Temperature-sensitive Escherichia coli mutant with an altered initiation codon of the uncG gene for the H+-ATPase gamma subunit.

A mutant of Escherichia coli showing temperature-sensitive growth on succinate was isolated, and its mutation in the initiation codon (ATG to ATA) of the uncG gene (coding for the gamma subunit of H+-ATPase F0F1) was identified. This strain could grow on succinate as the sole carbon source at 25 and 30 degrees C, but not at 37 or 42 degrees C. When this strain was grown at 25 degrees C on succinate or glycerol, its membranes had about 15% of the ATPase activity of wild-type membranes, whereas when it was grown at 42 degrees C, its membranes had about 2% of the wild-type ATPase activity. Membranes of the mutant grown at 25 or 42 degrees C could bind F1 functionally, resulting in about 40% of the specific activity of wild-type membranes. The gamma subunit was identified in an EDTA extract of membranes of the mutant grown at 25 degrees C, but was barely detectable in the same amount of extract from the mutant grown at 42 degrees C. These results indicate that initiation of protein synthesis from the AUA codon is temperature sensitive and that the gamma subunit is essential for assembly of F1 in vivo as shown by in vitro reconstitution experiments (S. D. Dunn and M. Futai, J. Biol. Chem. 255:113-118, 1980).     

Functional characterization of cleavage-defective mutants of encephalomyocarditis virus.

We characterized seven temperature-sensitive capsid cleavage (cleavage-defective) mutants of encephalomyocarditis virus. Our experimental approach was to monitor in vitro proteolysis reactions of either wild-type or cleavage-defective mutant capsid precursors mixed with cell-free translation products (containing the viral protease) of either wild-type or mutant viral RNA. The cell-free translation reactions and in vitro proteolysis reactions were done at 38 degrees C, because at this temperature cleavage of the capsid precursors was restricted in reactions containing cleavage-defective mutant viral RNA as the message, relative to those reactions containing wild-type viral RNA as the message. Wild-type or cleavage-defective mutant capsid precursors were prepared by adding cycloheximide to cell-free translation reactions primed with wild-type or mutant viral RNA, respectively, 12 min after the initiation of translation. In vitro proteolysis of wild-type capsid precursors with cell-free translation products of either wild-type or cleavage-defective mutant viral RNA led to similar products at 38 degrees C, indicating that the cleavage-defective mutant viral protease was not temperature sensitive. As a corollary to this, at 38 degrees C cleavage-defective mutant capsid precursors were not cleaved as completely as were wild-type capsid precursors by products of cell-free translation of wild-type viral RNA. The results from these in vitro proteolysis experiments indicate that all seven of the cleavage-defective mutants have capsid precursors with a temperature-sensitive configuration.     

Decline in mutation frequency in temperature-sensitive SV40 viruses before viral DNA synthesis.

Lesions that promote reversion from a temperature-sensitive to a wild-type phenotype were induced in temperature-sensitive late mutants of SV40 virus by UV irradiation. When cultures infected with UV-irradiated temperature-sensitive mutants were grown for various times at permissive temperature (35 degrees C) and then at restrictive temperature (39 degrees C), the reversion frequency declined just before the onset of semiconservative DNA synthesis when DNA synthesis began at 32 degrees C. This can be explained by competition between reactions that lead to the onset of viral DNA synthesis and reactions that repair the lesions before the onset of viral DNA synthesis.     

The C-terminal region of subunit 4 (subunit b) is essential for assembly of the F0 portion of yeast mitochondrial ATP synthase.

The role of the C-terminal part of yeast ATP synthase subunit 4 (subunit b) in the assembly of the whole enzyme was studied by using nonsense mutants generated by site-directed mutagenesis. The removal of at least the last 10 amino-acid residues promoted mutants which were unable to grow with glycerol or lactate as carbon source. These mutants were devoid of subunit 4 and of another F0 subunit, the mitochondrially encoded subunit 6. The removal of the last eight amino-acid residues promoted a temperature-sensitive mutant (PVY161). At 37 degrees C this strain showed the same phenotype as above. When grown at permissive temperature (30 degrees C) with lactate as carbon source, PVY161 and the wild-type strain both displayed the same generation time and growth yield. Furthermore, the two strains showed identical cellular respiration rates at 30 degrees C and 37 degrees C. However, in vitro the ATP hydrolysis of PVY161 mitochondria exhibited a low sensitivity to F0 inhibitors, while ATP synthesis displayed the same oligomycin sensitivity as wild-type mitochondria. It is concluded that, in this mutant, the assembly of the truncated subunit 4 in PVY161 ATP synthase is thermosensitive and that, once a functional F0 is formed, it is stable. On the other hand, the removal of the last eight amino-acid residues promoted in vitro a proton leak between the site of action of oligomycin and F1.     

Suppressor mutations in the chloroplast-encoded large subunit improve the thermal stability of wild-type ribulose-1,5-bisphosphate carboxylase/oxygenase

A temperature-conditional, photosynthesis-deficient mutant of the green alga Chlamydomonas reinhardtii, previously recovered by genetic screening, results from a leucine 290 to phenylalanine (L290F) substitution in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC ). Rubisco purified from mutant cells grown at 25 degrees C has a reduction in CO(2)/O(2) specificity and is inactivated at lower temperatures than those that inactivate the wild-type enzyme. Second-site alanine 222 to threonine (A222T) or valine 262 to leucine (V262L) substitutions were previously isolated via genetic selection for photosynthetic ability at the 35 degrees C restrictive temperature. These intragenic suppressors improve the CO(2)/O(2) specificity and thermal stability of L290F Rubisco in vivo and in vitro. In the present study, directed mutagenesis and chloroplast transformation were used to create the A222T and V262L substitutions in an otherwise wild-type enzyme. Although neither substitution improves the CO(2)/O(2) specificity above the wild-type value, both improve the thermal stability of wild-type Rubisco in vitro. Based on the x-ray crystal structure of spinach Rubisco, large subunit residues 222, 262, and 290 are far from the active site. They surround a loop of residues in the nuclear-encoded small subunit. Interactions at this subunit interface may substantially contribute to the thermal stability of the Rubisco holoenzyme.     

Temperature-sensitive mutants of bacteriophage SH-133 specific for the hydrogen bacterium Pseudomonas facilis: isolation, complementation, and partial characterization.

Seventeen temperature-sensitive mutants of bacteriophage SH-133 have been isolated following mutagenesis with UV-light, nitrosoguanidine, and ethyl methanesulfonate. The mutants were classified into 15 complementation groups according to their ability to complement each other at 32 degrees C, the nonpermissive temperature. Each mutant was studied with regard to the relationship between its ability to multiply in heterotrophically (H-) and autotrophically (A-) grown Pseudomonas facilis cells. At 27 degrees C, the permissive temperature, the plaque-forming ability of the 17 mutants and wild-type phage was reduced 10-fold in A-grown cells. At 32 degrees C, mutants belonging to 10 groups exhibited identical levels of multiplicity-dependent leak under both modes of growth. However, the infection of A-grown cells by mutants belonging to the remaining five groups resulted in as much as 500-fold inhibition of multiplicity-dependent leak when contrasted with the infection of cells grown heterotrophically. These observations indicate that the expression of five SH-133 phage cistrons is defective when multiplication proceeds under autotrophic metabolism. Seven mutants were found to differ from the wild-type phage with regard to thermal stability at 56 degrees C which suggests that they possess altered structural proteins. Four of the seven thermosensitive mutants exhibited reduced levels of multiplicity-dependent leak in A-grown cells. The data suggest that the reduction in plaque-forming ability of SH-133 in A-grown cells is caused by a defect in the expression of specific phage structural components.     

Peroxisomes in the methylotrophic yeast Hansenula polymorpha do not necessarily derive from pre-existing organelles.

We have identified two temperature-sensitive peroxisome-deficient mutants of Hansenula polymorpha (ts6 and ts44) within a collection of ts mutants which are impaired for growth on methanol at 43 degrees C but grow well at 35 degrees C. In both strains peroxisomes were completely absent in cells grown at 43 degrees C; the major peroxisomal matrix enzymes alcohol oxidase, dihydroxyacetone synthase and catalase were synthesized normally but assembled into the active enzyme protein in the cytosol. As in wild-type cells, these enzymes were present in peroxisomes under permissive growth conditions (< or = 37 degrees C). However, at intermediate temperatures (38-42 degrees C) they were partly peroxisome-bound and partly resided in the cytosol. Genetic analysis revealed that both mutant phenotypes were due to monogenic recessive mutations mapped in the same gene, designated PER13. After a shift of per13-6ts cells from restrictive to permissive temperature, new peroxisomes were formed within 1 h. Initially one--or infrequently a few--small organelles developed which subsequently increased in size and multiplied by fission during prolonged permissive growth. Neither mature peroxisomal matrix nor membrane proteins, which were present in the cytosol prior to the temperature shift, were incorporated into the newly formed organelles. Instead, these proteins remained unaffected (and active) in the cytosol concomitant with further peroxisome development. Thus in H.polymorpha alternative mechanisms of peroxisome biogenesis may be possible in addition to multiplication by fission upon induction of the organelles by certain growth substrates.     

Carotenes from Mutants of the Dinoflagellate, Crypthecodinium cohnii

SYNOPSIS.
The carotenoid compositions of 15 nitrosoguanidine-induced mutants of Crypthecodinium cohnii , a heterotrophic dinoflagellate, were determined by chromatographic and mass spectral analyses. Wild-type C. cohnii grown with irradiation of 250 W/cm² visible light at 27 C synthesizes β-carotene (33%) and γ-carotene (67%) amounting to 0.083 mg/g dry wt. There are 4 types of carotenoid-deficient mutants: (I) albinos which synthesize no C₄₀-carotonoids: (II) albinos blocked at the level of phytoene desaturation; (III) cream-colored cells which accumulate mainly §–carotene, with phytoene and/or β-zeacarotene also present; and (IV) light-orange strains which synthesize reduced amounts of β-carotene and γ-carotene.
Dark-grown wild-type cells produced 35% as much carotenoids as light-grown cells. Inhibition studies revealed that diphenylamine (3 γ) caused phytoene accumulation; nicotine at 0.9 mM blocked the final cyclization, to cause γ-carotene to accumulate in wild-type cells. Inhibition by adenine and guanine (1.5 mM) of carotenogenesis was demonstrated for the first time in any system. The effect of these purines was similar to that of diphenylamine addition: phytoene desaturation was largely inhibited.
The carotenogenic system in this dinoflagellate is similar to that of green algae and higher plants, and is under nuclear genetic control.     

Defined mutations in a small region of the brome mosaic virus 2 gene cause diverse temperature-sensitive RNA replication phenotypes

The central portion of the brome mosaic virus (BMV) 2a protein represents the most conserved element among the related RNA replication components of a large group of positive-strand RNA viruses of humans, animals, and plants. To characterize the functions of the 2a protein, mutations were targeted to a conserved portion of the 2a gene, resulting in substitutions between amino acids 451 and 484. After the temperature profile of wild-type BMV RNA replication was defined, RNA replication by nine selected mutants was tested in barley protoplasts at permissive (24 degrees C) and nonpermissive (34 degrees C) temperatures. Four mutants did not direct RNA synthesis at either temperature. Various levels of temperature-sensitive (ts) replication occurred in the remaining five mutants. For two ts mutants, no viral RNA synthesis was detected at 34 degrees C, while for two others, an equivalent reduction in positive- and negative-strand RNA accumulation was observed. For one mutant, positive-strand accumulation was preferentially reduced over negative-strand accumulation at 34 degrees C. Moreover, this mutant and another displayed preferential suppression of genomic over subgenomic RNA accumulation at both 24 and 34 degrees C. The combination of phenotypes observed suggests that the 2a protein may play a role in the differential initiation of specific classes of viral RNA in addition to a previously suggested role in RNA elongation.