Characterization of temperature-sensitive mutants of animal cells

An early increase in lymphocyte plasma membrane K⁺ transport is essential for PHA stimulated lymphocytes to divide. Little is known about the specific source and amount of energy required to support the increased transport by activated lymphocytes. Since ouabain, a cardiac glycoside, specifically inhibits the transport ATPase, we have measured the decrement in glycolysis and tricarboxylic acid cycle activity when untreated and PHA treated lymphocytes were exposed to ouabain. This metabolic decrement represents the portion of metabolism associated with monovalent cation transport and closely related processes. Since TCA cycle activity accounted for only 0.2% of glucose consumption, aerobic glycolysis was the major source of energy, i.e., ATP, for increased transport. Approximately one-third of the total lactate production in both control and PHA stimulated lymphocytes was ouabain-sensitive. Ouabain sensitive lactate production in control, 105 μmol/10¹⁰ cells/hour, increased 1.8-fold to 193 μmol/10¹⁰ cells/hour after PHA treatment. Active K⁺ influx in similar cell populations increased from 40 μmol/10¹⁰ cells/hour to 74 μmol/10¹⁰ cells/hour (1.9-fold) after PHA treatment. The increment in ouabain-sensitive energy production and K⁺ transport were closely correlated and, therefore, 0.38 moles of K⁺ are transported for each mole of ATP generated in both control and PHA treated cells. The increased requirement for transport related energy is provided by increasing the ouabain-sensitive ATP production rather than altering the efficiency of ATP transduction.     

Repair-defective mutants of Alteromonas espejiana, the host for bacteriophage PM2

The in vivo repair processes of Alteromonas espejiana, the host for bacteriophage PM2, were characterized, and UV- and methyl methanesulfonate (MMS)-sensitive mutants were isolated. Wild-type A. espejiana cells were capable of photoreactivation, excision, recombination, and inducible repair. There was no detectable pyrimidine dimer-DNA N-glycosylase activity, and pyrimidine dimer removal appeared to occur by a pathway analogous to the Escherichia coli Uvr pathway. The UV- and MMS-sensitive mutants of A. espejiana included three groups, each containing at least one mutation involved with excision, recombination, or inducible repair. One group that was UV sensitive but not sensitive to MMS or X rays showed a decreased ability to excise pyrimidine dimers. Mutants in this group were also sensitive to psoralen plus near-UV light and were phenotypically analogous to the E. coli uvr mutants. A second group was UV and MMS sensitive but not sensitive to X rays and appeared to contain mutations in a gene(s) involved in recombination repair. These recombination-deficient mutants differed from the E. coli rec mutants, which are MMS and X-ray sensitive. The third group of A. espejiana mutants was sensitive to UV, MMS, and X rays. These mutants were recombination deficient, lacked inducible repair, and were phenotypically similar to E. coli recA mutants.     

Genetic studies on temperature-sensitive mutants ofBacillus subtilis bacteriophage SPP 1

A total of 30ts mutants ofBacillus subtilis bacteriophage SPP1 were isolated and subjected to complementation test. On the basis of this test 21 mutants were classified into 4 functional groups; the classification of the remaining 9 mutants was unclear. The frequency of recombination by mutual crossing was determined in representatives of individual groups; this made it possible to place these mutants on a linear map comprising a total length of 7.62 recombination units.     

Phenotypic characteristics of temperature-sensitive mutants of bacteriophage T4 in gene 19]

The order of three thermosensitive mutations in gene 19 is determined and compared with the character of defective structures observed in an electrone microscope study of the lysates obtained in non-permissive and semi-permissive conditions. The degree of defectiveness of core polymerization increases if the mutation is located near the beginning of the gene. In one the combinations among these mutants the interallelic complementation is found. The mechanism of complementation is discussed on the basis of the electrone microscopic pattern. The polymerization of core protein in vivo starts from the base plate.     

PM2 bacteriophage plaque morphology mutants exhibit differences in DNA length and restriction endonuclease patterns

A large plaque (LP) and a small plaque (SP) variant of PM2 bacteriophage were isolated from a mixture of the two plaque variants and were grown separately in the appropriate host bacterium,Alteromonas espejiana. They have remained pure for approximately one year from the original isolation. Restriction endonuclease analyses revealed differences in theHaeIII restriction profile between the two variants.HaeIII fragment 1 of the SP DNA was found to be smaller than the corresponding fragment from the LP variant DNA, whereas fragment 7 from the SP DNA was slightly larger than the same fragment from LP DNA. Electron microscopy of heteroduplexes formed between the DNAs from the two variants revealed that the deletion in fragment 1 mapped very close to the junction betweenHaeIII fragments 1 and 13 on the physical map of PM2 DNA. The difference in DNA length between the two variants results from addition or deletion mutations.Deceased.     

Isolation and complementation analysis of temperature-sensitive mutants of the PS8 bacteriophage ofAgrobacterium tumefaciens

The isolation, method of complementation analysis and classification of temperature-sensitive (ts) mutants of phage PS8 (Agrobacterium tumefaciens) are described.     

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).     

Inactivation of bacteriophage PM2 during storage

The kinetics of bacteriophage inactivation in the medium that is optimal for its storage has been studied at temperatures from 4 to 55 degrees C. The plot of Arrhenius dependence of the constant of inactivation rate consists of the two linear parts with the energies of activation Ea = 25 kcal/mol for 4-37 degrees C and Ea = 91 kcal/mol for 37-55 degrees C. The DNA of inactivated bacteriophage remained mostly in superspiralized form and completely preserved its biological activity as tested by transfection in spheroplasts. The analysis of inactivation kinetics suggests ageing of virions cultivated at 4 degrees C. The addition of watersoluble antioxidant amoxipin did not change the inactivation kinetics. The addition of antioxidant ionol with twin-80 increased the inactivation that was paralleled by the bacteriophage DNA degradation.     

Membrane morphogenesis from cloned fragments of bacteriophage PM2 DNA that contain the sp6.6 gene

The formation of new membrane vesicles normally occurs during eukaryotic organellogenesis and maturation of bacteriophage PM2. This virus was studied as a simple model for membrane morphogenesis. Previous biochemical and genetic studies suggest that a major structural protein of PM2, sp6.6, is an integral membrane protein involved in viral membrane morphogenesis. To establish the necessity of sp6.6 in membrane formation, restriction fragments of PM2 that contained the sp6.6 coding sequence were cloned into several plasmid vectors for expression in Escherichia coli. A construction in pBR322 containing two HindIII fragments of PM2 DNA caused production of intracellular membrane vesicles of the same size as those produced in the course of natural infection of Alteromonas espejiana. Similar results were obtained with a smaller construct of HindIII fragments in the plasmid vector pPL-lambda. Expression of sp6.6 was detected via incorporation of 35S-labeled methionine after SDS-polyacrylamide gel electrophoresis and with a specific rabbit antiserum on immunoblots. Other constructs did not produce recognizable vesicles or sp6.6. These results are the first to suggest that a hydrophobic membrane protein can cause development of new membrane structure.     

Characterization of Bacillus subtilis mutants with a temperature-sensitive intracellular protease.

A colony screening procedure was devised to detect Bacillus subtilis mutants containing temperature-sensitive trypsin-like intracellular protease activity. The enzyme was characterized as a non-sulfhydryl serine protease on the basis of inhibitor studies. It was also inhibited by D- or L-histidine but not by any other amino acid tested. The long-term survival at 45 degrees C of these mutants in a minimal salts medium was decreased, with rapid lysis occurring within 24 h. A D-histidine function in long-term survival and inhibition accounted for the presence of additional protease mutants among survivors of histidine auxotrophs selected for their ability to utilize D-histidine. In addition to being lysed when incubated at 45 degrees C under nongrowth conditions, all of the protease mutants had a decreased rate of protein turnover and produced spores deficient in a major low-molecular-weight spore coat polypeptide. The morphology of the undercoat layers was altered, but there was no effect on spore heat resistance or on germination. The missing spore coat polypeptide appeared to be processed from a larger precursor by cleavage to produce N-terminal histidine. A defect in this protease could account for the lack of processing and thus the absence of this polypeptide in spore coats.     

Membrane attachment of the chromosome in Bacillus subtilis mutants temperature-sensitive in DNA replication

We have examined three mutants of Bacillussubtilis temperature sensitive in DNA initiation and one temperature sensitive in DNA elongation, in order to investigate whether these lesions can cause or can result in a detachment of the membrane-bound chromosomal region.Our results argue against any effect of the mutations examined on the association between the chromosome and the membrane.     

Characterization of bacteriophage lambda excisionase mutants defective in DNA binding

The bacteriophage lambda excisionase (Xis) is a sequence-specific DNA binding protein required for excisive recombination. Xis binds cooperatively to two DNA sites arranged as direct repeats on the phage DNA. Efficient excision is achieved through a cooperative interaction between Xis and the host-encoded factor for inversion stimulation as well as a cooperative interaction between Xis and integrase. The secondary structure of the Xis protein was predicted to contain a typical amphipathic helix that spans residues 18 to 28. Several mutants, defective in promoting excision in vivo, were isolated with mutations at positions encoding polar amino acids in the putative helix (T. E. Numrych, R. I. Gumport, and J. F. Gardner, EMBO J. 11:3797-3806, 1992). We substituted alanines for the polar amino acids in this region. Mutant proteins with substitutions for polar amino acids in the amino-terminal region of the putative helix exhibited decreased excision in vivo and were defective in DNA binding. In addition, an alanine substitution at glutamic acid 40 also resulted in altered DNA binding. This indicates that the hydrophilic face of the alpha-helix and the region containing glutamic acid 40 may form the DNA binding surfaces of the Xis protein.     

Control of membrane morphogenesis in bacteriophage PM2

The regulation of membrane formation in bacteriophage PM2 serves as a simple model for changes in membrane structure in eukaryotic cells. Prior to Pseudomonas host lysis, wild-type virions mature to an icosahedral morphology at the inner face of the cytoplasmic membrane. The proliminary charcterization of two temperature-sensitive mutants of PM2 is described. In cells infected at the restrictive temperature with ts 1, an abundance of “empty” virus-size membrane vesicles are seen. Synthesis of DNA is also reduced in ts 1 infected cells. The preponderance of vesicles is not sen in cells infected with wil-type virus or with ts 1 at the permissive temperature. The “empty” appearance of the viral membranes suggests that viral DNA is not encapsulated. The major viral capsid protein (MW 26,000) is located just out side the viral membrane and normallyl sediments with host and virus membranes; insted, large amounts of capsid protein can be precipitated from the supernatant with TCA. Compared to cells infected with wild type virus, cells infected with is 5 at th restrictive temperature produce inside the cell an aboundance of virus-soze membrane vesicles. Taken Together, These results with viral mutants suggest that formation of a viral membrane of the proper size does not require a DNA core around which to form, or an outer scaffolding of coat protein against which to form a spherical bilayer.     

Characterization of two temperature-sensitive mutants of Sigma virus (authors transl)

Summary Temperature-sensitive mutants have been isolated from Drosophila Sigma virus, a Rhabdovirus inducing CO₂ sensitivity in Drosophila melanogaster. We have studied the decay of infectious centers at non permissive temperature. The proportion of destroyed infectious centers is the same for the wild type, ts⁺, and for ts9. On the opposite, it is more important for ts 4. Temperature-sensitive function of ts 4 appears necessary to the viral genome replication. With the three clones, ts⁺, ts 4 and ts 9, we have obtained stabilized Drosophila females able to transmit Sigma virus to their whole progeny. We have tried to see in each case, if stabilized flies could transmit the virus to their progeny at non permissive temperature. Flies stabilized with ts⁺ and ts 9 can, flies stabilized with ts 4 cannot. Therefore two categories of mutants are defined: those that are transmitted hereditarily. at non permissive temperature, and not blocked in genome replication. Those that are blocked in genome replication and not transmitted. When the virus cannot replicate, the divisions in the germ line cells dilute the viral genomes. The consequence will be a real healing of germ line cells, and then a break in hereditary transmission by stabilized flies. All the results with temperature-sensitive mutants are coherent with this hypothesis.

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Étude de mutants thermosensibles du virus Sigma

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Mémoire présenté par F. Gros     

Characterization of Saccharomycopsis lipolytica mutants that express temperature-sensitive synthesis of isocitrate lyase

Four mutants specifically deficient in the activity of isocitrate lyase were independently isolated in the alkane yeast Saccharomycopsis lipolytica. Genetic analysis by means of protoplast fusion and mitotic haploidization revealed that the mutations were recessive and non-complementary at a single genetic locus, icl. icl is a structural gene for isocitrate lyase, because some revertants from icl-1 and icl-3 mutants produced thermolabile isocitrate lyase in comparison with the wild-type enzyme, and also because the gene dosage effect was observed on the specific activity of isocitrate lyase in icl+/icl-1 and icl+/icl-3 heterozygotes. The icl-3 mutation also gave rise to temperature-sensitive revertants that could grow on acetate at 23 degrees C but not at 33 degrees C, exhibiting temperature-sensitive synthesis as well as thermostable activity of isocitrate lyase. Studies on purified isocitrate lyase showed that this enzyme is tetrameric and that the enzyme synthesized at 23 degrees C by a temperature-sensitive synthesis mutant was indistinguishable from the wild-type enzyme with respect to the subunit molecular weight (59,000), the isoelectric pH (5.3), the thermostability, and the Km value for threo-Ds-isocitrate (0.2 mM). When induced by acetate at 33 degrees C, the temperature-sensitive synthesis mutant did not express isocitrate lyase activity but did synthesize polypeptides whose electrophoretic mobilities were equal to that of the purified mutant enzyme. Hence, the temperature-sensitive mutation assumed in the structural gene for isocitrate lyase might have prevented the maturation of the polypeptide chains synthesized at the restrictive temperature.     

Characterization of Bacillus subtilis mutants temperature-sensitive in the synthesis of ribonucleic acid

Two mutants of Bacillus subtilis temperature-sensitive in RNA synthesis were isolated. One mutation (rna-20) was demonstrated to be an allele of a previously identified gene (Riva et al., 1976). The other mutation (rna-16) identified a different gene and was mapped near aroI. The rna-16 mutation at the permissive temperature affected the spore outgrowth process. Purified RNA polymerase from rna-16 did not show any temperature sensitivity or structural defect.     

Characterization of temperature-sensitive mutants of simian rotavirus SA11: protein synthesis and morphogenesis.

The synthesis of viral polypeptides, distribution of viral antigens, and morphogenesis of viral structures have been examined in cells infected with temperature-sensitive (ts) mutants of SA11 representing 10 recombination groups. At the permissive temperature (31 degrees C) the synthesis of viral polypeptides and the distribution of viral antigens did not differ significantly from those of the wild type. At the nonpermissive temperature (39 degrees C) some mutants (tsB, -C, -E, -F, and -G) synthesized significantly smaller amounts of viral polypeptides and had a very diffuse distribution of viral antigen. Several of the mutants synthesized one or more electrophoretically aberrant polypeptide species at both 31 and 39 degrees C. All of the mutants, except tsF, assembled morphogenetic intermediates at 39 degrees C. Aberrant intermediates were assembled in all mutants at 31 and 39 degrees C. No specific morphogenic defect could be associated with any of the ts mutants.