Isolation and characterisation of a strain of Saccharomyces cerevisiae deficient in in vitro RNA polymerase B(II) activity.

Summary Two hundred strains of Saccharomyces cerevisiae temperature sensitive for RNA synthesis were selected and screened in crude extracts for DNA-dependent RNA polymerase activities. One strain was isolated which had only residual in vitro RNA polymerase B activity. In normal growth conditions total RNA, poly A⁺ RNA and protein synthesis were indistinguishable from those of the wild type strain at 23°C and after shift to 37°C. A temperature sensitive phenotype was detected only when rpoB containing strains were grown in adverse conditions. The mutant character showed mendelian segregation and was coexpressed with the wild type character in heterozygous diploids. Residual enzyme activity was characterised in crude extracts using synthetic polymers and natural templates in different ionic conditions.     

Phenotypic instability in a tif-1 mutant of Escherichia coli

Summary The mutant T44() of Escherichia coli K12, grown in the presence of adenine, develops an increased tolerance to streptomycin. In cultures grown on streptomycin, the ts character (tif) may temporarily be suppressed but, on further transfer, both the temperature-sensitive phenotype and streptomycin tolerance disappear. In a cell-free system, the relative efficiency of translation of MS2 and poly U messenger RNAs was, respectively, 75 and 50% lower in extracts from cultures grown at 37° with adenine than in extracts from 30° cultures. Similar results were obtained when adenine was added in vitro to an extract from a culture grown at 37° in the absence of adenine, using MS2 RNA as messenger. Moreover, the 37° extracts showed a much lower misincorporation of isoleucine into polyphenylalanine in the poly U system. In addition, the Mg⁺⁺ concentration required for optimal translational activity was higher for the 37° than for the 30° extracts. Extracts from a culture grown in L medium at 37° or from a tif ^-/F tif ⁺ merodiploid grown at 37° with adenine behaved similarly to that from the 30° culture when poly U was used as messenger RNA. It is suggested that the tif ⁺ gene product may play a regulatory role in ribosomal function and the pleiotropic nature of the tif-1 mutation could be due to impairment of translational activity augmented by elevated temperature or by adenine.     

F-Factor-mediated restriction of bacteriophage T7: protein synthesis in cell-free systems from T7-infected Escherichia coli F- and F+ cells.

A characteristic phenomenon in the F-factor-mediated inhibition of T7 phage is a virtual absence of T7 late protein synthesis in T7-infected Escherichia coli male cells, in spite of the presence of T7 late mRNA which is translatable in vitro when isolated from the cell. To determine whether the translational defect in T7-infected F+ cells is due to a T7 late mRNA-specific translational block, or to a general decrease of F+ cell translational activity, we compared the activities of cell-free, protein-synthesizing systems prepared from isogenic F- and F+ cells harvested at different times of T7 infection. The cell-free systems from uninfected F- and F+ cells translated T7late mRNA equally as well as MS2 RNA and T7early mRNA. The activity of cell-free systems from T7-infected F+ cells to translate MS2 RAN, T7 early mRNA, and T7 late mRNA decreased concomitantly at a much faster rate than that of T7-infected F- cells. Therefore, the abortive infection of F+ cells by T7 does not result from a T7 late mRNA-specific translational inhibition, although a general reduction of the translational activity appears to be a major factor for the inability of the F+ cells to produce a sufficient amount of T7 late proteins.     

Ribonucleic Acid Polymerase in a Thermosensitive Sporulation Mutant (ts-4) of Bacillus subtilis

Partially synchronized cultures of a Bacillus subtilis thermosensitive sporulation mutant (ts-4) and the 168 try⁻try⁻ (168tt) parental strain were infected with the virulent phage e at various times during their growth cycle at 30 and 42 C (permissive and restrictive temperatures, respectively). It was shown that at the restrictive temperature the burst size in the parental strain was two- to threefold lower than in the ts-4 mutant. No such difference was observed at the permissive temperature. However, the time at which this difference was observed excludes a correlation between the burst size and initiation of the sporulation process. It was further found that the capacity to transcribe in vitro phage e deoxyribonucleic acid by partially purified ribonucleic acid (RNA) polymerase from both strains decreased sharply if the source of enzyme was sporulating cells instead of vegetative ones. However, a similar decrease, although to a lesser extent, was observed with the RNA polymerase isolated from stationary-phase cells of the ts-4 mutant grown at the nonpermissive temperature, or with the enzyme derived from several other zero-stage sporulation mutants. At no time was a structural modification in the β subunits of the RNA polymerase observed during growth of the sporulating bacteria. We have also shown that, in addition to the relatively low specific activity of the RNA polymerase, the level of the intracellular protease activity is about 15-fold lower in the ts-4 mutant grown at the restrictive temperature than that of the parental strain grown at the same temperature. At the permissive temperature no such difference was observed between these two strains. However, the present data do not allow us to establish a correlation among the low content of intracellular protease, the weak specific activity of the RNA polymerase, and the loss of the sporulation capacity in the ts-4 mutant grown at the restrictive temperature.     

Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides. The involvement of sulphur metabolism

1. The ;initial' 5-aminolaevulinate synthetase activity, that is the activity observed immediately after cell disruption, in extracts prepared from unharvested semianaerobically grown Rhodopseudomonas spheroides, was twice that observed under the same assay conditions in extracts prepared from harvested cells. 2. The effect of oxygenation of a culture on the ;maximum' aminolaevulinate synthetase activity, that is the activity observed 1h after disruption of harvested cells, is markedly influenced by the contents of the growth medium. Oxygenation of organisms for 1h in the medium in which they have grown produces an 80-90% decrease in maximum activity, whereas similar treatment of organisms resuspended in fresh medium produces less than a 40% decrease. 3. This protective effect of fresh medium is absolutely dependent on the presence of sulphate. When cells are suspended in sulphate-deficient fresh medium, the maximum activity falls by 65-75% even without oxygenation. A high maximum activity is regenerated when sulphate is resupplied. 4. When organisms are oxygenated in the medium in which they have grown, the cellular contents of GSH+GSSG and cysteine+cystine fall very markedly and homolanthionine is formed. Both the fall in aminolaevulinate synthetase activity and the changes in sulphur metabolism are largely prevented by the addition of compounds which stimulate synthesis of cysteine de novo or inhibit the conversion of cysteine S into homocysteine S. 5. The maximum aminolaevulinate synthetase activity was directly proportional to the GSH+GSSG content of all cell preparations. In glutathione-depleted extracts the ;low'-activity enzyme could be re-activated in vitro by the addition of GSH, GSSG, cysteine or cystine, whereas in extracts with a high glutathione content the ;high'-activity enzyme was unaffected by these sulphur compounds. 6. The activation of low-activity enzyme with exogenous sulphur compounds was prevented by excluding air or by adding NADH. Studies with purified enzyme indicate that sulphur compounds do not interact directly with the enzyme, but that their effect is mediated by a number of other endogenous factors.     

Specific changes in the synthesis of mitochondrial DNA in chick embryo fibroblasts transformed by Rous sarcoma viruses

In chick-embryo fibroblasts infected with the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup A (wild type), or with a thermosensitive mutant of this virus, T5, the rates of mitochondrial DNA synthesis differ in cells that exhibit normal and malignant phenotypes. In wild type virus-infected cells grown at 36 or 41 degrees C, morphological transformation is expressed, the rate of 2-deoxy-D-[3H]glucose uptake is stimulated, and mitochondrial DNA synthesis in vivo is stimulated three- to fivefold over that in uninfected cells. In T5-infected cells these changes occur only at the permissive temperature (36 degrees C); a shift to the nonpermissive temperature (41 degrees C) causes the reversal of these effects, and the specific activity of purified mitochondrial DNA is characteristic of that from uninfected cells. In contrast, the specific activities of nuclear DNA purified from cells maximally transformed under the permissive conditions do not differ between wild type-infected and uninfected with the T5 virus. In parallel experiments with isolated mitochondria, the rate of mtDNA synthesis in vitro is again greater in mitochondria isolated from transformed cells. In addition, mitochondrial DNA synthesis in vitro in mitochondria from nontransformed and virus-transformed cells exhibits differential sensitivity to inhibition by mercaptoethanol. Furthermore, the ntDNAP polymerase activity in mitochondrial extracts prepared from cells with transformed phenotypes is about sevenfold higher than in extracts from cells with nontransformed phenotypes.     

Translational regulation in growing clonal human astrocytoma cells in culture

The in vivo and in vitro protein synthesis by polysomes prepared from Cox astrocytoma cells grown in the presence of 100 mM ethanol were examined during transition from exponential to stationary growth phase. A sharp decline of translational activities of Cox poly (A)+messenger RNAs (mRNAs) occurred during this transition. This decline was accentuated when cells were grown in the presence of ethanol. The observed decline in mRNA translational activity was investigated in vitro in a micrococcal nuclease treated, mRNA depleted postmitochondrial supernatant (PMS) fraction containing [³⁵S]methionine. The formation of the³⁵S-labeled 40S ternary complex in the absence of mRNA and of the³⁵S-labeled 80S initiation complex in the presence of Cox or brain poly (A)+mRNAs were reduced substantially when the source of PMS was from stationary phase or ethanol exposed cells. The sedimentation of peaks containing 40S ternary and 80S initiation complexes following sucrose density gradient analysis showed marked reductions in [³⁵S] methionine labeling during the transition to stationary phase and also following ethanol exposure. The reduced formation of initiation complexes suggests possible functional modifications of eukaryotic initiation factor-2 (eIF-2) present in the PMS fraction and of mRNAs under these conditions. Data suggest that cells initiate adaptive or protective mechanisms by reducing the rate of the initiation reaction following environmental alterations produced by ethanol.     

Cell-free translation of frog virus 3 messenger RNAs. Initiation factors from infected cells discriminate between early and late viral mRNAs

Cell-free protein-synthesizing extracts prepared from rabbit reticulocytes, wheat germ, or cultured baby hamster kidney cells efficiently translated frog virus 3 early mRNAs; in contrast, late mRNAs were translated poorly under similar conditions. However, the translational efficiency of the late viral mRNAs was markedly enhanced in cell-free extracts prepared from frog virus 3 (FV 3)-infected baby hamster kidney cells and in nuclease-treated rabbit reticulocyte extracts by the addition of a 0.5 M KCl wash from FV 3-infected cell ribosomes; the 0.5 M KCl wash (initiation factors) from uninfected cells had no such effect. Total cytoplasmic RNA from infected cells was fractionated according to size on sucrose gradients and fractions containing different concentrations, and relative proportions of early and late mRNAs were translated in either native or initiation factor-supplemented extracts. Under these conditions, the translation efficiency of early mRNAs was unchanged, while the translation of late mRNAs increased 2-7-fold. Thus, the in vitro discriminatory activity of the 0.5 M wash was not dependent on the complexity of the mRNAs present in the translation mixture. We show also that in native extracts, under conditions of blocked polypeptide chain elongation, early mRNAs are initiated preferentially. However, late as well as early mRNAs are initiated equally well in reticulocyte extracts under similar experimental conditions when supplemented with crude initiation factors from infected cells. These data support the conclusion that the translational enhancement of FV 3 mRNAs in vitro is mediated by a virus-specified or virus-modified initiation factor(s) and likely represents a regulatory mechanism of protein synthesis operative in vivo (Willis, D. B., Goorha, R., Miles, M., and Granoff, A. (1977) J. Virol. 24, 326-342).     

Functional modifications of the translational system in Bacillus subtilis during sporulation.

Extracts of sporulating cells were found to be defective in vitro translation of phage SP01 ribonucleic acid (RNA) and vegetative Bacillus subtilis RNA. The activity of washed ribosomes from sporulating cells was very similar to that of washed ribosomes from vegetative cells in translating polyuridylic acid, SP01 RNA, and vegetative RNA. The S-150 fraction from either vegetative or sporulating cells grown in Difco sporulation medium contained an apparent inhibitor of protein synthesis. The crude initiation factor fraction from ribosomes of sporulating cells was defective in promoting the initiation factor-dependent translation of SP01 RNA. The crude initiation factor preparations from sporulating cells were as active as the corresponding preparations from vegetative cells in promoting the initiation factor-dependent translation of either phage Qbeta or phage T4 RNA by washed Escherichia coli ribosomes. The crude initiation factors from sporulating cells were perhaps more active than those from vegetative cells in promoting the initiation factor-dependent synthesis of phage T4 lysozyme by E. coli ribosomes. The crude initiation factor preparations from either vegetative or stationary-phase cells of an asporogenous mutant showed similar ability to promote the in vitro translation of SP01 RNA.     

Regulation of catalase synthesis in Saccharomyces cerevisiae by carbon catabolite repression.

Summary A number of strains of Saccharomyces cerevisiae, wild type or respiratory deficient, were grown on glucose, galactose or raffinose. Specific activities of catalase T were about tenfold higher in late stationary wild type cells grown on glucose than in wild type cells harvested when glucose had just disappeared completely from the medium, or in respiratory deficient strains (rho^–, mit^–, pet) grown to stationary phase.Catalase A activity is completely absent in wild type cells grown to zero percent glucose or in respiratory deficient cells grown on glucose to stationary phase. High catalase A activity was detected in derepressed wild type cells and in a strain carrying the op 1 (pet 9) mutation, although this strain is unable to grow on nonfermentable carbon sources. All respiratory deficient strains tested have low, but significant catalase A activities after growth on galactose or raffinose.Wild type cells harvested during growth on glucose and rho^–-cells grown on low glucose to stationary phase contain enzymatically inactive catalase A protein. The apoprotein of the enzyme is apparently accumulated in rho^–-cells whereas glucose-repressed wild type cells seem to contain a mixture of apoprotein and heme-containing catalase A monomer.These results show that a source of chemical energy, probably ATP, is required for derepression of yeast catalase from catabolite repression. At least in the case of catalase A, energy produced by respiration is necessary if catabolite repression is caused by glucose. If less repressing sugars are utilized, ATP derived from fermentation appears sufficient for partial derepression. Formation of the active enzyme can apparently be influenced by carbon catabolite repression at different points: (1) at the level of protein synthesis, (2) at the stage of heme incorporation, (3) at the level of formation of the enzymatically active tetramer.     

Binding of the La autoantigen to the 5' untranslated region of a chimeric human translation elongation factor 1A reporter mRNA inhibits translation in vitro.

Human translation elongation factor 1A (EF1A) is a member of a large class of mRNAs, including ribosomal proteins and other translation elongation factors, which are coordinately translationally regulated under various conditions. Each of these mRNAs contains a terminal oligopyrimidine tract (TOP) that is required for translational control. A human growth hormone (hGH) expression construct containing the promoter region and 5' untranslated region (UTR) of EF1A linked to the hGH coding region (EF1A/hGH) was translationally repressed following rapamycin treatment in similar fashion to endogenous EF1A in human B lymphocytes. Mutation of two nucleotides in the TOP motif abolished the translational regulation. Gel mobility shift assays showed that both La protein from human B lymphocyte cytoplasmic extracts as well as purified recombinant La protein specifically bind to an in vitro-synthesized RNA containing the 5' UTR of EF1A mRNA. Moreover, extracts prepared from rapamycin-treated cells showed increased binding activity to the EF1A 5' UTR RNA, which correlates with TOP mRNA translational repression. In an in vitro translation system, recombinant La dramatically decreased the expression of EF1A/hGH construct mRNA, but not mRNAs lacking an intact TOP element. These results indicate that TOP mRNA translation may be modulated through La binding to the TOP element.     

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.     

Synchronous Cell Differentiation in Caulobacter crescentus

The growth of a stalked bacterium, Caulobacter crescentus, has been synchronized easily and reproducibly by a new method. When this bacterium is grown to a late log phase in nutrient broth at 30 C with aeration, swarmer cells are accumulated in the culture to 80% of the whole cell population. When this culture is inoculated into fresh pre-warmed broth at twentyfold dilution, it immediately initiates synchronous cell growth. Simultaneously, synchronous cell differentiation is monitored by the susceptibility of the cells to RNA phage infection. The swarmer cells accumulated in the late log phase of growth possess nearly the same susceptibility to RNA phage infection as those in the early log phase of growth while RNA phage-adsorbing capacity is lower in such swarmer cells. It is suggested that the swarmer cells accumulated in the late log phase of growth have lost some pili.     

In Vitro Packaging of UV Radiation-Damaged DNA from Bacteriophage T7

When DNA from bacteriophage T7 is irradiated with UV light, the efficiency with which this DNA can be packaged in vitro to form viable phage particles is reduced. A comparison between irradiated DNA packaged in vitro and irradiated intact phage particles shows almost identical survival as a function of UV dose when Escherichia coli wild type or polA or uvrA mutants are used as the host. Although uvrA mutants perform less host cell reactivation, the polA strains are identical with wild type in their ability to support the growth of irradiated T7 phage or irradiated T7 DNA packaged in vitro into complete phage. An examination of in vitro repair performed by extracts of T7-infected E.coli suggests that T7 DNA polymerase may substitute for E. coli DNA polymerase I in the resynthesis step of excision repair. Also tested was the ability of a similar in vitro repair system that used extracts from uninfected cells to restore biological activity of irradiated DNA. When T7 DNA damaged by UV irradiation was treated with an endonuclease from Micrococcus luteus that is specific for pyrimidine dimers and then was incubated with an extract of uninfected E. coli capable of removing pyrimidine dimers and restoring the DNA of its original (whole genome size) molecular weight, this DNA showed a higher packaging efficiency than untreated DNA, thus demonstrating that the in vitro repair system partially restored the biological activity of UV-damaged DNA.     

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.