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PubMed1996

Role of inhibitory CDC2 phosphorylation in radiation-induced G2 arrest in human cells.

The activity of the mitosis-promoting kinase CDC2-cyclin B is normally suppressed in S phase and G2 by inhibitory phosphorylation at Thr14 and Tyr15. This work explores the possibility that these phosphorylations are responsible for the G2 arrest that occurs in human cells after DNA damage. HeLa cell lines were established in which CDC2AF, a mutant that cannot be phosphorylated at Thr14 and Tyr15, was expressed from a tetracycline-repressible promoter. Expression of CDC2AF did not induce mitotic events in cells arrested at the beginning of S phase with DNA synthesis inhibitors, but induced low levels of premature chromatin condensation in cells progressing through S phase and G2. Expression of CDC2AF greatly reduced the G2 delay that resulted when cells were X-irradiated in S phase. However, a significant G2 delay was still observed and was accompanied by high CDC2-associated kinase activity. Expression of wild-type CDC2, or the related kinase CDK2AF, had no effect on the radiation-induced delay. Thus, inhibitory phosphorylation of CDC2, as well as additional undefined mechanisms, delay mitosis after DNA damage.

CDC2 Protein Kinase↗
PubMed1996

Analysis of CRP-CytR interactions at the Escherichia coli udp promoter.

Multiprotein complexes regulate the transcription of certain bacterial genes in a sensitive, physiologically responsive manner. In particular, the transcription of genes needed for utilization of nucleosides in Escherichia coli is regulated by a repressor protein, CytR, in concert with the cyclic AMP (cAMP) activated form of cAMP receptor protein (CRP). We studied this regulation by selecting and characterizing spontaneous constitutive mutations in the promoter of the udp (uridine phosphorylase) gene, one of the genes most strongly regulated by CytR. We found deletions, duplications, and point mutations that affect key regulatory sites in the udp promoter, insertion sequence element insertions that activated cryptic internal promoters or provided new promoters, and large duplications that may have increased expression by udp gene amplification. Unusual duplications and deletions that resulted in constitutive udp expression that depended on the presence of CytR were also found. Our results support the model in which repression normally involves the binding of CytR to cAMP-CRP to form a complex which binds to specific sites in the udp promoter, without direct interaction between CytR protein and a specific operator DNA sequence, and in which induction by specific inducer cytidine involves dissociation of CytR from cAMP-CRP and the RNA polymerase interaction with cAMP-CRP bound to a site upstream of then transcription start point. The stimulation of udp expression by CytR in certain mutants may reflect its stabilization of cAMP-CRP binding to target DNA and illustrates that only modest evolutionary changes could allow particular multiprotein complexes to serve as either repressors or transcriptional activators.

Bacterial Proteins↗
PubMed1997

A tricarboxylic acid cycle intermediate regulating transcription of a chloroaromatic biodegradative pathway: fumarate-mediated repression of the clcABD operon.

The ortho-cleavage pathways of catechol and 3-chlorocatechol are central catabolic pathways of Pseudomonas putida that convert aromatic and chloroaromatic compounds to tricarboxylic acid (TCA) cycle intermediates. They are encoded by the evolutionarily related catBCA and clcABD operons, respectively. Expression of the cat and clc operons requires the LysR-type transcriptional activators CatR and ClcR, respectively, and the inducer molecules cis,cis-muconate and 2-chloro-cis,cis-muconate, respectively. The regulation of the cat and clc promoters has been well studied, but the extent to which these operons are repressed by growth in TCA cycle intermediates has not been explored. We demonstrate by transcriptional fusion studies that the expression from the clc promoter is repressed when the cells are grown on succinate, citrate, or fumarate and that this repression is ClcR dependent and occurs at the transcriptional level. The presence of these organic acids did not affect the expression from the cat promoter. In vitro transcription assays demonstrate that the TCA cycle intermediate fumarate directly and specifically inhibits the formation of the clcA transcript. No such inhibition was observed when CatR was used as the activator on either the cat or clc template. Titration studies of fumarate and 2-chloromuconate show that the fumarate effect is concentration dependent and reversible, indicating that fumarate and 2-chloromuconate most probably compete for the same binding site on ClcR. This is an interesting example of the transcriptional regulation of a biodegradative pathway by the intracellular sensing of the state of the TCA cycle.

Bacterial Proteins↗
PubMed1969

Alteration of glucose metabolism of Arthrobacter crystallopoietes by compounds which induce sphere to rod morphogenesis.

Succinate and several other compounds which induce sphere to rod morphogenesis of A. crystallopoietes were found to suppress both catabolism and assimilation of glucose. Diauxic growth patterns resulted from growth on glucose plus any one of these compounds. Glutamate stimulated growth but was not an inducer of morphogenesis. With this compound, diauxic growth and suppression of glucose catabolism or assimilation did not occur. Glucose permease was studied with alpha-methylglucoside as substrate. The entry system for glucose was found to involve active transport and to have a K(m) of 8 x 10(-4)m. It was inducible, was repressed in succinate-grown cells, and was also inhibited by succinate. The exit system was constitutive and appeared to be less sensitive than the entry system to inhibition by azide. The properties of the glucose permease system may account for the slow growth of the organism on glucose and the preferred use of other substrates for growth. Studies of metabolic pathways for glucose metabolism indicated the operation of the Embden-Meyerhof-Parnas (EMP) and pentose phosphate pathways and of the tricarboxylic acid cycle. Cells grown on glucose plus limiting amounts of succinate or other inducers of morphogenesis metabolized the glucose only after exhaustion of the inducers. Under these circumstances, the organisms employed the EMP pathway to a greater extent than when growing on glucose as sole carbon source.

Arthrobacter↗
PubMed1973

Abnormal cell envelope ultrastructure of a Saccharomyces mutant with invertase formation resistant to hexoses.

The most obvious morphological characteristic of Saccharomyces mutant FH4C cells is the tendency to form clumps (production of invertase and alpha-glucosidase by this mutant is highly resistant to repression by hexoses). This peculiar feature arises from the abnormal cell envelope ultrastructure of the mutant. Clumps are formed as a result of the failure of the cell wall of the bud to separate from that of the mother cell. The cell wall also shows irregular thickening. There are many cells with a doughnut shape and with small budlike protrusions. Abnormal septation and wall invagination into vacuoles give rise to cells of differing sizes and irregular profiles. Many vesicles, tubules, and coiled membranous bodies originate from invaginations of the plasmalemma. These structures are frequently observed in the cell wall or the periplasmic space. The cells of mutant FH4C growing in 0.2 M glucose, unlike parent strain 303-67, contain many mitochondria. Large numbers of glycogen deposits are also found in many cells of FH4C.

Cell Membrane↗
PubMed1995

Identification of a low specificity, oxygen, heme, and growth phase regulated DNA binding activity in Saccharomyces cerevisiae.

Cytochrome c oxidase from Saccharomyces cerevisiae is subject to intricate control at the level of transcription of the various genes encoding its subunits. Expression of the subunit VI encoding gene, COX6, is glucose repressed, growth phase induced, and dependent on oxygen and heme availability. An upstream activation region for COX6, UAS6, was found to contain a glucose responsive region, a heme dependent region (HDS1), and a binding site for the transcription factor, BAF1. BAF1 was the only factor observed to form a protein complex with UAS6 in vitro. However, we found that binding of BAF1 was unaffected by oxygen or heme regulation. In the present communication, we have identified a DNA binding activity that was growth phase induced and dependent on oxygen and heme availability. This highly regulated activity was detected by its discrete binding to the heme responsive site, HDS1, in UAS6. Nonetheless, it appears to have weak DNA binding specificity.

Base Sequence↗
PubMed1977

Alterations of alkaline phosphatase activity during adaptation of Escherichia coli to phosphite and hypophosphite.

When Escherichia coli cells were grown in media containing either phosphite or hypophosphite as the sole source of phosphorus, the responded to this situation primarily in the same way as phosphate-limited cultures: The activity of alkaline phosphatase increased drastically, which under natural conditions would enable the cells to compensate for the shortage of phosphate. Subsequent transfers, however, resulted in a quite different response: While the phosphatase activity of phosphate-limited cells stays at a high derepressed level, its increase was followed by a gradual decline in organisms grown on phosphite of hypophosphite. After eight to ten transfers on these P-compounds, phosphatase activity was back to its initial, repressed, low level, indicating that the cells were fully adapted to these substrates. Adaptation to either PO3-3 or PO3-2 was completely abolished if the cells were again grown with PO3-3 as P-source, whereafter the entire process of adaptation had to be repeated. The observed adaptation pattern, reflected by the alterations of phosphatase activity, was qualitatively equal with PO3-3 and PO3-2, but quantitatively different, because the response to hypophosphite gave much higher values than the increase obtained with phosphite. Phosphite-adapted cells are not simultaneously adapted to hypophosphite, but their response to the latter was less intense than observed after direct transfers from PO3-4 to PO3-2. Adaptation to hypophosphite, however, led simultaneously to phosphite adaptation, so that these cells can utilize both P-compounds as a substitute for phosphate.

Adaptation, Physiological↗
PubMed1990

A developmentally regulated and cAMP-repressible gene of Dictyostelium discoideum: cloning and expression of the gene encoding cyclic nucleotide phosphodiesterase inhibitor.

A 1.6-kb genomic fragment containing the coding region for the inhibitor (PDI) of cyclic nucleotide phosphodiesterase (PD) was isolated and sequenced. The genomic sequence includes 510 nucleotides (nt) of 5'-noncoding sequence and the full coding sequence, which contains two small introns. From the deduced amino acid (aa) sequence we predict a 26-kDa protein that, in agreement with previous data, contains approximately 15% Cys residues. The PDI possesses a hydrophobic leader sequence, five potential glycosylation sites, and three internal repeats. Northern-blot analysis showed a single transcript of 0.95 kb. The gene encoding PDI (pdi) was expressed early in development with little transcript remaining following aggregation. The appearance of pdi transcript was inhibited by cAMP, but when cAMP was removed the transcript appeared within 30 min. When cAMP was applied to cells containing pdi mRNA, the transcript disappeared with a half-life of less than 30 min.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1976

Repression, derepression and activation of nitrogenase in Azotobacter vinelandii.

Ammonium ion repressed nitrogenase in cells fixing N2 gas. Immunological tests and electrophoresis in various gels show that components I (Fe-Mo-S protein) was completely repressed by ammonium, whereas component II (Fe-S protein) apoprotein was not markedly affected. Component II from ammonium-grown cells, however, was inactive since it did not cross react with component I to reduce C2H2 to C2H4. The inactive component II apoprotein is immunologically identical to its active counterpart from cells fixing N2. Identical protein patterns were also observed in various gel-electrophoresis systems. Oxygen-inactivated component II may be reactivated with FeSo4. This salt is preferable to ferrous ammonium sulphate which inactivated component I. Immunodiffusion under aerobic conditions shows that purified component I is composed of aggregated and non-aggregated forms which are antigenically distinct. The aggregate was dissociated by treatment with sodium dodecyl sulphate (SDS) into a single antigenic species which was further resolved into two subunits on SDS disc polyacrylamide gel electrophoresis.

Ammonium Chloride↗
PubMed1995

Repressible cation-phosphate symporters in Neurospora crassa.

The filamentous fungus Neurospora crassa possesses two nonhomologous high-affinity phosphate permeases, PHO-4 and PHO-5. We have isolated separate null mutants of these permeases, allowing us to study the remaining active transporter in vivo in terms of phosphate uptake and sensitivity to inhibitors. The specificity for the cotransported cation differs for PHO-4 and PHO-5, suggesting that these permeases employ different mechanisms for phosphate translocation. Phosphate uptake by PHO-4 is stimulated 85-fold by the addition of Na+, which supports the idea that PHO-4 is a Na(+)-phosphate symporter. PHO-5 is unaffected by Na+ concentration but is much more sensitive to elevated pH than is PHO-4. Presumably, PHO-5 is a H(+)-phosphate symporter. Na(+)-coupled symport is usually associated with animal cells. The finding of such a system in a filamentous fungus is in harmony with the idea that the fungal and animal kingdoms are more closely related to each other than either is to the plant kingdom.

Biological Transport↗
PubMed1992

Upstream activation element of the PH03 gene encoding for thiamine-repressible acid phosphatase in Saccharomyces cerevisiae.

The PH03 gene of Saccharomyces cerevisiae encodes thiamine-repressible acid phosphatase and requires the positively acting regulatory protein THI2 for its expression. Deletion analysis of the 5'-flanking region of PH03 gene revealed that an activating region located at nucleotide position -234 to -215 relative to the translation initiation codon is required for the expression and sensitivity to thiamine. A chemically synthesized DNA fragment covering -234 to -215 showed a significant level of expression when inserted in front of the PH03 promoter lacking the activating region. Electrophoretic mobility shift assay demonstrated the presence of proteins that bound to the above DNA fragment in the nuclear extract from cells grown in thiamine-free medium. These findings suggested that this region between -234 and -215 acts as an upstream activation element of the PH03 gene that can interact with regulatory proteins.

Acid Phosphatase↗
PubMed1994

Repression of ALA synthase by heme and zinc-mesoporphyrin in a chick embryo liver cell culture model of acute porphyria.

We characterize a liver cell culture model for acute hepatic porphyrias that recapitulates the biochemical features of the human syndrome. In chick embryo liver cells in primary culture exposed to glutethimide and 4,6-dioxoheptanoic acid, heme alone produced a transient dose-dependent decrease in delta-aminolevulinate synthase and a concomitant increase in heme oxygenase. The addition of low concentrations of zinc-mesoporphyrin (50-200 nM), an inhibitor of heme oxygenase, led to more prolonged decreases in activity of the synthase and to an additive effect with heme. These effects of zinc-mesoporphyrin were associated with prolonged inhibition of heme oxygenase. These results suggest that the treatment of choice of acute porphyric syndromes may be the combination of low doses of heme and zinc-mesoporphyrin or another similarly non-toxic inhibitor of heme oxygenase.

5-Aminolevulinate Synthetase↗
PubMed2004

Targeted histone deacetylase inhibition for cancer therapy.

The histone deacetylase inhibitors are a new class of cytostatic agents that inhibit the proliferation of tumor cells in culture and in vivo by inducing cell cycle arrest, differentiation and/or apoptosis. Histone acetylation and deacetylation play important roles in the modulation of chromatin topology and the regulation of gene transcription. Histone deacetylase inhibition induces the accumulation of hyperacetyl-ated nucleosome core histones in most regions of chromatin but affects the expression of only a small subset of genes, leading to transcriptional activation of some genes, but repression of an equal or larger number of other genes. Non-histone proteins such as transcription factors are also targets for acetylation with varying functional effects. Ace-tylation enhances the activity of some transcription factors such as the tumor suppressor p53 and the erythroid differentiation factor GATA-1 but may repress transcriptional activity of others including T cell factor and the co-activator ACTR. Recent studies in our laboratory and others have shown that the estrogen receptor alpha (ERalpha) can be hyperacetylated in response to histone deacetylase inhibition, suppressing ligand sensitivity and regulating transcriptional activation by histone deacetylase inhibitors. Conservation of the acetylated ERalpha motif in other nuclear receptors suggests that acetylation may play an important regulatory role in diverse nuclear receptor signaling functions. A number of structurally diverse histone deacetylase inhibitors have shown potent antitumor efficacy with little toxicity in vivo in animal models. Several compounds are currently in early phase clinical development as potential treatments for solid and hematological cancers both as monotherapy and in combination with cytotoxics and differentiation agents. This report reviews the biology and clinical development of histone deacetylase inhibitors for cancer therapy.

Acetylation↗
PubMed1999

Mimicry in primary rat hepatocyte cultures of the in vivo perivenous induction by phenobarbital of cytochrome P-450 2B1 mRNA: role of epidermal growth factor and perivenous oxygen tension.

Treatment of male rats with phenobarbital (PB) results in a perivenous and mid-zonal pattern of cytochrome P-450 (CYP)2B1 mRNA expression within the liver acinus. The mechanism of this zonated induction is still poorly understood. In this study sinusoidal gradients of oxygen and epidermal growth factor (EGF) besides those of the pituitary-dependent hormones growth hormone (GH), thyroxine (T4), and triiodothyronine (T3) were considered to be possible determinants for the zonated induction of the CYP2B1 gene in liver. Moreover, heme proteins seem to play a key role in oxygen sensing. Therefore, the influence of arterial (16% O2) and venous (8% O2) oxygen tension (pO2), and of the heme synthesis inhibitors CoCl2 and desferrioxamine (DSF) on PB-dependent CYP2B1 mRNA induction as well as the repression by EGF and, for comparison, by GH, T4, and T3, of the induction under arterial and venous pO2 were investigated in primary rat hepatocytes. Within 3 days, phenobarbital induced CYP2B1 mRNA to maximal levels under arterial pO2 and to about 40% of maximal levels under venous pO2. CoCl2 annihilated induction by PB under both oxygen tensions, whereas desferrioxamine and heme abolished the positive modulation by O2, suggesting that heme is a necessary component for O2 sensing. EGF suppressed CYP2B1 mRNA induction by PB only under arterial but not under venous pO2, whereas GH, T4, and T3 inhibited induction under both arterial and venous pO2. Thus, in hepatocyte cultures, an O2 gradient in conjunction with EGF mimicked the perivenous induction by PB of the CYP2B1 gene observed in the liver in vivo.

Animals↗
PubMed1986

Induction and repression of the urea amidolyase gene in Saccharomyces cerevisiae.

The DUR1,2 gene from Saccharomyces cerevisiae has been isolated on recombinant plasmids along with all DNA between the DUR1,2 and MET8 loci. DUR1,2 was found to encode a 5.7-kilobase transcript, which is consistent with our earlier suggestion that the DUR1 and DUR2 loci are two domains of a single multifunctional gene. Steady-state levels of the DUR1,2 transcript responded to induction and nitrogen catabolite repression in the same way as urea amidolyase activity. dal81 mutants (grown with inducer) contained barely detectable amounts of DUR1,2 RNA, whereas dal80 mutants (grown without inducer) contained the same amount as a wild-type induced culture. These observations support our earlier hypothesis that DUR1,2 is transcriptionally regulated, with control being mediated by the DAL80 and DAL81 gene products. We cloned the DUR1,2-Oh mutation and found it to be a Ty insertion near sequences required for complementation of dur1,2 mutations. The ROAM phenotype of the DUR1,2-Oh mutation is sharply different from that of cis-dominant, DUR80 mutations, which enhance DUR1,2 expression but do not affect the normal control pattern of the gene. There is evidence that DUR80 mutations may also be Ty insertions, which generate phenotypes that are different from those in DUR1,2-Oh mutations.

Carbon-Nitrogen Ligases↗
PubMedDate not supplied

[The ratio of coat protein to bacteriophage f2 RNA in the translational repressor complex].

One of the mechanisms underlying the regulation of the bacteriophage f2 RNA translation is the repression of the phage RNA-replicase formation by coat protein. This repression is due to the formation of a complex between f2 RNA and coat protein (complex I). In this work the mechanism of complex I formation as well as the effect of this complex on the f2 RNA-replicase formation was followed by inhibition of alanine incorporation into RNA-replicase polypeptide which was separated by polyacrylamide gel electrophoresis. The molar ratios of protein to f2 RNA in complex I were analyzed by sucrose gradient sedimentation. It was been found that complex I consists of six molecules of coat protein bound per one molecule of RNA. Ribonuclease digestion of the glutaraldehyde-fixed complex resulted in a mixture of products in which the hexamers of coat protein molecules were predominant. This indicates that the six molecules of coat protein bound to f2 RNA are neighbouring. It has been also shown that under conditions required for phage protein synthesis, coat protein occurs in solution is dimer. The results show that the translational repression of the RNA-replicase cistron is due to the cooperative attachment of three dimers of coat protein to phage template, forming a hexameric cluster on the RNA strand. The proposed mechanism of the complex I formation seems to be in good agreement with the sequence of events in the phage F2 life cycle. It is known that shortly after infection of the host cell the coat protein and phage RNA-replicase begin to be synthesised. According to our findings, the first portions of coat protein do not affect the translation of the RNA-replicase gene since at low concentration the coat protein occure in the form of monomers. At a later period of phage development, when the concentration of coat protein is sufficiently high to promote the formation of protein dimers, the translational repressor complex is formed and the RNA-replicase gene becomes inoperative.

Coliphages↗
PubMed1989

Transcriptional and posttranscriptional control of the Bacillus subtilis succinate dehydrogenase operon.

The amount of succinate dehydrogenase (SDH) in Bacillus subtilis varies with growth conditions. In this work we studied the steady-state level and the rate of decay of B. subtilis sdh mRNA under different growth conditions. In exponentially growing cells, the steady-state level of sdh mRNA was severalfold lower when glucose was present compared with growth without glucose, whereas the rate of decay of sdh mRNA was the same with and without glucose. Thus, glucose repression seems to act by decreasing sdh mRNA synthesis. When the bacteria entered the stationary phase, the steady-state level of sdh mRNA dropped about sixfold. At the same time, sdh mRNA half-life decreased from 2.6 to 0.4 min. This result indicates that transcription of the sdh operon is initiated at the same rate in exponentially growing and in stationary-phase cells. The start point of the sdh transcripts, as measured by primer extension, was the same under all conditions studied, suggesting that the sdh operon is solely controlled by the previously identified sigma 43-like promoter. The increase of SDH activity in stationary phase may be explained by reduced dilution of the SDH proteins as a result of the retarded growth rate. We suggest that enhanced degradation of the sdh transcript is a means by which the bacteria adjust expression to the demands of stationary phase.

Bacillus subtilis↗
PubMed1974

Mechanism of carbamyl phosphate inhibition of nitrogenase of Clostridium pasteurianum.

Carbamyl phosphate caused a maximal inhibition of 50% of the in vitro nitrogenase activity measured by acetylene reduction and dinitrogen reduction. The addition of 1 mM carbamyl phosphate to a N(2)-fixing culture caused a rapid decrease of 30% of the acetylene reduction activity and also repression of nitrogenase biosynthesis. However, carbamyl phosphate had no effect on the reductant-dependent adenosine triphosphate hydrolysis and H(2) evolution reactions catalyzed by nitrogenase. Studies on the binding of carbamyl phosphate to nitrogenase and each of its two components (azoferredoxin and molybdoferredoxin) indicated that optimal binding was obtained only in the presence of an operating nitrogenase system. Moreover, the binding seemed to be on the molybdoferredoxin component rather than azoferredoxin. From a Scatchard plot and a reciprocal plot of the data, the values of n = 2 and dissociation constant (K) of approximately 5 x 10(-5) M were obtained. The value for the dissociation constant was of the same order of magnitude as the endogenous level of carbamyl phosphate in a N(2)-fixing cell. The carbamyl phosphate pool in NH(3)-grown cells was twice that of N(2)-fixing cells.

Acetylene↗