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)

phi 227, a temperate phage from a group H streptococcus (Streptococcus sanguis), was propagated vegetatively in group H strain Wicky 4-EryR, and its characteristics were determined. A procedure dependent on multiplicity of infection, incubation time, and treatment of crude lysates with diatomaceous earth was found to optimize phage yield, resulting in titers of 1 X 10(10) to 2 X 10(10) PFU/ml. Without prior treatment with diatomaceous earth, subsequent purification procedures (methanol, ammonium sulfate, polyethylene glycol) gave recoveries of less than 1% of crude lysate titers. Adsorption of phi227 to host cells was relatively unaffected by the medium, but calcium (not substituted by magnesium) was required for formation of infectious centers. The phage receptor was present on purified cell walls, resisted trypsin and heat, and was removed ty hydrochloric acid, trichloracetic acid, and hot formamide: however, formamide-extracted material failed to inactivate phage, and the nature of the receptor is unknown. Single-step growth experiments showed a latent period of 39 min and a burst size of 100 PFU/infectious center; results were unaffected by omission of supplemental Ca2+, by supplementation with Mg2, addition of glucose, or changes of pH between 6.35 and 8.0; but increased temperature (40 to 43 degrees C) shortened the latent period and decreased the burst size. The latent period was prolonged in genetically competent host cells and in chemically defined medium; and in the latter, the burst size was smaller. Phage replication was sensitive to those metabolic inhibitors which inhibited the host streptococcus: these included rifampin, fluorodeoxyuridine, hydroxyurea, dihydrostreptomycin, and 6-P-hydroxyphenylazouracil. The data suggest that phi227 does not code for a rifampin-resistant RNA polymerase. However, in a rifampin-resistant host strain, phage replication and lysogen formation were both decreased suggesting that altered host core polymerase had less affinity for (some) promotors on the phi227 template. In transfection, a Ca2+-dependent stabilization step that was inhibited by Mg2+ was demonstrated; transformation was not affected by either Ca2+ or Mg2+, and the site and nature of the stabilization are unknown. More than one molecule of DNA was required for plaque formation. Biophysical characterization showed a type B phage of buoyant density (CsCl) 1.50, containing five proteins and 54.8% DNA. The duplex linear DNA had a molecular weight (calculated from contour length) of 23.2 X 10(6) and a guanine plus cytosine content (calculated from melting point) of 42.3 mol%. Similar characterizations of streptococcal phages, including biophysical data, have not been previously available.
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PMID:Characterization of group H streptococcal temperate bacteriophage phi 227. 1 33

An easy and efficient procedure for the immobilization of polynucleotide ligands to bisoxirane activated insoluble polysaccharides has been elaborated and is described in this paper. The resulting materials have been applied to the chromatography of DNA polymerase I, and RNA polymerase from E.coli. Because of their extraordinary stability to temperature, formamide, and alkaline conditions they seem to be particularly useful adsorbents for nucleic acid hybridization.
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PMID:Affinity adsorbents consisting of nucleic acids immobilized via bisoxirane activated polysaccharides. 2 16

We describe a method for linking RNA and DNA covalently to finely divided cellulose through a diazotized aryl amine, which reacts primarily with guanine and uracil (thymine) residues of single strands. The high efficiency of coupling and high capacity of the cellulose for nucleic acid make possible a product with as much as 67 mug of nucleic acid per mg of cellulose. The product is especially suitable for hybridization experiments where very low backgrounds are important, and it is stable in 99% formamide at 80 degrees C so that hybridized nucleic acid can be recovered easily. Full length linear Simian Virus 40 (SV40) DNA, produced by cleavage of SV40(I) DNA with S1 nuclease, can be coupled to diazo cellulose with an efficiency of 80-90%, and is effective in hybridization experiments with SV40 DNA, complementary RNA synthesized in vitro from SV40(I) DNA with E. coli RNA polymerase, and the SV40-specific fraction of total RNA from SV40-infected and transformed cells. In these experiments an excess of cellulose-bound DNA was used, and the efficiency of hybridization was about 90% when ribonuclease treatment of the hybrids was omitted.
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PMID:Nucleic acid hybridization using DNA covalently coupled to cellulose. 16 82

Polyacrylamide gel electrophoresis and tryptic peptide fingerprint analysis of the proteins made in a cell-free system derived from L-cells and immunoprecipitated with simian virus 40 (SV40) anti-T serum demonstrated that both SV40 large-T and small-T antigens are synthesized in vitro in response to mRNA isolated from productively infected CV1 CELLS. Sucrose density centrifugation in gradients containing 85% formamide showed that the mRNA's for both forms of T-antigen sediment at about 17.5S, with the mRNA for small-t sedimenting marginally, but reproducibly, ahead of the mRNA for large-T. Hybridization experiments using restriction endonuclease fragments Hae III-E and Hind II/III-B showed that all fractions active in the cell-free synthesis of both forms of T-antigen hybridized equally to both fragments. This suggests that the mRNA's for SV40 T-antigens are at least partly virus coded and that the bulk of the early SV40 mRNA contains sequence information from both ends of the early region. The data are consistent with the suggestion that the large-T mRNA is spliced. SV40 complementary RNA (the product of transcription of SV40 DNA using Escherichia coli RNA polymerase) was also translated in the L-cell system and gave two families of polypeptides which specifically immunoprecipitate with anti-T serum. One family (the small-t family) includes a polypeptide indistinguishable by gel electrophoresis and tryptic peptide fingerprinting from small-t isolated from cells. The other family (the 60K family) has a major component with molecular weight approximately 60,000 and includes other polypeptides with molecular weights ranging from approximately 14,000 to about 70,000. The 60K family has petides in common with large-T but not with small-T. Together, the peptides of the small-t and 60K families account for virtually all of the methionine peptides of SV40 large-T. We conclude from these results (i) that small-t is probably entirely, and large-T at least predominantly, virus coded; (ii) that the small-t and 60K families represent the translation products of two different portions of the early region of SV40 DNA (approximately 0.65 to 0.55 map units and 0.54 to 0.17 map units); and (iii) that although most, if not all, of the large-T and small-t peptides are present in the cell-free product, some feature of sequence arrangement of SV40 complementary RNA prevents the translation of full-length large-T and results instead in the synthesis of fragments. We suggest that the absence of a splice in the complementary RNA is responsible for this result.
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PMID:Cell-free synthesis of simian virus 40 T-antigens. 21

Simian Virus (SV40) transcriptional intermediates (T.I.) were isolated from infected cell nuclei incubated in vitro in the presence of the four ribonucleoside triphosphates. The nascent mRNA strands in the viral DNA-RNA hybrid molecules were hydrogen bonded to their template by 200-250 nucleotides on the average, as judged from the extent of their RNase resistance and the aspect of T.I. under electron microscope after treatment with 50 per cent formamide. The RNA polymerase involved (RNA polymerase II) synthesized up to full length transcripts at a rate of approximately 150 nucleotides/min. at 25 degrees C. Each SV40 infected cell was found to contain about 200 active T.I. molecules at the peak of late transcription. The DNA in the T.I. molecules was exclusively form I DNA only in cell infected with the tsA30 mutant of SV40 that had been transferred to non-permissive temperature in order to arrest DNA replication, but both form I DNA and molecules behaving as replicative intermediates (R.I.) in wild type infected cells.
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PMID:Characterization of simian virus 40 transcriptional intermediates in infected CV1 cell nuclei. 23 Aug 57

Radioactively labeled RNAs were synthesized from cellulose-bound cDNA templates using Escherichia coli RNA polymerase. Hybridization of this RNA to excess unlabeled cDNA approached 100%, indicating the complementarity of product and template. The average length of the RNA product, as determined by formamide gels, was approximately 40% of the template length. Hybridization of unlabeled globin RNA produced by this technique to labeled globin cDNA indicated the population of RNA sequences represented at least 80% of the template sequences. Approximately 30% of the RNA product by mass contains poly(A) tails as determined by binding to oligo(dT)-cellulose. The template can be reused for several cycles of synthesis with little loss of synthetic capability and therefore, can amplify the amount of mRNA initially used to produce the template.
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PMID:Synthesis of RNA from cellulose-bound complementary DNA. 32 4

RNA polymerase B and DNA polymerase alpha were highly enriched simultaneously from calf thymus. It was shown that the preparation exhibits RNA-synthesizing activity, which is able to stimulate in vitro DNA synthesis by DNA polymerase alpha by its preceding RNA synthesis. A part of the DNA was found to be covalently attached to RNA in Cs2SO4 equilibrium gradients after denaturation by formamide.
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PMID:RNA-primed DNA synthesis by an enzyme preparation of calf thymus containing highly enriched RNA polymerase B and DNA polymerase. 54 71

1. As a prerequisite for analyzing the effect of estrogen on transcription in chick oviduct, we describe suitable methods for the synthesis (under conditions restricting reinitiation), and isolation of RNA transcripts from oviduct nuclei in vitro, utilizing mercurated UTP (Hg-UTP) as an RNA precursor and chromatography on sulphydryl-Sepharose (SH-Sepharose) to recover mercurated RNA (Hg-RNA). The techniques described include treatment of Hg-RNA with p-hydroxymercuribenzoate, to improve the efficiency of binding to SH-Sepharose, and elution of Hg-RNA from SH-Sepharose after treatment with 60% formamide at 90 degrees C, to eliminate contamination by aggregated nucleic acid. 2. RNA synthesized by endogenous form B RNA polymerase (using either UTP or Hg-UTP as precursor) was recovered in nuclear lysates in the form of 30--85-S heterogeneous RNA . protein complexes, and after removal of protein, was 10--12 S in size. 3. The nature of RNA transcripts synthesized in vitro was examined by hybridization. More than 90% of the RNA was complementary to "unique" DNA sequences, and 50--60% of the hybridized RNA could be competed with homologous, steady-state nuclear RNA, indicating a significant degree with homologous, steady-state nuclear RNA, indicating a significant degree of homology between in vitro transcripts and in vivo RNA. The level of homology was similar whether RNA synthesis was performed in low salt, or in high salt in the presence of heparin. Possible reasons for only partial competition in these experiments are discussed. 4. Withdrawal of estrogen from chicks leads to a 50% reduction in endogenous RNA polymerase activities in nuclei within 48 h. Similar levels of competition with Hg-RNA transcripts for "unique" DNA were obtained using oviduct nuclear RNAs isolated before or after estrogen withdrawal, and even with liver nuclear RNA. Thus, in oviduct, those sequences present in primary transcripts, and analyzed under our experimental conditions, are present in different hormonal states and also in other chick tissues.
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PMID:Estrogen withdrawal in chick oviduct. Characterization of RNA synthesized in isolated nuclei using a mercurated precursor. 69 29

Purified pig thyroid chromatin has been transcribed in vitro with Escherichia coli RNA polymerase. The transcript, analyzed by DNA-RNA hybridization, shows two major kinetic components: 40% of the transcript is copied by repetitive sequences present 100 times per haploid genome; another 25% anneals to DNA with a rate constant Kh 10-4 M - S-1, typical of single-copy sequences. The transcript annealed at cot = 40 M - S to fractions of 2000-nucleotide DNA, when banded in neutral CsCl gradient only hybridizes to the heavy side of the main band. At cot = 3000 M - S, another hybridizing fraction appears on the light side of the main band of the gradient. The reassociation properties of these fractions show that the heavy DNA fraction is reiterated about 100 times per haploid genome, whereas the light DNA appears as a unique sequence, associated to small repetitive elements. The transcript, analyzed by formamide/sucrose gradient, shows two peaks with sedimentation coefficients of 10 S and 4 S, respectively. The 10-S RNA, hybridized to native 2000-nucleotide-length DNA, has a Kh of 10-4 M - S-1 and a cot1/2 of 10(3) M - S, typical of single-copy sequences.
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PMID:Repetitive and non-repetitive sequences in the transcript in vitro of porcine thyroid chromatin. 91 90

Nuclei isolated from human cells productively infected with adenovirus 2 have been shown to synthesize four low molecular weight RNA species which hybridize efficiently to viral DNA. One species corresponds to the 5.5S or VA RNA (Ohe, Weissman, and Cooke, 1969), and is designated V156. The other three species are novel and have been designated V200, V140, V130, since they are approximately 200, 140, and 130 nucleotides in length, respectively. These viral RNAs retain their distinct electrophoretic properties after denaturation with formamide. RNA species with electrophoretic mobilities similar to those of the V200, V156, and V140 RNAs have been found in the cytoplasmic fraction of cells at late times after adenovirus infection. In isolated nuclei, the V200, V156, V140, and V130 RNAs are all synthesized by DNA-dependent RNA polymerase III, since synthesis is sensitive to high but not to low concentrations of alpha-amanitin. The synthesis of these low molecular weight RNAs continues for a prolonged period of time in isolated nuclei, suggesting that reinitiation occurs. Adenovirus 2 DNA fragments obtained by digestion with restriction endonucleases Eco RI and Sma I were used to map the location of the DNA sequences which encode the RNAs. All the low molecular weight RNAs hybridized to a region of the genome between o.18 and 0.38 fractional lengths from the left end of the adenovirus genome, suggesting that the respective DNA sequences are clustered. Other nonviral low molecular weight RNAs are synthesized in nuclei isolated from infected cells. These include the cellular 5S rRNA species which was minitored by its hybridization to purified 5S DNA from Xenopus laevis.
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PMID:Low molecular weight viral RNAs transcribed by RNA polymerase III during adenovirus 2 infection. 95 87


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