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:3.1.26.9 (ribonuclease)
6,589 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The muscle wasting which occurs in animals bearing a transplantable tumour is accompanied by a decrease in the level of protein synthesis and a loss in RNA. This paper examines the behaviour of RNA polymerases I and II (EC 2.7.7.6) in nuclei isolated from skeletal muscle of rats bearing a Walker 256 carcinoma. Marked decreases were observed in template-engaged RNA polymerase I and II activities and in free RNA polymerase I activity. Free RNA polymerase II activity was unaltered. When assays were carried out at high (NH4)2SO4 concentration or in the presence of heparin the diminished RNA polymerase I activity was still apparent, but heparin and high ionic strength overcame the inhibition of RNA polymerase II. Loss of RNA polymerase I activity was associated with a decrease in the number of template-engaged enzyme molecules and in the polynucleotide elongation rate. The number of template-engaged RNA polymerase II molecules was unaltered by tumour growth, but the polynucleotide elongation rate was significantly reduced. No evidence was obtained for any alteration in ribonuclease activity in nuclei or whole muscles of tumour-bearing rats. These results demonstrate an effect of the tumor on transcription in skeletal muscle of its host.
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PMID:Response of skeletal muscle RNA polymerases I and II to tumour growth. 316 55

Transcription by purified mammalian RNA polymerase II in vitro leads to extensive formation of DNA-RNA hybrids between nascent RNA and the template DNA strand. This is especially clear during transcription of 3'-extended (dC-tailed) DNA templates where the nontranscribed DNA strand is progressively displaced as transcription proceeds [Kadesch, T. R., & Chamberlin, M. J. (1982) J. Biol. Chem. 257, 5286-5295]. Addition of small amounts of a HeLa cell extract to such a transcription system enhances renaturation of the template DNA and displacement of the nascent RNA, as measured by the sensitivity of the RNA to pancreatic ribonuclease. Using this latter assay, we have purified a protein factor (renaturase) 250-fold from HeLa cell extracts using chromatography on DEAE-cellulose, DNA-cellulose, and hydroxylapatite. Renaturase preparations facilitate complete renaturation of the template DNA duplex during transcription by RNA polymerase II and lead to concurrent displacement of the nascent RNA. Current preparations are free from all but traces of deoxyribonuclease or ribonuclease. The active component has a molecular weight of about 30000 as estimated by preparative density gradient sedimentation. We have examined the structure of transcribing RNA polymerase II complexes in the presence and absence of renaturase, using the electron microscope and the Williams polylysine technique [Williams, R. C. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 2311-2315]. In the presence of renaturase, the DNA template is fully renatured, and a ternary complex in which the nascent RNA is displaced during transcription is seen.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Studies on transcription of 3'-extended DNA templates by mammalian RNA polymerase II. Partial purification and characterization of a factor from HeLa cells that facilitates renaturation of the DNA template. 399 13

Evidence is presented that isoproterenol treatment of rat C6 glioma cells, under conditions that increase glioma cell cAMP levels, causes the phosphorylative modification of several RNA polymerase II subunits. RNA polymerase II in control and isoproterenol-stimulated 32Pi-labeled confluent glioma cells was immunoprecipitated from ribonuclease-treated nuclear extracts with hen anti-calf RNA polymerase II antiserum conjugated to Sepharose. The immunoprecipitated RNA polymerase II was analyzed for 32P-labeled subunits by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Using this technique, we have shown that isoproterenol causes a time-dependent increase of phosphate incorporation into RNA polymerase II subunits of 214,000, 180,000, 140,000, 35,000, 28,000, and 16,500 daltons. Phosphate incorporation occurred exclusively on serine in all of the six subunits. About 0.5-2 mol of phosphate/mol of RNA polymerase II subunit were incorporated. Dibutyryl cAMP (10(-3)M) mimics the stimulatory action of isoproterenol and mediates increased phosphate incorporation into the six subunits. (RS)-propranolol (10(-4)M) prevents the isoproterenol-mediated phosphorylative changes. These data indicate that isoproterenol, via cAMP, mediates a transient structural modification of RNA polymerase II subunits in rat C6 glioma cells which may possibly lead to a modulation of RNA polymerase II function(s).
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PMID:Phosphorylation of rat C6 glioma cell DNA-dependent RNA polymerase II in vivo. Identification of phosphorylated subunits and modulation of phosphorylation by isoproterenol and N6,O2'-dibutyryl cyclic AMP. 609 70

Ternary transcription complexes have been formed with a HeLa cell extract, a specific DNA template, and nucleoside triphosphates. The assay depends on the formation of sarkosyl-resistant initiation complexes which contain RNA polymerase II, template DNA, and radioactive nucleoside triphosphates. Separation from the other elements in the in vitro reaction is achieved by electrophoresis in agarose - 0.25% sarkosyl gels. The mobility of the ternary complexes in this system cannot be distinguished from naked DNA. Formation of this complex is dependent on all parameters necessary for faithful in vitro transcription. Complexes are formed with both the plasmid vector and the specific adenovirus DNA insert containing a eucaryotic promoter. The formation of the complex on the eucaryotic DNA is sequence-dependent. An undecaribonucleotide predicted from the template DNA sequence remains associated with the DNA in the ternary complex and can be isolated if the chain terminator 3'-0-methyl GTP is used, or after T1 ribonuclease treatment of the RNA, or if exogenous GTP is omitted from the in vitro reaction. This oligonucleotide is not detected in association with the plasmid vector. Phosphocellulose fractionation of the extract indicates that at least one of the column fractions required for faithful runoff transcription is required for complex formation. A large molar excess of abortive initiation events was detected relative to the level of productive transcription events, indicating a 40-fold higher efficiency of transcription initiation vs. elongation.
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PMID:RNA polymerase II ternary transcription complexes generated in vitro. 619 89

In experiments to determine the mechanism of glucocorticoid induced decreases in thymic transcription, adrenalectomized rats were injected with hydrocortisone (50 mg/kg) or vehicle. Thymic nuclei were used to prepare chromatins and soluble nuclear extracts containing RNA polymerase II for cross-over experiments. With calf thymus DNA or rat thymic chromatins as templates limiting RNA polymerase II from rats treated with hydrocortisone 3 h previously had 130% of the [3H]UMP incorporating activity of RNA polymerase II from control vehicle treated rats. In contrast, limiting RNA polymerase II from rats treated with hydrocortisone 12 h previously had 40-50% of the [3H]UMP incorporating activity of RNA polymerase II from controls. When limiting calf thymus DNA or rat thymic chromatins were used in 12 h cross-over experiments. Individual RNA polymerases II produced equal [3H]UMP incorporations, but RNA polymerase II activity from hydrocortisone treated rats was again only 50% of control values. Thus with template saturation, RNA polymerase II from hydrocortisone treated rats could not transcribe rat thymic chromatin templates to the level achieved by RNA polymerase II from control rats. This suggests that the activity, rather than the amount, of RNA polymerase II from hydrocortisone treated rats is reduced. Double reciprocal plots of [3H]UMP incorporation on rat chromatins with increasing concentrations of RNA polymerases II were made at 12 h. The apparent Km for RNA polymerase II from animals treated with hydrocortisone was identical to that of RNA polymerase II from controls, but the Vmax of RNA polymerase II from hydrocortisone treated animals was reduced. These data suggest the presence of an inhibitor of transcription or an RNA polymerase II defective in its capacity to initiate and/or elongate RNA transcripts. Further experiments demonstrated that these effects were not due to steroid induced changes in ribonuclease or protease activities.
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PMID:Studies on the mechanism of glucocorticoid hormone induced alterations in rat thymic transcription--I. Evidence from reconstituted cross-over transcription assays that sequential increases and decreases in transcription are due to changes in the activity of RNA polymerase II rather than in the activity of chromatin template. 667 54

Total HeLa cell extract was separated into multiple components using first DEAE-Sephadex and then phosphocellulose column chromatography. Four major fractions, DE175, DE500, P100, and P1000, from HeLa cells are found to be essential for accurate and efficient transcription of cloned ovalbumin DNA fragments in the reconstituted system. DE175 serves as the source for RNA polymerase II and DE500 minimizes the synthesis of small molecular size RNA. P100 enhances the levels of specific transcription, while P1000 is absolutely required for correct initiation. Chick oviduct crude extract was also fractionated into multiple components according to the same procedure. Similar efficiency of specific initiation could be obtained when an individual fraction (DE175, DE500, P100, or P1000) from oviduct cells was exchanged for a fraction from HeLa cells. These results indicate that chick oviduct tissue also contains general transcription factors like that of HeLa cells and these factors can be fractionated according to the same procedure. In this fractionation scheme, we were able to separate the bulk of RNase activity into the P350 fraction which was not required for initiation of ovalbumin RNA transcription. Thus, this reconstituted system is suitable for studying in vitro transcription in a homologous system derived from tissue extracts, even though a substantial amount of cellular ribonuclease may be associated with it.
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PMID:Transcription factors from oviduct and HeLa cells are similar. 689 17

Highly purified yeast RNA polymerase III ternary complexes were found to possess a hydrolytic chain retracting activity that cleaves nascent RNA from its 3'-OH end. Most of the shortened transcripts were capable of resuming RNA chain elongation, indicating that they remain stably associated with the enzyme-DNA complex. Analysis of the products of cleavage indicated that retraction primarily occurred in dinucleotide increments, but that mononucleotides were also excised at lower frequency. The ribonuclease activity was totally dependent on the presence of a divalent cation and was stimulated by the addition of non-cognate ribonucleotides. The inclusion of ATP in the reaction enhanced both the rate and extent of transcript cleavage. Evidence suggesting that the hydrolytic activity is intrinsic to RNA polymerase III and factor-independent is also presented. Transcript cleavage by RNA polymerase III ternary complexes appears to be more closely related to the intrinsic nucleolytic activity of vaccinia virus RNA polymerase ternary complexes than to TFIIS-dependent cleavage that has been described for RNA polymerase II ternary complexes.
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PMID:Hydrolytic cleavage of nascent RNA in RNA polymerase III ternary transcription complexes. 750 90

The effects of cis-diamminedichloroplatinum(II) (cis-DDP) and trans-DDP adducts on mammalian transcription in vivo have been investigated. A plasmid containing the beta-galactosidase (beta-gal) reporter gene was modified with either of the two platinum compounds and transfected into human or hamster cell lines. A 2-3 fold higher level of transcription was observed in both cell lines from plasmids containing trans-DDP adducts as compared to plasmids modified by cis-DDP. This difference in transcriptional activity was not decreased in human and rodent nucleotide excision repair deficient cell lines, indicating that more efficient excision repair of the trans-DDP adducts was not the cause of its lower ability to block transcription in this assay. For this conclusion to be valid, it is assumed that trans-DDP adducts are repaired primarily by the nucleotide excision repair pathway, as is the case with the adducts of cis-DDP. The possibility that trans-DDP adducts are preferentially bypassed by RNA polymerase was examined by monitoring the elongation of beta-gal mRNA on damaged templates in vivo. Nascent beta-gal mRNA transcripts were recovered from excision repair deficient xeroderma pigmentosum A cells transfected with platinated plasmids, and the extent of RNA synthesis was measured by using ribonuclease protection. Fourfold more trans-DDP than cis-DDP adducts were required to inhibit transcription elongation by 63%. RNA polymerase II bypassed cis- and trans-DDP DNA adducts with efficiencies of 0-16% and 60-70%, respectively. These data provide insight into the differential toxicity of the two platinum isomers.
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PMID:DNA adducts of cis-diamminedichloroplatinum(II) and its trans isomer inhibit RNA polymerase II differentially in vivo. 757 87

In the Solanaceae, self-incompatibility is controlled by a single, multi-allelic ('S') locus. One product of this locus is a ribonuclease, the S-RNase, which is expressed predominantly in mature pistils and has recently been shown to cause allele-specific pollen rejection in transgenic plants. Hybrid Nicotiana plumbaginifolia x N. alata plants were used to test the effects of antisense suppression of the SA2-RNase from N. alata using three different gene constructs: two driven by RNA polymerase II-transcribed promoters, and the third, containing a truncated soybean tRNA (met-i) gene, transcribed by RNA polymerase III. All three constructs caused suppression of S-RNase activity in the transgenic plants. Unexpectedly, the CaMV 35S promoter was more effective for antisense suppression than the tissue specific tomato ChiP promoter. Antisense suppression of S-RNase correlated with low sense SA2 transcript levels and high antisense SA2 transcript levels. Untransformed hybrids that contained the N. alata SA2 allele were incompatible with N. alata SA2 pollen, while transgenic plants with suppressed SA2 gene expression accepted the pollen. The utility of this hybrid plant system for studying some aspects of antisense gene suppression is discussed.
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PMID:Antisense suppression of S-RNase expression in Nicotiana using RNA polymerase II- and III-transcribed gene constructs. 757 73

RNA polymerase II molecules that transcribe the late strand of the 5.3-kb circular polyomavirus genome stall just upstream of the DNA replication origin, in a region containing multiple binding sites for polyomavirus large T antigen. Stalling of RNA polymerases depends on the presence of functional large T antigen and on the integrity of large T antigen binding site A. To gain insight into the interaction between DNA-bound large T antigen and RNA polymerase II, we mapped the position of stalled RNA polymerases by analyzing nascent RNA chains associated with these polymerases. Elongation of RNA in vitro, followed by hybridization with a nested set of DNA fragments extending progressively farther into the stalling region, allowed localization of the 3' end of the nascent RNA to a position 5 to 10 nucleotides upstream of binding site A. Ribonuclease treatment of nascent RNAs on viral transcription complexes, followed by in vitro elongation and hybridization, allowed localization of the distal end of stalled RNA polymerases to a position 40 nucleotides upstream of binding site A. This RNA footprint shows that elongating RNA polymerases stall at a site very close to the position of DNA-bound large T antigen and that they protect approximately 30 nucleotides of nascent RNA against ribonuclease digestion.
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PMID:RNA footprint mapping of RNA polymerase II molecules stalled in the intergenic region of polyomavirus DNA. 776 4


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