene at low temperature.     

Isolation and characterization of a temperature-sensitive dnaK mutant of Escherichia coli B.

A temperature-sensitive dnaK mutant (strain MT112) was isolated from Escherichia coli B strain H/r30RT by thymineless death selection at 43 degrees C. By genetic mapping, the mutation [dnaK7(Ts)] was located near the thr gene (approximately 0.2 min on the may). E. coli K-12 transductants of the mutation to temperature sensitivity were assayed for their susceptibility to transducing phage lambda carrying the dnaK and/or the dnaJ gene. All of the transductants were able to propagate phage lambda carrying the dnaK gene. When macromolecular synthesis of the mutant was assayed at 43 degrees C, it was observed that both deoxyribonucleic acid and ribonucleic acid syntheses were severely inhibited. Thus, it was suggested that the conditionally defective dnaK mutation affects both cellular deoxyribonucleic acid and ribonucleic acid syntheses at the nonpermissive temperature in addition to inability to propagate phage lambda at permissive temperature.     

Assembly of the mitochondrial ribosomes in a temperature-conditional mutant of Saccharomyces cerevisiae defective in the synthesis of the var1 protein

An investigation of the role of the var1 protein in the assembly of the yeast mitochondrial ribosomes was carried out in a temperature conditional mutant, strain h56, which contains a mutation (tsv1) just upstream of the structural gene for the var1 protein. The mutation results in a marked decrease in the synthesis of the var1 protein at the permissive temperature of 28 degrees C and an apparently complete absence of var1 synthesis at the restrictive temperature of 36 degrees C. Long-term growth of strain h56 at the non-permissive temperature was found to result in the loss of the small (37 S) ribosomal subunit and the appearance of a novel 30 S ribonucleoparticle. Both the small (37 S) and the large (54 S) mitochondrial ribosomal subunits were found to be assembled in strain h56 for at least 3 h after transfer to the non-permissive temperature.     

A temperature-sensitive mutation affecting synthesis of outer arm dyneins in Tetrahymena thermophila.

We have characterized a novel, temperature-sensitive mutation affecting motility in Tetrahymena thermophila. Mutants grew and divided normally at the restrictive temperature (38 degrees C), but became nonmotile. Scanning electron microscopic analysis indicated that nonmotile mutants contained the normal number of cilia and that the cilia were of normal length. Transmission electron microscopic analysis indicated that axonemes isolated from nonmotile mutants lacked outer dynein arms, so the mutation was named oad 1 (outer arm deficient). Motile mutants shifted to 38 degrees C under conditions that prevent cell growth and division (starvation) remained motile suggesting that once assembled into axonemes at the permissive temperature (28 degrees C) the outer arm dyneins remain functional at 38 degrees C. Starved, deciliated mutants regenerated a full complement of functional cilia at 38 degrees C, indicating that the mechanism that incorporates the outer arm dynein into developing axonemes is not affected by the oad 1 mutation. Starved, nonmotile mutants regained motility when shifted back to 28 degrees C, but not when incubated with cycloheximide. We interpret these results to rule out the hypothesis that the oad 1 mutation affects the site on the microtubules to which the outer arm dyneins bind. Axonemes isolated from mutants grown for one generation at 38 degrees C had a mean of 6.0 outer arm dyneins, and axonemes isolated from mutants grown for two generations at 38 degrees C had a mean of 3.2 outer arm dyneins. Taken together, these results indicate that the oad 1 mutation affects the synthesis of outer arm dyneins in Tetrahymena.     

A Temperature-Sensitive Mutation Affecting Synthesis of Outer Arm Dyneins in Tetrahymena thermophila

ABSTRACT. We have characterized a novel, temperature-sensitive mutation affecting motility in Tetrahymena thermophila . Mutants grew and divided normally at the restrictive temperature (38°C), but became nonmotile. Scanning electron microscopic analysis indicated that nonmotile mutants contained the normal number of cilia and that the cilia were of normal length. Transmission electron microscopic analysis indicated that axonemes isolated from nonmotile mutants lacked outer dynein arms, so the mutation was named oad I ( outer arm defficient ). Motile mutants shifted to 38° C under conditions that prevent cell growth and division (starvation) remained motile suggesting that once assembled into axonemes at the permissive temperature (28° C) the outer arm dyneins remain functional at 38° C. Starved, deciliated mutants regenerated a full complement of functional cilia at 38° C, indicating that the mechanism that incorporates the outer arm dynein into developing axonemes is not affected by the oad I mutation. Starved, nonmotile mutants regained motility when shifted back to 28° C, but not when incubated with cycloheximide. We interpret these results to rule out the hypothesis that the oad I mutation affects the site on the microtubules to which the outer arm dyneins bind. Axonemes isolated from mutants grown for one generation at 38° C had a mean of 6.0 outer arm dyneins, and axonemes isolated from mutants grown for two generations at 38° C had a mean of 3.2 outer arm dyneins. Taken together, these results indicate that the oad I mutation affects the synthesis of outer arm dyneins in Tetrahymena .     

Isolation and characterization of a temperature-sensitive conditional mutant of Escherichia coli altered for the control of phosphate-regulated proteins

We have identified a conditional mutation which confers a ple?otropic phenotype to Escherichia coli cells: no growth at temperature higher than 36 degrees C, an altered control of the synthesis of several phosphate-regulated polypeptides (including alkaline phosphatase, sn-glycerol-3-phosphate binding protein, phosphate binding protein and outer membrane porin protein PhoE) after growth at 36 degrees C and a wild-type phenotype at 30 degrees C. This mutation was located at minute 89.5 on the E. coli chromosome in a gene we have called cpr for conditional phosphate-regulated.     

Lack of UV-induced respiration shutoff in a recF strain of Escherichia coli: temperature conditional suppression at 30 degrees C by the sfrA mutation

A mutation in the recF gene of Escherichia coli results in a radiation-sensitive strain. The RecF pathway and the RecBC pathway account for nearly all of the conjugative recombination occuring in E. coli. recBC cells are radiation-sensitive and carry only out a small amount of recombination but these deficiencies are suppressed by an sbcB as recombination is shunted to the RecF pathway. A recBC sbcB recF strain is very radiation-sensitive and is devoid of recombination ability. These deficiencies are suppressed by the srfA mutation; srfA is a recA allele. UV-induced respiration shutoff is a recA⁺, lexA⁺ and recBC⁺ dependent. We report in this paper that respiration does not shutoff in a recF strain at 37 and 30°C. an srfA mutation suppresses this lack of respiration shutoff effect in a recF srfA mutant at 30°C but not at 37°C; no suppression by this mutation occurs at either temperature in a recF recBC sbcB strain. An srfA strain also does not shut off its respiration at 37°C and shows a temperature conditional UV-induced respiration shutoff response at 30°C. The srfA mutation is thought to cause an altered RecA protein to be produced and we suggest that at 37° This altered protein is temperature sensitive. We conclude from the results in this paper that the recF gene product is required for UV-induced respiration shutoff and that the RecA protein plays a special role in the induction process.     

Regulatory role of external calcium on Pythium porphyrae (Oomycota) zoospore release,development and infection in causing red rot disease of Porphyra yezoensis (Rhodophyta)

Growth at the restrictive temperature (42 degrees C) of Aspergillus nidulans B120, carrying the conditional-lethal mutation sod(VI)C1, was partially improved by the addition of 1.0 M sorbitol to the medium. The mutant grown at 42 degrees C, with osmotic stabilizer, showed abnormal hyphal morphology, a decrease in beta-1,3-glucan synthase activity as well as cell wall sugar content, but an increase in chitin synthase activity and N-acetyl-glucosamine content. The mutation also affected the secretion of extracellular protease. The temperature-dependent osmo-sensitive phenotype of a Saccharomyces cerevisiae alpha-COP mutation can be rescued by the A. nidulans sod(VI)C(+) gene. These results indicate that the sod(VI)C1 mutation affects proper processing of secretory proteins destined for the surface of cells or beyond.     

Myristic acid auxotrophy caused by mutation of S. cerevisiae myristoyl- CoA:protein N-myristoyltransferase

The S. cerevisiae myristoyl-CoA:protein N-myristoyltransferase gene (NMT1) is essential for vegetative growth. NMT1 was found to be allelic with a previously described, but unmapped and unidentified mutation that causes myristic acid (C14:0) auxotrophy. The mutant (nmt1-181) is temperature sensitive, but growth at the restrictive temperature (36 degrees C) is rescued with exogenous C14:0. Several analogues of myristate with single oxygen or sulfur for methylene group substitutions partially complement the phenotype, while others inhibit growth even at the permissive temperature (24 degrees C). Cerulenin, a fatty acid synthetase inhibitor, also prevents growth of the mutant at 24 degrees C. Complementation of growth at 36 degrees C by exogenous fatty acids is blocked by a mutation affecting the acyl:CoA synthetase gene. The nmt1-181 allele contains a single missense mutation of the 455 residue acyltransferase that results in a Gly451----Asp substitution. Analyses of several intragenic suppressors suggest that Gly451 is critically involved in NMT catalysis. In vitro kinetic studies with purified mutant enzyme revealed a 10-fold increase in the apparent Km for myristoyl-CoA at 36 degrees C, relative to wild-type, that contributes to an observed 200-fold reduction in catalytic efficiency. Together, the data indicate that nmt-181 represents a sensitive reporter of the myristoyl-CoA pools utilized by NMT.     

Effects of high-temperature treatments on development,viability, and heat-shock response in a temperature-sensitive cell-lethal mutant of Drosophila melanogaster

Pulses of various durations at temperatures between 29 and 38°C were applied to developing larvae of Drosophila melanogaster carrying the temperature-sensitive cell-lethal mutation 1 (1)ts726. The results show that it is not possible to reduce the time required for the induction of abnormalities in the mutant by treating larvae with heat pulses at temperatures higher than 29°C. Instead, treatment with high temperature leads to fewer abnormalities than 29°C treatments. Furthermore with high temperature treatments, the mutation has less effect on viability than is seen at 29°C. It is suggested that 1 (1)ts726 leads to abnormalities and death by a temperature-induced imbalance between different physiological or development events, rather than by interfering with the ability of the cell or the organism to withstand high temperature in general.     

The biological activity of Humanin analogs correlates with structure stabilities in solution

A single mutation has resulted in large differences in neuroprotective activity of a 24 amino acid Humanin (HN). A mutation of Ser7Ala (S7A-HN) resulted in loss of activity, while a mutation of Ser14Gly (S14G-HN) resulted in about 1000-fold increase. The mechanism of the effects conferred by these mutations have been totally unclear, although our recent structure analysis suggested a possibility of the effect of mutation on the structure stability. Here, we have studied the effects of buffer and temperature on the structure of these three HN peptides. These peptides showed a similar disordered structure at 10 °C in 10 mM phosphate, pH 6.0. They were also similar in phosphate-buffered saline (PBS) as long as the temperature was kept low at 10 °C. However, a large difference was observed in both phosphate buffer and PBS between the peptides, when the temperature was raised to a physiological temperature of 37 °C. While S14G-HN showed small changes in both solutions at 37 °C, the less active HN and inactive S7A-HN showed much larger changes under the identical conditions. In addition, it appeared that structure change at 37 °C was faster for S7A-HN than HN. These results show that the structure stability at 37 °C increases in the order of S7A-HN, HN and S14G-HN, in correlation with their neuroprotective activities.     

Isolation of a dnaA mutant of Bacillus subtilis defective in initiation of replication: amount of DnaA protein determines cells'' initiation potential.

The dnaA gene is essential for initiation of chromosomal replication in Escherichia coli. A gene homologous with the E. coli dnaA was found in the replication origin region of the Bacillus subtilis chromosome. We have now isolated a temperature sensitive mutant of the B. subtilis dnaA by in vitro mutagenesis of the cloned gene. At a nonpermissive temperature, 49 degrees C, DNA replication stops completely after 60% increase in a rich medium, while cell mass continues to increase exponentially at 2.5 times the rate at 30 degrees C. A ratio of gene frequency between purA (origin marker) and metB (terminus marker) changes gradually from 2.7 at 30 degrees C to 1.0 in 45 min at 49 degrees C, indicating completion of the ongoing replication cycle. Upon the temperature shift down to 30 degrees C after the incubation at 49 degrees C for 60 min, DNA replication resumes without delay, and the purA/metB ratio increases rapidly to 6, i.e. consecutive initiation of more than two rounds of replication. Addition of chloramphenicol at the time of the temperature shift down did not inhibit the increase in the purA/metB ratio, while rifampicin inhibited the re-initiation completely. The mutation is a single base change from C to T in the dnaA gene resulting in an amino acid substitution from Ser to Phe in the DnaA protein. The mutation was responsible for both temperature sensitive growth and the defect in initiation of chromosomal replication. We observed a remarkable correlation between the amount of DnaA protein and the amount of initiation potential accumulated during incubation at the non-permissive temperature.     

Analysis of a newly-isolated temperature sensitive maternal effect mutation of Drosophila melanogaster.

A mutation located near the tip of the X chromosome in Drosophila melanogaster has been isolated, and its developmental effects described. This mutation (1(1)ts-1 is temperature sensitive, and at permissive temperature (18 degrees C) develops normally. However, zygotes from females raised or aged at restrictive temperature (28 degrees C) never hatch, regardless of the embryonic genotype. Midgut formation is abnormal in lethal zygotes and dorsal closure is probably incomplete. Temperature shift experiments have shown that the zygotic lethality is governed by a temperature sensitive period in oocytes of stage seven or older. If viable 1(1)ts-1 embryos are shifted to restrictive temperatures, they develop as far as the pupal stage, but never eclose. The temperature sensitive period for pupal lethality includes the last 2.5 days of pupal development and does not involve a maternal effect.     

Modulation of gene expression by promoter mutants of the lambdacI857/pRM/pR system

Gene expression driven by the p(R) promoter of the lambdacI857/p(RM)/p(R) system results from inactivation of the temperature-sensitive CI857 repressor. The CI857 repressor, whose gene is transcribed by the divergently orientated p(RM) promoter, is destabilised at temperatures above 30 degrees C. In this study, the lambdacI857/p(RM)/p(R) system was modified by the introduction of a single (A-32G) and a double mutation (A-32G and T-41C). The mutated lambdap(R) expression modules, 32G and 32G/41C, tightly repressed the highly lethal phage PhiX174 lysis gene E at temperatures up to 37 and 39 degrees C, respectively. Expression of protein E and subsequent lysis of Escherichia coli was still induced by a temperature up-shift to 42 degrees C. The impact of the mutations on gene expression levels driven by the lambdap(R) and p(RM) promoters was evaluated at various temperatures using the lacZ reporter gene. Results indicate that the A-32G mutation confers a lambdap(R) promoter-down phenotype. The additional increase in the temperature stability of the 32G/41C expression system is due to the T-41C mutation leading to a higher p(RM) activity. The described lambdap(R) expression modules can be used to obtain a defined expression level at a given temperature and to tightly repress in particular highly lethal genes at different bacterial growth temperatures.     

Isolation and preliminary characterization of a phosphonoacetic acid-resistant and temperature-sensitive mutant of herpes simplex virus type 1.

A group of 43 phosphonoacetic acid (PAA)-resistant mutants of herpes simplex virus type 1 was isolated after the mutagenesis of infected cells with nitrosoguanidine. One of these mutants, designated PAA1rts1, was found to be temperature sensitive (ts), that is, unable to replicate at 39.5 degrees C, the nonpermissive temperature. Recombination analysis of PAA1rts1 indicated that the PAA1r mutation and the ts1 mutation are loosely linked and are located on two separate genes. PAA1rts1 showed a defect in viral DNA synthesis at 39.5 degrees C, which presumably can be attributed to the production of a PAA-resistant and thermolabile DNA polymerase. PAA1rts1 was also defective in the shutoff of host DNA synthesis at the restrictive temperature.     

Two distinct effects on neurotransmission in a temperature-sensitive SNAP-25 mutant.

Vesicle fusion in eukaryotic cells is mediated by SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). In neurons, the t-SNARE SNAP-25 is essential for synaptic vesicle fusion but its exact role in this process is unknown. We have isolated a SNAP-25 temperature-sensitive paralytic mutant in Drosophila, SNAP-25(ts). The mutation causes a Gly50 to Glu change in SNAP-25's first amphipathic helix. A similar mutation in the yeast homologue SEC9 also results in temperature sensitivity, implying a conserved role for this domain in secretion. In vitro-generated 70 kDa SNARE complexes containing SNAP-25(ts) are thermally stable but the mutant SNARE multimers (of approximately 120 kDa) rapidly dissociate at 37 degrees C. The SNAP-25(ts) mutant has two effects on neurotransmitter release depending upon temperature. At 22 degrees C, evoked release of neurotransmitter in SNAP-25(ts) larvae is greatly increased, and at 37 degrees C, the release of neurotransmitter is reduced as compared with controls. Our data suggest that at 22 degrees C the mutation causes the SNARE complex to be more fusion competent but, at 37 degrees C the same mutation leads to SNARE multimer instability and fusion incompetence.     

The Characteristics and Genetic Map Location of a Temperature Sensitive DNA Mutant of E. COLI K12

A temperature sensitive strain of E. coli K12 has been isolated in which residual DNA synthesis occurs at the 40 degrees C restrictive temperature; syntheses of RNA, protein and DNA precursors are not directly affected. The mutation has been designated dna-325 and is located at 89 min on the E. coli map in the same region where the dnaC locus is found. dnaC mutants are considered to be defective in DNA initiation. Some of the data are consistent with the view that the dna-325 mutation is temperature sensitive in the process of DNA initiation rather than DNA chain elongation: (1) more than two cell divisions occur after a shift to 40 degrees C; (2) upon a shift down to 30 degrees C, cell division occurs again only after the DNA content of the cells has doubled; (3) 80% more DNA is made at 30 degrees C in the presence of chloramphenicol after prior inhibition of DNA synthesis at 40 degrees C. These three observations indicate that rounds of DNA replication were completed at 40 degrees C. Also (4) infective lambda particles can be made at 40 degrees C long after bacterial DNA replication has ceased. It appears however that some DNA initiation can occur at 40 degrees C since (1) a limited amount of DNA synthesis does occur at 40 degrees C after prior alignment of the chromosomes by amino acid starvation at 30 degrees C, and (2) after incubation in bromouracil at the restrictive temperature, heavy DNA is found with both strands containing bromouracil.