A temperature-sensitive mammalian cell mutant exhibiting micronucleation

A temperature-sensitive mutant (ts 39) of murine leukemic cells was shown to undergo micronucleation upon exposure to the non-permissive temperature. The formation of micronucleate cells appeared to be preceded by an increase in the fraction of mitotic cells. Since this phenomenon resembles micronucleation induced by colcemid, it is possible that ts 39 cells may be defective in microtubule assembly.     

Characterization of RNA synthesis in an Escherichia coli mutant with a temperature-sensitive lesion in stable RNA synthesis

Previous experiments with Escherichia coli strain 2S142 have shown that the synthesis of stable RNA is preferentially blocked at the restrictive temperature. In this paper, we have examined the capacity of this mutant strain to synthesize RNA in vitro. Growth of the strain for as short a period as 10 min at 42 degrees C resulted in a 40 to 60% loss of RNA synthetic capacity and a fourfold decrease in percent rRNA synthesized in toluenized cell preparations. The time course for the loss and recovery of this RNA synthetic capacity correlated very well with the changes in RNA synthesis observed in vivo. We found no difference in temperature sensitivity of the purified RNA polymerase from the mutant and the parental strains. Moreover, there was no detectable alteration in the amount of enzyme, specific activity of the enzyme, or electrophoretic mobility of the subunits when the mutant strain was grown at 42 degrees C. The capacity for rRNA synthesis was also measured with the Zubay in vitro system (Reiness et al., Proc. Natl. Acad. Sci. 72:2881-2885, 1975). Supernatant fractions (S-30) prepared from cells grown at 30 degrees C were capable of up to 31.2% rRNA synthesis, using phi 80d3 DNA as template. S-30 fractions from cells grown at 42 degrees C synthesized 8.6% rRNA. The bottom one-third of the S-100 fraction and the ribosomal salt wash from 30 degrees C cells contained one or more factors which partially restored preferential rRNA synthesis in S-30 fractions from cells grown at 42 degrees C. Preliminary evidence suggests that the factor(s) is protein in nature.     

A temperature-sensitive mutant of E. coli exhibiting slow processing of exported proteins

A temperature-sensitive E. coli mutant with a mutation in the spc ribosomal protein operon was found to have a conditional defect in the processing of precursor proteins destined for the periplasmic space or the outer membrane. At high temperatures, significant amounts of precursor proteins having unprocessed signal sequences are detected in the mutant cell by pulse-labeling. The precursors are processed at very slow rates during a subsequent chase. Genetic analysis indicates that the mutation impairs the function of a gene, termed secY, located at the promoter-distal part of the spc operon. The secY gene is distinct from those genes previously known to specify ribosomal proteins, yet it is within the spc operon. It is suggested that the product of the secY gene is a component of the cellular apparatus that is essential for protein secretion across the cytoplasmic membrane. The gene secY is probably identical with prlA, previously identified as a suppressor of signal sequence mutations.     

Deoxyribonucleic acid synthesis in a temperature-sensitive Escherichia coli dnaH mutant, strain HF4704S.

The dnaH mutant strain HF4704S, isolated by Sakai et al. (1974), was examined for its effect on phiX174 deoxyribonucleic acid (DNA) synthesis. It was found to carry two mutations affecting DNA synthesis. One mutation had no affect on phiX174 DNA synthesis, but did affect the ability of the mutant cells to form colonies on agar medium at 41 degrees C, and caused host DNA synthesis to cease after 1 h at 41 degrees C. The mutant marker cotransduced with ilvD at a frequency of about 9%. It seems likely that this mutation is in the dnaA gene. The second mutation affected the ability of the mutant cells to form colonies on agar medium supplemented with only 2 mug of thymine per ml, and affected both host and phiX174 DNA synthesis in medium supplemented with only 2 mug of thymine per ml. Both effects could be overcone by adding excess exogenous thymine. We were not able to unambiguously determine the map position of this mutant locus. Our data show that the DNA synthesis phenotype of the mutant strain HE4704S is governed by both these mutations, neither of which directly affects the replication of phiX174 DNA.     

Non-coordinate synthesis of RNA polymerase beta beta'' subunits in a temperature-sensitive beta''-subunit mutant of Escherichia coli

Summary On exposure to high temperature of a temperature-sensitive RNA polymerase subunit (rpoC92) mutant of Escherichia coli, selective reduction was observed in the rate of synthesis of a group of proteins including RNA polymerase subunit. The finding that the synthesis of subunit but not subunit was specifically repressed in this mutant grown at non-permissive temperature indicates that the functionally intact RNA polymerase is required for the synthesis of subunits be coordinated. In addition, the assembly of newly synthesized RNA polymerase subunits was inefficient in this mutant at the steps where altered subunit was involved, and the unassembled enzyme subunits were rapidly and preferentially degraded. During recovery to non-restricted growth, the synthesis of both and subunits was transiently enhanced in parallel leading to recovery of the intracellular concentration of functional RNA polymerase.     

Escherichia coli K-12 mutant forming a temperature-sensitive D-serine deaminase.

A single-site mutant of Escherichia coli K-12 able to grow in minimal medium in the presence of D-serine at 30 C but not at 42 C was isolated. The mutant forms a D-serine deaminase that is much more sensitive to thermal denaturation in vitro at temperatures above but not below 47 C than that of the wild type. No detectable enzyme is formed by the mutant at 42 C, however, and very little is formed at 37 C. The mutant enzyme is probably more sensitive to intracellular inactivation at high temperatures than the wild-type enzyme. The mutation lies in the dsdA region. The mutant also contains a dsdO mutation, which does not permit hyperinduction of D-serine deaminase synthesis.     

Gene expression in a temperature-sensitive gyrB mutant of Escherichia coli.

The effect of gyrase inactivation on gene expression was studied by examining the activities of different promoters in a temperature-sensitive gyrB mutant of Escherichia coli. The relative activities of promoters affected by cAMP-binding protein (CAP), e.g., the lac promoter, are not reduced by gyrase inactivation but can, on the contrary, be enhanced. This stimulation depends on the promoter location or its structure. The tnaA promoter is activated when located near the origin of replication, suggesting a differential effect of gyrase inactivation on various chromosomal domains. Only silent or mutant promoters such as the non-functional wild-type bgl or the lacIq can be activated. No differential effect of gyrase inactivation on the lambda pL and the trp promoters carried by the phage can be detected.     

The nature of ompA mutants of Escherichia coli K12 exhibiting temperature-sensitive bacteriophage resistance

Summary A class of ompA mutants of Escherichia coli, exhibiting temperature-sensitive resistance towards phages using the OmpA protein as receptor, was analysed. The mutants produce detectable levels of the protein at 42°C but not at 30°C (Manning and Reeves 1976). They were found to have a deletion (one isolate) or insertions (three isolates) upstream of the coding part of the ompA gene. Several previously characterized mutants possessing insertions or a deletion in the non-translated 5 area of the gene also exhibited a similar temperature-sensitive phage resistance. This cold-sensitive phenotype is explained in terms of the recent discovery that the stability of ompA mRNA is regulated by the rate of cell growth (Nilsson et al. 1984).     

Genetic analysis of a streptomycin-resistant Escherichia coli mutant temperature-sensitive for nonsense suppression

Summary Continuing the genetic and biochemical characterization of the streptomycin-resistant Escherichia coli mutant LD1, we confirmed that LD1 is temperature-sensitive for suppression of nonsense codons, and that this phenotype of the mutant and its streptomycin-resistance are genetically linked and are probably caused by a single mutation, strA (LD1). We also isolated a spontaneous revertant, called LD1-R, which partially relieves the restriction of nonsense suppression caused by the strA (LD1) mutation. LD1-R is derived by an additional mutation (revA) which is closely linked to strA (LD1). We further demonstrate that the weak suppression of a lacZ_UGA mutation in a suppressor-free strain, which probably takes place by normal tRNA^Trp, can be detected by the use of the chromagenic substance x-gal (5-Bromo-4-chloro-3-indolyl--d-Galactopyranoside).