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)

Purified RNA polymerase I was phosphorylated by the endogenous protein kinase or dephosphorylated by alkaline phosphatase and used as antigen in a radioimmunoassay with sera from systemic lupus erythematosus patients or serum from an immunized rabbit. Enzyme incubated in the absence of ATP or phosphatase served as control. Three to seven times more of the autoantibodies in the patients' sera reacted with phosphorylated RNA polymerase I than with control enzyme. The reactivity of the dephosphorylated enzyme with lupus autoantibodies was only 50-60% of that observed with control enzyme. Neither phosphorylation nor dephosphorylation of the enzyme had an effect on its reaction with the rabbit antibodies. The effect of phosphorylation on the reaction of each RNA polymerase I subunit (S1-S8; Mr = 190,000-17,000) with the patients' antibodies was determined by an immunoblot procedure following resolution of the subunits on polyacrylamide gels. Prior phosphorylation of the enzyme resulted in a dramatic increase in binding of each patient's antibodies to all polymerase subunits with the exception of S4. Anti-S4 antibody was not detected with either phosphorylated or control enzyme. Strikingly, antibodies in each patients' sera reacted with S6 only after its phosphorylation. Similarly, anti-S5 antibodies in the serum of one patient were only detected with phosphorylated RNA polymerase I. The present data suggest that at least a significant fraction of the anti-RNA polymerase I autoantibodies in the sera of systemic lupus erythematosus patients might be directed against phosphorylated sites on the enzyme and that phosphorylation may have a role in the production of this and other autoimmunogenic nuclear components which are hallmarks of this disease.
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PMID:Phosphorylation of RNA polymerase I augments its interaction with autoantibodies of systemic lupus erythematosus patients. 650 Dec 73

A simple method for the purification of human placental nuclei is described. Nuclei were isolated by homogenizing tissue in standard saline citrate solution in the presence of zinc chloride to stabilize the nuclear membranes, NP40 as non-ionic detergent and sodium bisulphite for inhibition of proteolytic activity. Nuclei purification was achieved by low-speed centrifugation through a discontinuous sucrose gradient. The purified nuclei were evaluated by morphological criteria using phase contrast and electron microscopy. The extent of contamination by cytoplasmic debris was estimated by Papanicolaou's staining technique. Biochemical criteria include measurements of alkaline phosphatase activity as a plasma membrane enzyme marker and DNA-dependent RNA polymerase activity for the functional integrity of nuclear components. Transcriptionally active nuclei were obtained but the yield of nuclei was low; however, this low yield is compensated by the high degree of purity, the simplicity of the method and the functional and morphological integrity of the purified nuclei.
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PMID:Low-speed purification of human placental nuclei. 652 84

The effects of bile-duct ligation on hepatic and intestinal (jejunum) alkaline phosphatase activities were studied using rats and guinea pigs. In ligated rats, the enzyme activity was increased 4.1-fold in the liver after 24 h and 2.8-fold in the intestine after 12 h. In guinea pigs, the hepatic and intestinal enzyme activities were increased 2.3-fold and 1.5-fold after 100 and 24 h, respectively. The intestinal activity was induced sooner after ligation than hepatic activity. The induction of alkaline phosphatase was inhibited by prior treatment of animals with amanitin, an inhibitor of RNA polymerase activity. This result indicates that the induction is associated with de novo enzyme synthesis. The content of cyclic AMP in liver and intestine increased immediately after ligation. The increase in alkaline phosphatase activities was also inhibited by pretreatment with chlorpromazine, an inhibitor of adenylate cyclase activity. Hence, cellular cyclic AMP may be implicated in playing a role in the induction of alkaline phosphatase by bile-duct ligation.
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PMID:A possible mechanism for the changes in hepatic and intestinal alkaline phosphatase activities in bile-duct-ligated rats or guinea pigs. 661 80

A fast and simple assay for T7 RNA polymerase based upon chemiluminescent detection of the synthesized, digoxigenin-labeled RNA on a nylon membrane with anti-digoxigenin coupled alkaline phosphatase and CSPD as substrate is described. Activity of RNA polymerase is determined with high sensitivity by quantifying the emitted light of the microplate-formatted dot-blot membrane with a photon counting microplate luminometer and a specially designed filter adapter. The described method is one example for the application of this new adapter to measure luminescent membrane filters.
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PMID:Fast chemiluminescent measurement of RNA polymerase activity based on photon counting technology. 750 3

The expression pattern of tissue nonspecific alkaline phosphatase (TNAP) in the developing neural tube of mouse is reported. Homogeneous AP activity in the neuroepithelium becomes prominent at E8.5. At E9.5, distinctly AP-positive cells appear in the brain and spinal cord area. At stages E10.5 to E12.5, AP positivity is observed between the mesencephalon and the rhombencephalon, along the entire spinal cord and cranial nerves emerging from the myelencephalon. At E13.5, strongly AP positive fibers become prominent in the pons. At E14.5, AP expression in brain tissue is considerably reduced and there is a complete absence of AP activity in the nerve cells and glial cells of adult brain. The choroid plexus remains distinctly positive for AP expression until the adult stage. Northern blot analysis and reverse-transcriptase polymerase chain reaction amplification of RNA indicate that this AP activity results from the expression of the Akp-2 locus. This AP expression pattern is distinct from those reported for the expression of GD3, nestin, Hox 2.3, and Wnt-1 during brain development. We conclude that AP is a useful marker of a subpopulation of neuroectodermal cells present in the neural tube as early as E8.5, at which stages there are no other AP positive intraembryonic cells except PGCs.
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PMID:Stage-specific expression of alkaline phosphatase during neural development in the mouse. 753 63

The transcription initiation factor, TFIIF, is essential not only for the initiation of transcription but also for efficient elongation of mRNA synthesis by mammalian RNA polymerase II and is extensively phosphorylated in vivo. The possible regulation of TFIIF activity by protein phosphorylation was investigated by comparing the biochemical properties of alkaline phosphatase-treated HeLa TFIIF with those of native or bacterially expressed factor. Alkaline phosphatase treatment decreased the size of the large subunit (RAP74) of TFIIF to that of the recombinant protein but did not change the size of the small subunit (RAP30). Both the transcription initiation and elongation stimulating activities of the alkaline phosphatase-treated TFIIF decreased to 15-20% of the native form under conditions in which the amount of TFIIF was rate-limiting for transcription. Furthermore, phosphatase-treated TFIIF assembled the DBPolF complex and bound to RNA polymerase II less efficiently than the native protein. When hybrid TFIIFs were reconstituted using native or recombinant subunits, a native form of RAP74 stimulated both transcription and DBPolF complex formation activity regardless of whether native or recombinant RAP30 was used. We propose that TFIIF activity is regulated by protein phosphorylation, particularly of the RAP74 subunit. The functional role of RAP74 in assembling the preinitiation complex and modulating TFIIF activity is discussed.
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PMID:Regulation of the human general transcription initiation factor TFIIF by phosphorylation. 796 96

Interphase cytosol extracts prepared from Xenopus laevis eggs are active in RNA polymerase III (Pol III) transcription. Addition of recombinant B1 cyclin to these extracts activates mitotic protein kinases that repress transcription. Affinity-purified p34cdc2-cyclin B kinase (mitosis-promoting factor) is sufficient to effect this repression in a simplified Pol III transcription system. This mitotic repression involves the direct phosphorylation of a component of the Pol III transcription initiation factor TFIIIB, which consists of the TATA box-binding protein (TBP) and associated Pol III-specific factors. The transcriptional activity of the TFIIIB-TBP fraction can be modulated in vitro by phosphorylation with mitotic kinases and by dephosphorylation with immobilized alkaline phosphatase.
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PMID:Mitotic repression of RNA polymerase III transcription in vitro mediated by phosphorylation of a TFIIIB component. 827 69

The Fab region of an IgG2b antibody (AM7B2.1) reactive to the herbicide atrazine was cloned into a plasmid vector using the polymerase chain reaction and two sets of degenerate oligonucleotide primers designed to mimic the amino acid variation at the N-termini of kappa L-chains and gamma H-chains. These primers also provide a secretion signal fused precisely to the antibody gene sequence for secretion of the mature antibody. A further set of universal oligonucleotide primers was developed for the direct sequencing of the VH and CH1 regions of gamma H-chains and the VL and CL regions of kappa L-chains without subcloning and were used to determine the sequence of this antibody. The kappa L-chain was found to not possess a conserved Cys residue at position 23 and the implications of this observation are discussed. The cloned genes were expressed in Escherichia coli using a commercially available T7 RNA polymerase-based plasmid. The clones were also expressed in a T7 RNA polymerase-based system containing an attenuated version of the T7 RNA polymerase promoter, plus a lac promoter placed in an antisense orientation, to enhance plasmid stability. The expressed products were confirmed as atrazine reactive by binding to an atrazine derivative conjugated with alkaline phosphatase.
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PMID:Cloning, sequencing and expression of the Fab fragment of a monoclonal antibody to the herbicide atrazine. 830 47

An indirect enzyme linked immunoassay (ELISA) has been developed to measure the amount of RNA polymerase I (E.C.2.7.7.6) in silkmoth tissue cell extracts. Subunit specific monoclonal antibodies (MABs) were immobilized on the solid substrate by a variation of the widely used Protein-Avidin-Biotin-Capture (PABC) technique. The use of the commercially available biotinylated anti-mouse antibody as a bridge to bind the monoclonal antibody eliminates the need for the biotinylation of the monoclonal antibody in the laboratory. The RNA polymerase in solution was captured by the monoclonal antibody and was measured by the successive binding of rabbit polyclonal antibody and alkaline phosphatase conjugated anti-rabbit antibody. This procedure is more reliable, reproducible and leads to greater sensitivity compared to the direct binding of the monoclonal antibody to the microtiter plate. RNA polymerase I captured by the antibodies from tissue extracts was measured at levels of 0.5 ng/well. This assay system can be utilized as a general procedure to quantitate the levels of proteins present at very low levels and that are found in different isoforms containing multiple and/or shared subunits.
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PMID:A modified PABC immunoassay for the quantitation of DNA dependent RNA polymerase I: a procedure applicable to other proteins present in minute amounts and/or isoforms. 834 15

Eight mutations lying within the promoter-proximal one-fifth of the spoIIA locus of Bacillus subtilis were studied. Two of these mutations (spoIIAA42 and spoIIAA69) were previously characterized at the DNA level, five more (spo-562, spo-565, spo-567, spo-568, and spo-569) were isolated in our laboratory several years ago but not fully characterized, and the eight (an in-frame deletion confined to spoIIAA, the first gene in the spoIIA operon) was constructed for this study. DNA sequencing showed that spo-569 was a transitions in the -35 region of the spoIIA promoter; the remaining point mutations were all G:C to A:T transitions in spoIIAA, with spo-565 having two transitions, one of which was identical to that in spo-562. All the spoIIAA mutations except spo-562 led to the replacement of Gly residues. The incidence of sporulation, the rate of synthesis of sporulation-associated alkaline phosphatase, and the rate of expression of the forespore-specific genes gpr and spoIIIG were determined for isogenic strains carrying the eight mutations. All the mutations except spoIIAA42 and spo-569 (which were slightly leaky) made the strains asporogenous, and all except spo-562 and spo-569 abolished the synthesis of alkaline phosphatase and the expression of gpr and spoIIIG. spo-562 allowed alkaline phosphatase synthesis and gpr and spoIIIG expression to occur at about 15% of the wild-type rates but with normal kinetics. spo-59 allowed appreciable gpr and spoIIIG expression during exponential growth; we attribute this expression to transcription by RNA polymerase containing sigma G and suggest that a spo-569 strain makes insufficient SpoIIAB to inhibit sigma G in growing cells.
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PMID:Molecular and phenotypic characterization of promoter-proximal mutations in the spoIIA locus of Bacillus subtilis. 836 48


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