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Requirements for macromolecular synthesis in the establishment of beta-galactosidase repression in zygotes.

Inhibitors of protein synthesis do not consistently prevent formation of the lac operon repressor, according to several published reports, although direct evidence indicates that the repressor is a protein. Inhibition of ribonucleic acid (RNA) synthesis has never been shown to block lactose repression. These results have raised the possibility that repressor is synthesized in some unusual fashion. We have studied the effect of various inhibitors upon the establishment of repression in zygotes, utilizing conditions which minimize catabolite repression. Inhibition of protein synthesis by either chloramphenicol treatment or tryptophan deprivation blocked repressor formation in our experiments. Sodium borate and 6-azauracil are compounds reported to be specific inhibitors of RNA synthesis, and their behavior in control experiments is consistent with this specificity. Both delayed the establishment of repression. Thymine deprivation, either by starvation of a thymine auxotroph or by treatment with 5-fluorodeoxyuridine, did not delay the onset of repression. We conclude that repressor formation requires RNA synthesis and probably utilizes the usual protein-forming mechanisms.

Aminopterin↗

A segment of mRNA encoding the leader peptide of the CPA1 gene confers repression by arginine on a heterologous yeast gene transcript.

The expression of the yeast gene CPA1, which encodes the small subunit of the arginine pathway carbamoylphosphate synthetase, is repressed by arginine at a translational level. CPA1 mRNA contains a 250-nucleotide-long leader which includes a 25-codon upstream open reading frame (uORF). Oligonucleotide site-directed mutagenesis of this uORF as well as sequencing of constitutive cis-dominant mutations has suggested that the leader peptide product of the CPA1 uORF is an essential negative element for repression of the CPA1 gene by arginine. In this work, a series of deletions affecting the regions 5' and 3' to the uORF in the leader sequence was constructed. The arginine-dependent repression of CPA1 was little affected in these constructions, indicating that these regions are not essential for the regulatory response. This conclusion was further supported by the finding that inserting the mRNA segment encoding the leader peptide sequence of CPA1 in the leader sequence of another gene, namely, GCN4, places this gene under arginine repression. Similarly, the behavior of fusions of the leader sequence of CPA1 with those of ARG4 or GAL10 confirmed that the regions of this leader located upstream and downstream from the uORF are dispensable for the regulation by arginine. Finally, a set of substitution mutations which modify the uORF nucleotide sequence while leaving unchanged the corresponding amino acid sequence was constructed. The mutations did not affect the repression of CPA1 by arginine. The data presented in this paper consequently agree with the conclusion that the leader peptide itself is the main element required for the translational repression of CPA1.

Amino Acid Sequence↗

trans-acting factors affecting carbon catabolite repression of the hut operon in Bacillus subtilis.

In Bacillus subtilis, CcpA-dependent carbon catabolite repression (CCR) mediated at several cis-acting carbon repression elements (cre) requires the seryl-phosphorylated form of both the HPr (ptsH) and Crh (crh) proteins. During growth in minimal medium, the ptsH1 mutation, which prevents seryl phosphorylation of HPr, partially relieves CCR of several genes regulated by CCR. Examination of the CCR of the histidine utilization (hut) enzymes in cells grown in minimal medium showed that neither the ptsH1 nor the crh mutation individually had any affect on hut CCR but that hut CCR was abolished in a ptsH1 crh double mutant. In contrast, the ptsH1 mutation completely relieved hut CCR in cells grown in Luria-Bertani medium. The ptsH1 crh double mutant exhibited several growth defects in glucose minimal medium, including reduced rates of growth and growth inhibition by high levels of glycerol or histidine. CCR is partially relieved in B. subtilis mutants which synthesize low levels of active glutamine synthetase (glnA). In addition, these glnA mutants grow more slowly than wild-type cells in glucose minimal medium. The defects in growth and CCR seen in these mutants are suppressed by mutational inactivation of TnrA, a global nitrogen regulatory protein. The inappropriate expression of TnrA-regulated genes in this class of glnA mutants may deplete intracellular pools of carbon metabolites and thereby result in the reduction of the growth rate and partial relief of CCR.

Bacillus subtilis↗

The Pasteur effect and catabolite repression in an oxidative yeast, Kluyveromyces lactis.

The presence of the Pasteur effect in Kluyveromyces lactis grown in glucose was shown by azide-stimulated glucose fermentation. Extracts from these cells contained ATP-sensitive phosphofructokinase activity. Cells grown on succinate oxidized glucose slowly at first without azide-stimulated rates of fermentation. Phosphofructokinase in these cells was ATP-insensitive. The activity of NAD+-isocitrate dehydrogenase in cell extracts did not require AMP activation. These results suggested the presence of a Pasteur effect in glucose-grown but not in succinate-grown K. lactis, mediated by (a) ATP inhibition of phosphofructokinase (b) possibly via feedback control of glucose transport, but not by AMP activation of isocitrate dehydrogenase. Azide inhibition of the Pasteur effect during growth of the cells did not lead to catabolite repression of respiratory activity. The results therefore suggest that the Pasteur effect does not inhibit the development of a Crabtree effect in oxidative yeasts.

Adenosine Triphosphate↗

Involvement of the lac regulatory genes in catabolite repression in Escherichia coli.

1. Acute transient catabolite repression of beta-galactosidase synthesis, observed when glucose is added to glycerol-grown cells of Escherichia coli (Moses & Prevost, 1966), requires the presence of a functional operator gene (o) in the lactose operon. Total deletion of the operator gene abolished acute transient repression, even in the presence of a functional regulator gene (i). 2. Regulator constitutives (i(-)) also show transient repression provided that the operator gene is functional. Regulator deletion mutants (i(del)), with which to test specifically the role of the i gene, have not so far been available. 3. The above mutants, showing various changes in the lactose operon, show no alteration in the effect of glucose on induced tryptophanase synthesis. Glucose metabolism, as measured in terms of the release of (14)CO(2) from [1-(14)C]glucose and [6-(14)C]glucose, also showed no differences between strains exhibiting or not exhibiting transient repression. This suggests no change in the operation of the pentose phosphate cycle, a metabolic activity known to be of paramount importance for glucose repression of beta-galactosidase synthesis (Prevost & Moses, 1967). 4. Chronic permanent repression by glucose of beta-galactosidase synthesis (less severe in degree than acute transient repression) persists in strains in which transient repression has been genetically abolished. Constitutive alkaline-phosphatase synthesis, which shows no transient repression, also demonstrates chronic permanent repression by glucose. 5. Chloramphenicol repression also persists in mutants with no transient repression, and also affects alkaline phosphatase. It is suggested that chronic permanent repression and chloramphenicol repression are non-specific, and that they do not influence beta-galactosidase synthesis via the regulatory system of the lactose operon.

Alkaline Phosphatase↗

Role of the regulator-gene product (repressor) in catabolite repression of beta-galactosidase synthesis in Escherichia coli.

1. The specific role of the lac repressor (i-gene product) in transient catabolite repression evoked by the introduction of glucose into the medium has been investigated in Escherichia coli by using mutants of the i-gene. 2. A temperature-sensitive mutant (i(TL)) is normally inducible and demonstrates transient repression when grown at 32 degrees . At 42 degrees it is about 20% constitutive and transient catabolite repression is abolished. 3. A strain carrying an amber suppressor-sensitive mutation in the i-gene is phenotypically constitutive and also fails to show transient catabolite repression. 4. Insertion of Flaci(+) into this strain restores both inducibility and transient repression. 5. It is concluded that the i-gene product interacts with the catabolite co-repressor in such a way that its affinity for the operator is increased.

Enzyme Induction↗

Synthesis of the beta and beta' subunits of Escherichia coli RNA polymerase is autogenously regulated in vivo by both transcriptional and translational mechanisms.

Numerous experiments have indicated that the synthesis of RNA polymerase (beta beta' alpha 2 sigma 70) in Escherichia coli is autogenously regulated. In the present study, we have examined expression of the rpoB and rpoC genes which encode the beta and beta' subunits of RNA polymerase. These genes are the distal cistrons of the rplKAJLrpoBC ribosomal protein-RNA polymerase transcription unit. Both transcriptional (operon) and translational (gene) fusions of either rpoB or rpoC to the lacZ reporter were used to monitor their in vivo expression by inserting single copies of these fusions into the bacterial chromosome on integration-proficient lambda vectors. In order to examine the expression of the rpoBC genes in response to varied intracellular concentrations of the RNA polymerase subunits, a series of expression plasmids carrying the rpoB, rpoC, rpoA (alpha) and rpoD (sigma 70) genes in different combinations were then introduced into these cells. Elevated concentrations of either beta or beta' were found to repress the expression of both rpoB and rpoC at the translational level. However, the simultaneous increase in the concentration of all the subunits that comprise holoenzyme repressed the transcription of rpoBC. To determine the site of this repression, additional operon fusions were utilized which placed lacZ downstream of each of the transcriptional regulatory sites of this gene cluster, including two promoters, rplKp and rplJp, and a transcriptional attenuator in the rplL-rpoB intercistronic region. Expression from these fusions showed that transcriptional repression is achieved primarily by reducing initiation at both upstream promoters, coupled with a small increase in termination at the attenuator.

DNA-Directed RNA Polymerases↗

HIV-1 expression is posttranscriptionally repressed in Drosophila cells.

The long terminal repeats (LTRs) of the human immunodeficiency virus (HIV) the Rous sarcoma virus (RSV) and the copia Drosophila retrotransposon were compared in their capacity to direct expression of the bacterial cat (chloramphenicol acetyltransferase) gene in human, murine, and Drosophila cell lines. The results indicate that HIV and RSV LTR expression is post transcriptionally repressed in the Drosophila cells while copia LTR expression is post-transcriptionally repressed in the human and murine cells.

Animals↗

Regulation of lac operon expression: reappraisal of the theory of catabolite repression.

The physiological state of Escherichia coli with respect to (permanent) catabolite repression was assessed by measuring the steady-state level of beta-galactosidase in induced or in constitutive cells under a variety of growth conditions. Four results were obtained. (i) Catabolite repression had a major effect on fully induced or constitutive expression of the lac gene, and the magnitude of this effect was found to be dependent on the promoter structure; cells with a wild-type lac promoter showed an 18-fold variation in lac expression, and cells with the lacP37 (formerly lac-L37) promoter exhibited several hundred-fold variation. (ii) Exogenous adenosine cyclic 3',5'-monophosphoric acid (cAMP) could not abolish catabolite repression, even though several controls demonstrated that cAMP was entering the cells in significant amounts. (Rapid intracellular degradation of cAMP could not be ruled out.) (iii) Neither the growth rate nor the presence of biosynthetic products altered the degree of catabolite repression; all variation could be related to the catabolites present in the growth medium. (iv) Slowing by imposing an amino acid restriction decreased the differential rate of beta-galactosidase synthesis from the wild-type lac promoter when bacteria were cultured in either the absence or presence of cAMP; this decreased lac expression also occurred when the bacteria harbored the catabolite-insensitive lacP5 (formerly lacUV5) promoter mutation. These findings support the idea that (permanent) catabolite repression is set by the catabolites in the growth medium and may not be related to an imbalance between catabolism and anabolism.

Culture Media↗

Reductive pentose phosphate-independent CO2 fixation in Rhodobacter sphaeroides and evidence that ribulose bisphosphate carboxylase/oxygenase activity serves to maintain the redox balance of the cell.

Whole-cell CO2 fixation and ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) activity were determined in Rhodobacter sphaeroides wild-type and mutant strains. There is no obvious difference in the levels of whole-cell CO2 fixation for the wild type, a form I RubisCO deletion mutant, and a form II RubisCO deletion mutant. No ribulose 1,5-bisphosphate-dependent CO2 fixation was detected in a form I-form II RubisCO double-deletion mutant (strain 16) or strain 16PHC, a derivative from strain 16 which was selected for the ability to grow photoheterotrophically with CO2 as an electron acceptor. However, significant levels of whole-cell CO2 fixation were detected in both strains 16 and 16PHC. Strain 16PHC exhibited CO2 fixation rates significantly higher than those of strain 16; the rates found for strain 16PHC were 30% of the level found in photoheterotrophically grown wild-type strain HR containing both form I and form II RubisCO and 10% of the level of the wild-type strain grown photolithoautotrophically. Strain 16PHC could not grow photolithoautotrophically in a CO2-H2 atmosphere; however, CO2 fixation catalyzed by photoheterotrophically grown strain 16PHC was repressed by addition of the alternate electron acceptor dimethyl sulfoxide. Dimethyl sulfoxide addition also influenced RubisCO activity under photolithoautotrophic conditions; 40 to 70% of the RubisCO activity was reduced without significantly influencing growth. Strain 16PHC and strain 16 contain nearly equivalent but low levels of pyruvate carboxylase, indicating that CO2 fixation enzymes other than pyruvate carboxylase contribute to the ability of strain 16PHC to grow with CO2 as an electron acceptor.

Carbon Dioxide↗

Effect of amino sugars on catabolite repression in Escherichia coli.

N-acetylglucosamine was found to be a good repressor source for catabolite repression of the beta-galactosidase system in Escherichia coli. It was found capable of increasing the severity of repression by glucose or gluconate when included in the medium with either of these substrates. N-acetylglucosamine was shown to be assimilated under these conditions, but had no effect on culture growth rates. Its influence on catabolite repression was not altered by growth in the presence of inhibiting levels of penicillin. These findings indicated that catabolite repression may be associated with certain reactions of amino sugar metabolism. A working model has been formulated along these lines and will be used to explore this possible relationship further.

Carbon Isotopes↗

Regulation of 3-deoxy-D-arabino-heptulosonic 7-phosphate acid synthetase activity in relation to the synthesis of the aromatic vitamins in Escherichia coli K-12.

Both in vivo and in vitro experiments on wild-type Escherichia coli K-12 and mutant strains possessing only single 3-deoxy-d-arabino-heptulosonic 7-phosphate acid (DAHP) synthetase isoenzymes indicated that, under conditions when all three isoenzymes are fully repressed, sufficient chorismate is still formed for the synthesis of aromatic vitamins. Under repressed conditions both DAHP synthetase (phe) and (trp), but not DAHP synthetase (tyr), were shown to contribute to vitamin production.

Aldehyde-Lyases↗

Isolation and characterization of transposon-induced chlorate resistant mutants of the cyanobacterium Anabaena species PCC 7120.

Mutants of Anabaena sp. PCC 7120 resistant to chlorate were isolated using transposon mutagenesis. The Anabaena population of 5 x 10(7) cells ml(-1) and log phase Escherichia coli cultures in undisturbed conditions produced maximum exconjugants. Nitrate-promoted growth and cellular constituents observed in the parent were absent in the mutants. Nitrate repressed heterocyst formation and N2-fixation in the parent, but had little or no effect on the mutants.

Anabaena↗

Regulation of histidase synthesis in intergeneric hybrids of enteric bacteria.

Regulation of the expression of the histidase coded by hutk of Klebsiella aerogenes in Salmonella typhimurium and in Escherichia coli and of the expression of the histidase coded by huts of S. typhimurium in E. coli was investigated. The hutk histidase was found to be sensitive to catabolite repression in K. aerogenes and in E. coli, but insensitive to catabolite repression in S. typhimurium; huts histidase has previously been shown to be catabolite sensitive in all three organisms. The expression of both hutk and huts histidase in E. coli was activated by nitrogen starvation. Apparently, the glutamine synthetase of E. coli may activate the formation of some glutamate- and ammonia-producing enzymes.

Ammonia-Lyases↗

Cyclic adenosine 3',5'-monophosphate levels and activities of adenylate cyclase and cyclic adenosine 3',5'-monophosphate phosphodiesterase in Pseudomonas and Bacteroides.

A modified Gilman assay was used to determine the concentrations of cyclic adenosine 3',5'-monophosphate (cAMP) in rapidly filtered cells and in the culture filtrates of Pseudomonas aeruginosa, Escherichia coli K-12, and Bacteroides fragilis. In P. aeruginosa cultures, levels of cAMP in the filtrate increased with the culture absorbance (3.5 to 19.8 X 10(-9) M) but did not vary significantly with the carbon source used to support growth. Intracellular concentrations (0.8 to 3.2 X 10(-5) M) were substantially higher and did not vary appreciably during growth or with carbon source. Sodium cAMP (5 mM) failed to reverse the catabolite repression of inducible glucose-6-phosphate dehydrogenase (EC 1.1.1.49) synthesis caused by the addition of 10 mM succinate. Exogenous cAMP also had no discernible effect on the catabolite repression control of inducible mannitol dehydrogenase (EC 1.1.1.67). P. aeruginosa was found to contain both soluble cAMP phosphodiesterase (EC 3.1.4.17) and membrane-associated adenylate cyclase (EC 4.6.1.1) activity, and these were compared to the activities detected in crude extracts of E. coli. B. fragilis crude cell extracts contain neither of these enzyme activities, and little or no cAMP was detected in cells or culture filtrates of this anaerobic bacterium.

3',5'-Cyclic-AMP Phosphodiesterases↗

Inductive and repressive effects of rifampicin on rabbit liver microsomal cytochrome P-450.

New Zealand White rabbits were treated with rifampicin at a dose of 50 mg/kg for 4 days. The total amount of microsomal hepatic cytochrome P-450 was not modified in treated, with respect to control, animals. However, further studies involving SDS-PAGE analysis, monooxygenase activity measurements and radial immunodiffusion assays indicated that rifampicin strongly affects the level of two P-450 isoenzymes. An LM3 form was induced; this form, apparently associated with erythromycine demethylase activity and hydroxylation of progesterone preferentially in position 6 beta, was shown to be immunologically and functionally different from LM3a and LM3b. On the other hand, an LM4 form, typically induced by beta-naphthoflavone, was repressed. The concomitant inductive/repressive effect of rifampicine on two cytochrome P-450 isoenzymes makes this drug a very atypical inducer, at least in the rabbit.

Animals↗

Activation of protein kinase B/Akt is sufficient to repress the glucocorticoid and cAMP induction of phosphoenolpyruvate carboxykinase gene.

A rat hepatoma cell line, H4IIE, was stably transfected with a tamoxifen regulatable Akt-1 construct. Treatment of these cells with tamoxifen caused a rapid stimulation of Akt enzymatic activity that was comparable with the activity observed with the endogenous Akt after insulin stimulation. Prior studies have extensively documented that insulin can repress the glucocorticoid and cAMP-stimulated increase in phosphoenolpyruvate carboxykinase (PEPCK) gene transcription. Activation of this regulatable Akt with tamoxifen was found to mimic the dominant inhibitory effect of insulin on PEPCK gene transcription. Dose response curves to insulin and tamoxifen demonstrated that this response was very sensitive to Akt activation although the maximal response observed with tamoxifen activation was slightly less than that observed with insulin, indicating that the response to insulin may also involve other signaling cascades. The regulation of PEPCK transcription via Akt was, like that previously described for insulin, not dependent upon 70 kDa S6 kinase activity in that it was not inhibited by rapamycin. Finally, the expression of a kinase dead Akt was able to partially inhibit the ability of insulin to stimulate this response. In summary, the present results indicate that activation of Akt alone is sufficient to repress the glucocorticoid and cAMP-stimulated increase in PEPCK gene transcription.

Animals↗

Microbial water relations. Effects of solute concentration on the respiratory activity of sugar-tolerant and non-tolerant yeasts.

The respiratory activity of the sugar-tolerant (osmophilic) yeast, Saccharomyces rouxii, and the non-tolerant species, Sacchromyces cerevisiae, were compared after growth in a complex basal medium, the medium supplemented with polyethylene glycol (mol. wt 200) to give a water activity of 0-95, and the medium supplemented with glucose (24 and 36%, w/v). The properties compared were Qo2 (glucose), NADH oxidase activity of isolated mitochondrial fractions, and cytochrome content. When grown in the basal medium S. cerevisiae was somewhat more active than S. rouxii by all criteria. Growth in the media supplemented were high glucose concentrations produced catabolite repression of respiration in S. cerevisiae but not in S. rouxii. The implications of this difference for polyol biosynthesis and the water relations of the sugar-tolerant species are discussed.

Cytochromes↗