Neurospora crassa temperature-sensitive mutant apparently defective in protein synthesis.

A temperature-sensitive mutant of Neurospora was isolated which appeared to be defective in the initiation of protein synthesis. The defect in mutant 34Cts was apparently due to a single gene mutation, and was recessive in heterokaryons. Conidial germination was normal and hyphal growth was nearly so in the mutant at 20 C, but both were greatly inhibited at 35 C. After 15 min at 35 C there was a reduced rate of protein synthesis, followed by decreases in ribonucleic acid and deoxyribonucleic acid synthesis. The percentage of ribosomes in polysomes declined at 35 C and the average size of polysomes decreased. Because the decrease in protein synthesis, it was believed that some part of the translational system may be affected by the mutation. Mutant 34Cts was given the designation psi-1.     

Mutants of Escherichia coli Unable to Make Protein at 42 C

Members of a collection of mutants of Escherichia coli unable to form colonies on nutrient agar at 42 C have been characterized on the basis of their growth response to a shift from 32 to 42 C in liquid medium. Forty-four mutants, which show an abrupt, nonlethal cessation of growth when moved to the restrictive temperature, have been characterized with respect to the effect of the mutation responsible for temperature sensitivity on deoxyribonucleic acid, ribonucleic acid, and protein synthesis. In 12 mutants, the mutation causing temperature sensitivity of growth primarily affects protein synthesis, in each case through an altered aminoacyl-transfer ribonucleic acid synthetase. Mutants with temperature-sensitive glutamyl-, phenylalanyl-, and valyl-transfer ribonucleic acid synthetases have been obtained, and the genes specifying these enzymes have been mapped by conjugation and transduction. Another mutant has been shown to possess a temperature-sensitive tryptophanyl-transfer ribonucleic acid synthetase, but this is not responsible for inability to grow at 42 C on media containing tryptophan.     

Ultrastructural Studies of Sporulation in a Conditionally Temperature-Sensitive Ribonucleic Acid Polymerase Mutant of Bacillus subtilis

Morphological studies of a conditionally temperature-sensitive ribonucleic acid polymerase mutant of Bacillus subtilis have revealed that sporulation is inhibited at stage II when the cells are grown at 47.5 C. Growth and sporulation occur normally at 30 C with the mutant. The mutant grows normally at 47.5 C but is prevented from sporulating at the nonpermissive temperature by an abnormal septation during forespore membrane formation which prevents the subsequent engulfment process (stage III). The mutation affects the normal functioning of ribonucleic acid polymerase at the nonpermissive temperature resulting in abortive sporulation.     

L-arabinose isomerase formation in a conditional mutant of gene araA of Escherichia coli B-r

A temperature-sensitive mutant of Escherichia coli in which the synthesis of l-arabinose isomerase is blocked during growth at 42 C was found to possess the following properties. (i) The mutation occurred in the structural gene for the isomerase, gene araA. (ii) During growth at elevated temperatures the mutant accumulates a product which is a precursor to the active enzyme. (iii) The precursor produced at 42 C is slowly converted to active enzyme at 28 C in the absence of protein and ribonucleic acid synthesis. It is concluded that the mutation results in a change in the structure of isomerase which causes formation of active enzyme to be thermolabile at a step beyond the level of translation.     

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.     

Regulation of Cell Division in Escherichia coli: Characterization of Temperature-Sensitive Division Mutants

A temperature-sensitive division mutant of Escherichia coli was isolated by using differential filtration to select for filaments at 42 C and normal cells at 30 C. Cells shifted from 30 to 42 C stop dividing almost immediately, suggesting the temperature-sensitive element is required for cell division late in the cell cycle. Cells returned to 30 from 42 C divide abruptly, suggesting accumulation of division potential at 42 C. Inhibitors of protein, deoxyribonucleic acid, and ribonucleic acid synthesis do not block division during the recovery period at 30 C. Cycloserine does not stop cell division, vancomycin shows some effect on cell division, whereas penicillin completely stops cell division during this period. The addition of high concentrations of NaCl to filaments at 42 C results in a burst of cell division. The final cell number is equivalent to the control which is grown at 30 C if sufficient salt is added (11 g/liter, final concentration). After the original burst, cell division ceases at the nonpermissive temperature even at increased osmolality. Chloramphenicol, puromycin, vancomycin, and penicillin prevent division during the recovery in the presence of NaCl. Kinetic data indicate division potential decays to a reversible inactive intermediate which rapidly decays to an irreversible inactive form. Conversion of division potential to the inactive form is correlated with a 100- to 1,000-fold derepression of the synthesis of division potential. The mutation appears to involve a stage in cross-wall synthesis which is required during the terminal stages of division.     

Biochemical and genetic characterization of a temperature-sensitive, tryptophanyl-transfer ribonucleic acid synthetase mutant of Bacillus subtilis

A temperature-sensitive, 5-fluorotryptophan (5FT)-resistant mutant of Bacillus subtilis was isolated which forms an altered tryptophanyl transfer ribonucleic acid synthetase [l-tryptophan: sRNA ligase (AMP), EC 6.1.1.2]. The mutant grows well at 30 C but not at 42 C. At the latter temperature, protein and ribonucleic acid (RNA) synthesis are abolished while deoxyribonucleic acid (DNA) synthesis proceeds for a considerable time. Tryptophanyl-transfer RNA (tRNA) synthetase activity is not detectable in the extracts of the mutant grown at 30 C whether this activity is measured by the attachment of l-tryptophan to tRNA or the l-tryptophan-dependent exchange of (32)P-pyrophosphate with adenosine triphosphate. Mixing experiments with extracts from the wild type and the mutant have ruled out the presence of an inhibitor or the absence of an activator as possible causes. Attempts to retrieve enzyme activity in vitro by various means (different conditions for cell disruption, addition of l-tryptophan, and adenosine triphosphate to the extraction buffer containing glycerol) were unsuccessful. The mutation in the locus of the tryptophanyl tRNA synthetase (trpS) was mapped on the bacterial chromosome by transformation and transduction. It is located between argC and metA. All temperature-resistant transformants recover wild-type levels of tryptophanyl tRNA synthetase activity and sensitivity to 5FT. Spontaneous revertants to temperature resistance are 5FT sensitive, but their levels of tryptophanyl tRNA synthetase activity and the thermolability of this enzyme in cell-free extracts varies. These revertants do not support the growth of a presumed nonsense mutant of phase SPO-1. Transduction experiments with phage PBS-1 indicated that reversion must be the result of an event at the site of the original mutation or at a site extremely close to it.     

Characterization of Mutants of Escherichia coli Temperature-Sensitive for Ribonucleic Acid Regulation: an Unusual Phenotype Associated with a Phenylalanyl Transfer Ribonucleic Acid Synthetase Mutant

A mutant strain AA-522, temperature-sensitive for protein synthesis, was isolated from a stringent strain (CP-78) of Escherichia coli K-12. The mutant strain has a relaxed phenotype at the nonpermissive growth temperature. Protein synthesis stops completely at 42 C, whereas the rate of ribonucleic acid (RNA) synthesis is maintained at 20% of the 30 C rate. Sucrose-gradient centrifugation analysis of RNA-containing particles formed at 42 C indicated the presence of “relaxed particles.” These particles possess 16S and 23S RNA and are precursors to normal 50S and 30S ribosomal subunits. A search for the temperature-sensitive protein responsible for the halt in protein synthesis implicated phenylalanyl transfer RNA (tRNA) synthetase. Essentially no enzyme activity is detected in vitro at 30 or 40 C. Analysis of phenylalanyl tRNA synthetase activity in revertants of strain AA-522 indicated the presence of intragenic suppressor mutations. Revertants of strain AA-522 analyzed for the relaxed response at 42 C were all stringent; strain AA-522 was stringent at 30 C. These data indicate that a single mutation in phenylalanyl tRNA synthetase is responsible for both a block in protein synthesis and the relaxed phenotype at 42 C.     

Temperature-Sensitive Mutant of Bacillus subtilis That Accumulates Membrane-Associated Protein Inclusions

At 47 C, a temperature-sensitive mutant of Bacillus subtilis 168 accumulates membrane-associated protein inclusions and exhibits a pleiotropic phenotype indicative of a defect in lipid synthesis. The mutant bacteria cease growing at 47 C, and the turbidity of the culture gradually declines. The lack of growth is not due to the death or lysis of the cells, since viability does not decrease for about 1 hr and the "lysis" can be delayed for several hours by increasing the osmotic pressure of the medium. Synthesis of deoxyribonucleic acid and ribonucleic acid stops at 47 C although a residual synthesis of protein occurs. When the temperature is raised, the mutant fails to increase the proportion of 17:0 branched-chain fatty acids and to decrease the proportion of 18:0 and 18:1 fatty acids. The membrane-associated inclusions can be seen by phase-contrast or electron microscopy and remain attached to protoplast membranes during isolation. The inclusions are mostly protein and are digested with Pronase.     

Temperature-sensitive deoxyribonucleic acid replication in a dnaC mutant of Bacillus subtilis.

A temperature-sensitive mutant of Bacillus subtilis is defective in deoxyribonucleic acid (DNA) synthesis, contains a lesion in the dnaC locus, and is not primarily an initiation mutant. The amount of DNA synthesized by this mutant at temperatures above 40 C decreases with increasing temperature. DNA synthesis resumes within 20 min after the temperature is lowered to 30 C. In the presence of chloramphenical, DNA synthesis begins at a reduced rate after the temperature is lowered to 30 C. Spores germinated at 46 C cannot initiate DNA replication. The capacity for residual DNA synthesis is stable at the restrictive temperature during inhibition of DNA synthesis. When the temperature is lowered to 30 C after a period of incubation at 43 C, DNA synthesis starts at the origin of the chromosome as well as at preexisting growing points. Similar DNA synthesis patterns are found in mutant cells in vivo and after toluene treatment.     

Strain of Escherichia coli with a temperature-sensitive mutation affecting ribosomal ribonucleic acid accumulation.

A mutant of Escherichia coli has been isolated that has a temperature-sensitive mutation that results in specific loss of ribosomal ribonucleic acid (RNA) synthesis and some reduction in messenger RNA synthesis. When the strain was grown in glucose medium at a restrictive temperature, RNA accumulation ceased, but both messenger RNA and protein synthesis continued for an extended time. Because carbon metabolism was slowed drastically when strain AA-157 was placed at the restrictive temperature, this phenotype can be compared with carbon depletion conditions present during diauxic lag. However, the phenotype of mutant AA-157 differs from shift-down conditions in that guanosine-3',5'-tetraphosphate levels are unaffected; therefore, a different site is affected. This mutant strain (AA-157) thus shows many characteristics similar to an aldolase mutant previously reported (Böck and Neidhardt, 1966). However, the mutation occurred in a different position on the E. coli genetic map, and furthermore, aldolase was not temperature sensitive in strain AA-157. In this paper we present a study of macromolecular biosynthesis in this mutant.     

Temperature-sensitive Yeast Mutant Defective in Ribonucleic Acid Production

A single, recessive mutation in a nuclear gene confers a temperature-sensitive growth response in a mutant of Saccharomyces cerevisiae, ts(-) 136. The mutant grows normally at 23 C, but exhibits a rapid and preferential inhibition of ribonucleic acid (RNA) accumulation after a shift to 36 C, demonstrating a defect in stable RNA production. Cultures of the mutant which were shifted from 23 to 36 C display the following phenomena which indicate that messenger RNA (mRNA), as well as stable RNA production, is defective. The entrance of pulse-labeled RNA into cytoplasmic polyribosomes is even more strongly inhibited than is net RNA accumulation. The rate of protein synthesis, at first unaffected, decreases slowly; this decrease is paralleled by the decay of polyribosomes to monoribosomes with a half-time of 23 min. The polyribosomes which remain after a 30-min preincubation of the mutant at 36 C are active in polypeptide synthesis in vivo, whereas the monoribosomes which accumulate are not. Furthermore, ribosomes isolated from a culture of the mutant preincubated for 1 hr at 36 C are inactive in polypeptide synthesis in vitro, but can be restored to full activity by the addition of polyuridylic acid as mRNA. We conclude that mutant ts(-) 136 is defective either in the synthesis of all types of cytoplasmic RNA, or in the transport of newly synthesized RNA from the nucleus to the cytoplasm, and that the mRNA of a eucaryotic organism (yeast) is metabolically unstable, having a half-life of approximately 23 min at 36 C.     

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.     

Protein synthesis during germination of Allomyces macrogynus mitospores.

Protein synthesis in Allomyces marcogynus mitospores began at the time of encystment and was required for further germination. The rate of protein synthesis in germinating spores decreased in the absence of ribonucleic acid synthesis relative to the uninhibited control. There was a concomitant decrease in polysome content, suggesting that some of the messenger ribonucleic acid synthesized during early germination is translation.     

Conditional Mutants of Meiosis in Yeast

Three temperature-sensitive mutants, spo1-1, spo2-1, and spo3-1, were characterized with respect to their behavior in sporulation medium at a restrictive temperature. The time of expression of the functions defective in the mutants was determined by temperature-shift experiments during the sporulation process. In addition, each mutant was examined for the following: (i) its ability to undergo the nuclear divisions of meiosis; (ii) deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein synthesis; (iii) protein turnover; and (iv) colony-forming ability after exposure to sporulation medium. Mutant spo1-1 is defective in a function which confers a temperature-sensitive period which extends over 32% of the sporulation cycle. The temperature-sensitive period of mutant spo2-1 occupies 34% of the cycle, whereas the temperature-sensitive period of mutant spo3-1 extends over 2% of the sporulation cycle. Cytological evidence indicates that all three mutants initiate but do not complete the meiotic nuclear divisions. The DNA content of sporulation cultures of mutants spo1-1 and spo3-1 did not increase to the wild-type level; DNA synthesis in spo2-1 was normal. All three strains exhibit a loss of colony-forming ability during incubation in sporulation medium at the restrictive temperature. RNA and protein synthesis and protein turnover occur in the mutants.     

Characterization of arithmetic deoxyribonucleic acid synthesis at restrictive temperature in a dnaE mutant of Escherichia coli K-12.

The dna-293 mutation is shown to be a dnaE allele. The linear deoxyribonucleic acid synthesis previously observed in this mutant has been further characterized. The production of small deoxyribonucleic acid intermediates and their subsequent joining were identical in the mutant and its dnaE+ parent at 42.5 degrees C. Though the mutant cells continued to divide at the nonpermissive temperature, the rate of division was reduced. The data are consistent with a lack of production of replicationally active deoxyribonucleic acid polymerase III at the restrictive temperature.     

Initiation of Deoxyribonucleic Acid Replication in Germinating Spores of Bacillus subtilis 168 Carrying the dnaB(Ts)134 Mutation

The nature of the deoxyribonucleic acid synthesis reported by others to occur at 45 degrees C in germinating spores of the temperature-sensitive deoxyribonucleic acid initiation mutant of Bacillus subtilis 168, TsB134, has been investigated. Density transfer experiments, using 5-bromouracil, show that a normal round of replication can occur in a significant fraction of the spore population under such conditions. No repair synthesis is detectable. The possibility raised by this finding, that initiation of the first round of replication during spore outgrowth is unique in that its initiation is determined prior to germination, has been investigated by comparing the behavior of germinating spores of isogenic strains of B. subtilis 168, one carrying and the other without the dnaB (Ts)134 mutation. It is shown that deoxyribonucleic acid synthesis in the Ts strain is very sensitive to temperature in the vicinity of 45 degrees C. At a slightly higher temperature, 49 degrees C, initiation of the first round of replication in the Ts strain is completely (greater than 96%) blocked, but it proceeds normally in the Ts(+) strain. Thus, it is concluded that, after the germination of a spore, the action of the dnaB134 gene product is an obligatory requirement for initiation of the first round of replication. The initiation of replication that can occur in spores of the original TsB134 strain germinating at 45 degrees C is presumably due to incomplete inactivation of the dnaB134 gene product under such conditions.     

Biochemical and genetic characterization of a mutant of Escherichia coli with a temperature-sensitive valyl ribonucleic acid synthetase

B?ck, August (Purdue University, Lafayette, Ind.), Lia Eidlic Faiman, and Frederick C. Neidhardt. Biochemical and genetic characterization of a mutant of Escherichia coli with a temperature-sensitive valyl ribonucleic acid synthetase. J. Bacteriol. 92:1076-1082. 1966.-To test our conclusion that Escherichia coli mutant I-9 possesses a valyl soluble ribonucleic acid (sRNA) synthetase that functions in vivo at 30 C but not at 37 C, measurements were made by use of the periodate method, of the level of charged valyl sRNA in this strain. A shift of temperature from 30 to 40 C resulted in a rapid discharging of valyl sRNA coordinate with the cessation of protein synthesis; at the same time, other species of sRNA, such as those for leucine, became fully charged. Identical results were obtained with a derivative of I-9 with relaxed ribonucleic acid (RNA) control. When P1 phage were grown on wild cells and then used at low multiplicities of infection to transduce temperature-resistant growth into I-9, complete cotransduction of normal valyl sRNA synthetase occurred. By means of the interrupted-mating technique, the structural gene for valyl sRNA synthetase was located on the E. coli chromosome map and found to be near thr, one-fifth of the length of the chromosome removed from the structural genes for the isoleucine-valine biosynthetic enzymes. Therefore, (i) the major valyl sRNA synthetase activity of I-9 appears to be temperature-sensitive in vivo, (ii) relaxed amino acid control over RNA synthesis does not appear to be a consequence of a normal charging of sRNA with a substitute molecule, and (iii) one structural gene for valyl sRNA synthetase is located on the E. coli chromosome not closely linked to the cistrons for the valine-biosynthetic enzymes.     

Characterization of Some Poliovirus Temperature-Sensitive Mutants and Poliovirus-Related Particle Formation Under Nonpermissive Conditions

Investigation of 15 poliovirus temperature-sensitive (ts) mutants by using physiological tests [formation of virus-specific antigen and ribonucleic acid (RNA) under nonpermissive conditions] permitted us to divide them into three groups. From each group, one mutant was selected (ts 2, 5, 11), and a comparative study of poliovirus-related particle (5, 10, 73, and 150S) formation under permissive (36 C) and nonpermissive (40 C) conditions was carried out. The ts 2 and ts 11 are mutants with greatly reduced RNA synthesis which at 40 C produce particles with a sedimentation constant of 5S, and the ts 5 (RNA(+)) mutant produces both 5 and 10S particles. The relationship between different temperature-sensitive defects in the mutants is discussed. The results obtained indicate a possible role of 5S protein structures in morphogenesis of poliovirus.     

Specific inhibition of outgrowth of Bacillus subtilis spores by novobiocin.

Spores of a Bacillus subtilis mutant temperature sensitive in deoxyribonucleic acid (DNA) replication proceeded through outgrowth at the nonpermissive temperature to the same extent as the wild-type parent spores. In contrast, the DNA synthesis inhibitor novobiocin completely prevented spore outgrowth while displaying a marginal effect on logarithmic growth during one generation time. Inhibition of outgrowth by novobiocin occurred in the absence of DNA replication, as demonstrated in an experiment with spores of the temperature-sensitive DNA synthesis mutant at the restrictive temperature. Novobiocin inhibited the initial rate of ribonucleic acid synthesis to the same extent in germinated spores and in exponentially growing cells. A novobiocin-resistant mutant underwent normal outgrowth in the presence of novobiocin. Therefore, novobiocin inhibition was independent of its effect on chromosome replication per se.