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:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

Evidence suggesting the presence in rat liver nuclear extracts of a new RNP complex of 70-110S has been provided [Hatzoglou, M., Adamtziki, E., Margaritis, L. and Sekeris, C.E (1985) Exp. Cell. Res. 157, 227-241]. Biochemical features unique to this RNP were its stability to salt and RNase digestion and the presence of a pair of polypeptides of 72/74 kDa. By producing antibodies against the 72/74 kDa polypeptides these proteins have been defined as integral components of the 70-110S RNP complex. They comprise two immunologically related polypeptides with an exclusively nucleoplasmic localization, giving a speckled pattern in a diffuse background, similar, but not identical, to the Sm antigen. The 70-110S RNP complex, referred to as large heterogeneous nuclear RNP (LH-nRNP), has a simple protein pattern that includes, in addition to the 72/74 kDa proteins, three stably associated polypeptides of apparent molecular size 110, 61 and 59 kDa. The bulk of its RNA component represents a discrete RNA population of 10-20S, belonging to a subset of the RNA detected within immunopurified HeLa hnRNP complexes. These RNA species are RNA polymerase II transcripts of greater stability relative to the bulk of hnRNA, containing oligo(A) or poly(A) sequences. Immunodepletion and/or antibody addition studies in HeLa splicing extracts using antibodies with specificity for the 72/74 kDa proteins revealed a rather strong inhibition of splicing activity, suggesting participation of the LH-nRNP complex in in vitro splicing.
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PMID:Two immunologically related polypeptides of 72/74 kDa specify a novel 70-100S heterogeneous nuclear RNP. 765 36

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

In previous studies we have shown that specific nuclear pre-mRNAs and their splicing products, as well as the general population of nuclear poly(A)+ RNA, are found packaged in 200 S large nuclear ribonucleoprotein (lnRNP) particles that represent the splicing machinery in vivo. The lnRNP particles contain all U small nuclear ribonucleoproteins (snRNPs) required for splicing, as well as several proteins including non-snRNP splicing factors. Here we show that upon addition of EDTA to sucrose gradient-fractionated 200 S particles, part of their components (e.g. part of the U snRNPs) are no longer associated with pre-mRNAs, which are now packaged in 70 S particles. This 200 S to 70 S transition makes the pre-mRNA more susceptible to digestion by RNase. The effect of EDTA is reversible, as back addition of Mg2+ results in the reconstitution into 200 S lnRNP particles of: (1) all five snRNPs required for splicing; (2) the SR proteins; and (3) CAD mRNA, as a representative of nuclear RNA polymerase II transcripts. Remarkably, electron microscopy of the reconstituted particles shows a compact structure, 50 nm in diameter, that is indistinguishable from the original undissociated particles. We conclude that Mg2+ is required for the integrity of the 200 S lnRNP particles.
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PMID:Magnesium cations are required for the association of U small nuclear ribonucleoproteins and SR proteins with pre-mRNA in 200 S large nuclear ribonucleoprotein particles. 786 77

An in vitro transcription system from Candida utilis is described. The template used is a hybrid plasmid containing Saccharomyces cerevisiae CYC1 promoter linked to a synthetic 377-bp G-minus casette (1). In vitro transcriptions are carried out in the presence of RNase. T1. Under these conditions only the transcripts that are resistant to RNase T1 accumulate. Using this protocol, it has been shown that in the absence of cytosolic factors RNA polymerase II (pol II) from C. utilis initiated RNA synthesis randomly. But both C. utilis and S. cerevisiae cell-free extracts could direct pol II from C. utilis to initiate transcription accurately. Results also indicated that the general transcription factors are functionally interchangeable between S. cerevisiae and C. utilis.
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PMID:Accurate transcription initiation by RNA polymerase II from Candida utilis. 798 59

Most DNA topoisomerase II (topo II) in cell-free extracts of 0-2-h old Drosophila embryos appears to be nonnuclear and remains in the supernatant after low-speed centrifugation (10,000 g). Virtually all of this apparently soluble topo II is particulate with a sedimentation coefficient of 67 S. Similar topo II-containing particles were detected in Drosophila Kc tissue culture cells, 16-19-h old embryos and extracts of progesterone-matured oocytes from Xenopus. Drosophila topo II-containing particles were insensitive to EDTA, Triton X-100 and DNase I, but could be disrupted by incubation with 0.3 M NaCl or RNase A. After either disruptive treatment, topo II sedimented at 9 S. topo II-containing particles were also sensitive to micrococcal nuclease. Results of chemical cross-linking corroborated those obtained by centrifugation. Immunoblot analyses demonstrated that topo II-containing particles lacked significant amounts of lamin, nuclear pore complex protein gp210, proliferating cell nuclear antigen, RNA polymerase II subunits, histones, coilin, and nucleolin. Northern blot analyses demonstrated that topo II-containing particles lacked U RNA. Thus, current data support the notion that nonnuclear Drosophila topo II-containing particles are composed largely of topo II and an unknown RNA molecule(s).
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PMID:An RNase-sensitive particle containing Drosophila melanogaster DNA topoisomerase II. 808 68

We have previously reported that the subgroup C adenovirus E2 early (E2E) RNA polymerase II promoter can specify efficient in vitro transcription by RNA polymerase III. We now show that promoter proximal sequences of the E2E transcription unit are also transcribed by RNA polymerase III in nuclei isolated from adenovirus-infected cells. Small E2E RNA species that possessed the same properties as in vitro synthesized RNA polymerase III E2E transcripts were detected in cytoplasmic RNA populations from infected cells by using blotting, primer extension, and RNase protection assays. The 3' termini of these RNAs were mapped to thymidine-rich sequences typical of RNA polymerase III termination sites. These results demonstrate that a single gene can be transcribed by both RNA polymerase II and RNA polymerase III in vivo.
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PMID:In vivo transcription from the adenovirus E2 early promoter by RNA polymerase III. 810 99


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