Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To examine the possible role of the vaccinia virus glutaredoxin as a cofactor for viral ribonucleotide reductase, viral growth, DNA synthesis, and dNTP pools were measured in infections of B-SC-40 monkey kidney cells with wild type vaccinia virus and with mutants of vaccinia that lacked a functional reductase or glutaredoxin. In infections of untreated host cells, the lack of viral ribonucleotide reductase or glutaredoxin had only small effects upon virus growth. When host cells were pretreated with alpha-amanitin, which blocks host RNA polymerase II but not viral transcription, viral DNA synthesis was markedly reduced in infections with either of the mutants when compared with wild type infections. Relative to dNTP levels in wild type infections, pools of dCTP, but not of the other dNTPs, were significantly reduced in infections of amanitin-treated cells with either mutant. The parallel depletion of dCTP in the two mutant suggests that the role of glutaredoxin may be to function as a cofactor for viral ribonucleotide reductase. The data suggest that both viral proteins become essential for DNA replication only when levels of the corresponding host cell proteins are depleted.
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PMID:Roles of vaccinia virus ribonucleotide reductase and glutaredoxin in DNA precursor biosynthesis. 749 96

In order to examine whether splicing can occur cotranscriptionally in mammalian nuclei, we mapped exon-intron boundaries on nascent RNA chains transcribed by RNA polymerase II. A procedure that allows fractionation of nuclei into a chromatin pellet containing DNA, histones, and ternary transcription complexes and a supernatant containing the bulk of the nonhistone proteins and RNAs that are released from their DNA templates was developed. The transcripts of the genes encoding DBP, a transcriptional activator protein, and HMG coenzyme A reductase recovered from the chromatin pellet and the supernatant were analyzed by S1 nuclease mapping. The large majority of the RNA molecules from the pellet appeared to be nascent transcripts, since, in contrast to the transcripts present in the supernatant, they were not cleaved at the polyadenylation site but rather contained heterogeneous 3' termini encompassing this site. Splicing intermediates could be detected among nascent and released transcripts, suggesting that splicing occurs both cotranscriptionally and posttranscriptionally. Our results also indicate that polyadenylation is not required for the splicing of the last DBP intron. In addition to allowing detailed structural analysis of nascent RNA chains, the physical isolation of nascent transcripts also yields reliable measurements of relative transcription rates.
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PMID:Physical isolation of nascent RNA chains transcribed by RNA polymerase II: evidence for cotranscriptional splicing. 752 61

11 beta-Hydroxysteroid dehydrogenase (11 beta-HSD), responsible for the interconversion of hormonally active cortisol to inactive cortisone, dictates specificity for the mineralocorticoid receptor (MR) in the distal nephron and colon. Two isoforms of human 11 beta-HSD have been cloned, an NADP(H)-dependent (type 1) dehydrogenase/oxo-reductase enzyme, and a high-affinity NAD-dependent (type 2) unidirectional dehydrogenase. Using the reverse-transcriptase polymerase chain reaction (RT-PCR) amplification of RNA extracted from human adult tissues, type 1 11 beta-HSD mRNA was found in decidua, placenta, liver, lung, spleen, kidney medulla, cerebellum and pituitary, but was absent in kidney cortex, sigmoid and rectal colon, salivary gland and thyroid. In contrast, type 2 11 beta-HSD mRNA was found only in placenta and in the classical mineralocorticoid target tissues, kidney cortex, kidney medulla, sigmoid and rectal colon, salivary gland, and colonic epithelial cell lines (AAC1 and RGC28). In situ hybridization studies of renal cortex, cortico-medullary junction and medulla using a 35S-labeled antisense cRNA probe for type 2 human 11 beta-HSD, revealed specific localization of type 2 11 beta-HSD mRNA expression exclusively to renal cortical and medullary collecting ducts. Type 1 and type 2 isoforms of human 11 beta-HSD are expressed in a distinct tissue-specific fashion, in keeping with the proposed differences in their physiological roles. Type 2 11 beta-HSD is found predominantly in mineralocorticoid target tissues where it serves to protect the MR in an autocrine fashion.
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PMID:Detection of human 11 beta-hydroxysteroid dehydrogenase isoforms using reverse-transcriptase-polymerase chain reaction and localization of the type 2 isoform to renal collecting ducts. 754 19

Monodehydroascorbate radical (MDA) reductase, an FAD-enzyme, is the first enzyme to be identified whose substrate is an organic radical and catalyzes the reduction of MDA to ascorbate by NAD(P)H. Its cDNA has been cloned from cucumber seedlings (Sano, S., and Asada, K. (1994) Plant Cell Physiol. 35, 425-437), and a plasmid was constructed in the present study that allowed a high level expression in Escherichia coli of the cDNA-encoding MDA reductase using the T7 RNA polymerase expression system. The recombinant MDA reductase was purified to a crystalline state, with a yield of over 20 mg/liter of culture, and it exhibited spectroscopic properties of the FAD similar to those of the enzyme purified from cucumber fruits during redox reactions with NADH and MDA. The red semiquinone of the FAD of MDA reductase was generated by photoreduction. p-Chloromercuribenzoate inhibited the reduction of the enzyme-FAD by NADH, and dicumarol suppressed electron transfer from the reduced enzyme to MDA. The specificity of electron acceptors of the recombinant enzyme appeared to be similar to that of MDA reductase, even though the amino acid sequence encoded by the cDNA was somewhat different from that of the enzyme purified from cucumber fruits. The Km values for NADH and NADPH of the recombinant enzyme indicated a high affinity of the enzyme for NADH. The reaction catalyzed by the enzyme did not exhibit saturation kinetics with MDA up to 3 microM. A second order rate constant for the reduction of the enzyme-FAD with NADH was 1.25 x 10(8) M-1 s-1, as determined by a stopped-flow method, and its value decreased with increases in ionic strength, an indication of the enhanced electrostatic guidance of NADH to the enzyme-FAD.
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PMID:Molecular characterization of monodehydroascorbate radical reductase from cucumber highly expressed in Escherichia coli. 754 69

N-Substituted indan-1.3-diones have proven to be potent cytotoxic agents effective against the growth of single cell leukemia tumors and cell lines derived from solid tumors. A number of the derivatives were active against growth of solid tumors e.g. colon, lung bronchogenic and osteosarcoma for which few effective agents are available to inhibit their growth. These agents inhibited DNA and RNA synthesis of L1210 cells. The de novo purine synthetic pathway was inhibited at PRPP amido transferase and IMP dehydrogenase. The pyrimidine synthetic pathway was inhibited at aspartate transcarbamylase. Other sites which demonstrate minor inhibition were DNA polymerase alpha, r- and t-RNA polymerase, ribonucleoside reductase, dihydrofolate reductase, nucleoside kinases and thymidylate synthetase. In addition d(NTP) pool levels were reduced by the drugs. L1210 DNA strand scission was evident after exposure to drugs for 24 hr. at 100 microM.
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PMID:Cytotoxicity and mode of action of substituted indan-1, 3-diones in murine and human tissue cultured cells. 784 49

The expression of the Escherichia coli torCAD operon, which encodes the anaerobically expressed trimethylamine N-oxide (TMAO) reductase respiratory system, requires the presence of TMAO in the medium. The response regulator, TorR, has recently been identified as the regulatory protein that controls the expression of the torCAD operon in response to TMAO. The torC regulatory region contains four direct repeats of a decameric consensus motif designated the tor boxes. Alteration by base substitutions of any of the four tor boxes in a plasmid containing a torC'-lacZ fusion dramatically reduces TorR-dependent torC expression. In addition, deletion of the distal tor box (box1) abolishes torC induction whereas the presence of a DNA fragment starting three bases upstream from box1 suffices for normal torC expression. Footprinting and gel-retardation experiments unambiguously demonstrated that TorR binds to the torC regulatory region. Three distinct regions are protected by TorR binding. One of approximately 24 nucleotides covers the first two tor boxes (box1 and box2); the second is located upstream from the -35 promoter sequence and includes the third tor box (box3); the last is found downstream from the -35 sequence and corresponds to the fourth tor box (box4). Binding to the upstream tor boxes (box1 and box2) appears to be stronger than binding to the downstream tor boxes (box3 and box4) since only the upstream region is protected at the lower concentration of TorR used in the footprinting experiments. We propose a model in which multiple binding sites (i.e. the tor boxes) contribute to the formation of a nucleoprotein complex, but only one particular proximal site positions TorR properly so that it interacts with RNA polymerase.
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PMID:Binding of the TorR regulator to cis-acting direct repeats activates tor operon expression. 859 46

The Escherichia coli gene murB, encoding the enzyme uridine-5'-diphospho-N-acetyl-2-amino-2-deoxy-3-O-lactylglucosenicoti namide adenine dinucleotide phosphate oxidoreductase (EC 1.1.1.158) (EP-reductase), the second enzyme in the peptidoglycan biosynthetic pathway, has been amplified using PCR technology with the Kohara recombinant lambda phage E11C11 (534) as template. The synthetic gene was subcloned into the NdeI and BamHI restriction sites of the expression vector pT7-7, designed to utilize T7 RNA polymerase to direct transcription of the target gene, in a two-step procedure. The first step involved the directional insertion of the 590-bp NdeI to BamHI restriction fragment of murB into the pT7-7 vector to give the plasmid pT7-7-murB-590. The construction of the desired overproducing plasmid was completed by the bidirectional insertion of the 442-bp BamHI to BamHI restriction fragment of murB into a similarly restricted pT7-7-murB-590 plasmid followed by restriction digestion to select the properly oriented insert, pT7-7-murB. Overexpression of EP-reductase from the E. coli strain BL 21 (DE 3) containing the pT7-7-murB gene, after induction, allowed the production of 36 mg of target protein per 3 wet grams of E. coli cells. The EP-reductase was purified in a single step utilizing dye-ligand chromatography to yield 30 mg of pure protein. The availability of these levels of reductase will allow the mechanism of this pivotal enzyme to be thoroughly studied as a potential target for the design of a new generation of antibiotics. In addition, the EP-reductase generated in this study has been utilized as a coupling enzyme to assay the first enzyme in the peptidoglycan biosynthetic pathway, UDP-N-acetylglucosamine enolpyruvyl transferase, and these results are also presented.
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PMID:Overproduction and one-step purification of Escherichia coli UDP-N-acetylglucosamine enolpyruvyl reductase. 874 27

The 3,5-isoxazolidinediones and 2-isoxazolin-5-ones demonstrated potent cytotoxicity against the growth of human Tmolt3 T cell leukemia, murine P388 and L1210 leukemias, as well as human HeLa-S3 uterine carcinoma and glioma tumor cell growth. The specificity of the 3,5-isoxazolidinedione and 2-isoxazoline-5-one derivatives as cytotoxic agents varied with the histological type of tumor cell. Selected compounds were active against solid HeLa uterine. KB nasopharynx, skin A431, SW-480 adenocarcinoma, osteosarcoma and glioma growth. Selected compounds demonstrated in vivo antineoplastic activity against Ehrlich ascites carcinoma growth. In L-1210 leukemia cells, the agents blocked DNA and protein synthesis at 25, 50 and 100 microM over 60 min. The agents were effective in reducing rate limiting enzymes in the de novo purine and pyrimidine pathways. In addition they suppressed dihydrofolate reductase and ribonucleoside reductase activities with moderate inhibition of DNA and RNA polymerase activities. DNA itself was not a target of the agents.
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PMID:Synthesis and cytotoxic action of 3,5-isoxazolidinediones and 2-isoxazolin-5-ones in murine and human tumors. 916 49

This review describes a range of pH responses. Some are only induced if relevant DNA is brought to an appropriately supercoiled configuration by DNA gyrase and bent by the action of, for example, integration host factor (IHF). Bending may allow transcription by bringing activators into juxtaposition with RNA polymerase, which is CysB-associated in several of the responses. Control of arginine decarboxylase (AdiA) synthesis at acid pH is of the above type, with dependence on the presence of gyrase, H-NS, IHF and CysB; acid induction of LysU has similar requirements but also needs Lrp; lysine decarboxylase (CadA) formation at acid pH is controlled quite differently, needing the CadC activator and interaction of lysine/lysine permease; H-NS probably reverses induction by CadC. The Hyd components of formic hydrogenlyase are induced by acid under anaerobiosis; a transcriptional activator is involved and Fur may also function in regulation. Acid tolerance induced at low pH in log-phase cells needs CysB and PhoE but not DNA gyrase; tolerance is reduced by NaCl but not affected by Fe3+, Fe2+, glucose/cAMP or by lrp, him, fur, hns or nhaA/B lesions. Alkali tolerance (habituation), induced at pH0 8.5-9.0, probably involves DNA supercoiling and bending; the induction process needs IHF, CysB, PhoE, NhaA, TonB and Fur and is glucose-repressed; tolerance may result from Na+ efflux catalysed by the NhaA antiporter, which is induced at pH0 9.0. Alkali sensitivity induced at pH0 5.5 also requires gyrase, IHF and CysB, but H-NS, Lrp, NhaA and OmpC are also needed and induction is abolished by NaCl. Salt-induced acid sensitivity results from PhoE formation and is blocked by glucose (reversed by cAMP), FeCl3 and hns and relA lesions, the effect of relA being envZ-suppressed. Acid sensitivity induction (ASI) at pH0 9.0 needs H-NS, is inhibited by FeCl3 and amiloride, and is associated with alkyl hydroperoxide reductase synthesis. Leucine-induced acid sensitivity needs gyrase, CysB, H-NS, Fur, OmpA and RelA, is inhibited by Fe3+, Fe2+, tetracycline, glucose and nalidixic acid, but not by chloramphenicol; increased outer membrane proton passage may result from OmpA modification.
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PMID:Regulatory components, including integration host factor, CysB and H-NS, that influence pH responses in Escherichia coli. 917 36

Plasma cholesterol levels increase after birth, and to a greater extent in breast-fed versus formula-fed infants. This increase is believed to be due to the high fat and cholesterol content of the infant diet, but little is known about the effects of early diet on the expression of proteins involved in regulating cholesterol metabolism. This study examined changes in the expression of hepatic proteins regulating cholesterol metabolism during development. Newborn piglets were fed sow milk or one of four formulas for 18 days. The formulas had similar levels of palmitic acid (16:0) as in milk, supplied as palm olein oil with 16:0 esterified predominantly to the sn-1,3 position or as synthesized triglyceride (TG) with 16:0 esterified mainly to the sn-2 position of glycerol, each with no cholesterol (<0.10 mmol/L) or 0.65 mmol/L cholesterol added. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of mRNA levels was used to assess the effects of diet on hepatic hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase, low-density lipoprotein (LDL) receptor, and 7alpha-hydroxylase (C7H). LDL receptor mRNA levels showed no appreciable difference between milk- and formula-fed piglets. However, the levels of HMG-CoA reductase and C7H mRNA were higher (P < .05) in all formula-fed versus milk-fed piglets, irrespective of the formula TG source or cholesterol content. The lower levels of HMG-CoA reductase and C7H mRNA in milk-fed piglets were accompanied by higher (P < .05) plasma total, high-density lipoprotein (HDL), and apolipoprotein (apo) B-containing cholesterol. These studies show that the levels of hepatic HMG-CoA reductase and C7H mRNA, but probably not LDL receptor mRNA, are altered by early diet.
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PMID:Early diet influences hepatic hydroxymethyl glutaryl coenzyme A reductase and 7alpha-hydroxylase mRNA but not low-density lipoprotein receptor mRNA during development. 944 Apr 72


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