The Use of Mercurated Nucleoside Triphosphate as a Probe in Transcription Studies <i>in vitro</i>

Beebee, Trevor J. C.; Butterworth, Peter H. W.

doi:10.1111/j.1432-1033.1976.tb10580.x

Cited by 27 publications

(20 citation statements)

References 22 publications

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“…In vitro synthesis in isolated nuclei or chromatin was carried out in the presence of 5-mercuriuridine triphosphate (Hg-UTP). The rate of RNA synthesis is essentially the same either in the presence of Hg-UTP or ordinary U T P as measured by incorporation of [3H]GTP in accord with previous observations by other investigators (Dale et al, 1973;Smith and Huang, 1976;Crouse et al, 1976;Beebee and Butterworth, 1976;Biessmann et al, 1976). Addition of saturating amounts of E .…”

Section: In Vivo Content Of Viral Sequences In Pulse-labeled Rnasupporting

confidence: 82%

“…Many of these studies suggest that chromatin and nuclei appear to retain their in vivo template specificities in transcription of specific genes (Axel et al, 1973;Gilmour and Paul, 1973;Astrin, 1973;Shih et al, 1973;Steggles et al, 1974;Rymo et al, 1974;Jacquet et al, 1974;Wilson et al, 1975;Gilmour et al, 1975;Marzluff and Huang, 1975;Stein et al, 1975;Tsai et al, 1976). Recent reports on the use of mercurated nucleotide triphosphate as the substrate for RNA synthesis in isolated nuclei or chromatin and purification of the Hg-tagged RNA by sulfhydryl affinity column have been met with interest (Smith and Huang, 1976;Crouse et al, 1976;Beebee and Butterworth, 1976;Biessmann et al, 1976). It seems to offer a novel method of resolving the newly synthesized in vitro RNA from any preexisting nuclear and chromatin RNA, and to permit the use of specific radioactive cDNA probes to analyze the in vitro products.…”

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confidence: 97%

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In vitro transcription of Moloney leukemia virus genes in infected cell nuclei and chromatin: elongation of chromatin associated ribonucleic acid by Escherichia coli ribonucleic acid polymerase

Shih¹,

Young²,

Parks³

et al. 1977

Biochemistry

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The in vitro transcription of viral specific DNA sequences in nuclei and chromatin isolated from mouse cells chronically infected with Moloney murine leukemia virus (Mo-MuLV) has been studied. The in vitro RNA synthesized by Escherichia coli RNA polymerase has been isolated by sulfhydryl affinity column following reaction in the presence of 5-mercuriuridine triphosphate. By comparison of the Crt curves of the in vitro RNA with that of 70S viral RNA, the content of viral sequences is found to be 1.3% in nuclei product and 0.24% in chromatin product which is lower than the 2.5% found in chromatin associated RNA. This latter value, however, is very close to the in vivo viral RNA content in pulse-labeled [3H]RNA of the infected cells. Unexpectedly, it is observed that over 20% of the chromatin associated RNA prelabeled in vivo with [5-3H]uridine is elongated and tagged with Hg atoms during RNA synthesis catalyzed by the exogenous E. coli RNA polymerase in the presence of Hg-UTP. The elongation reaction is dependent on the presence of all four nucleotide triphosphates and appears to be due to E. coli RNA polymerase per se. It is suggested that most of the viral specific sequences observed in the in vitro RNA products are very likely initiated and derived from the chromatin associated species. The implication of the present findings for in vitro RNA synthesis in nuclei and chromatin as related to regulation of gene expression is discussed.

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Section: In Vivo Content Of Viral Sequences In Pulse-labeled Rnasupporting

confidence: 82%

mentioning

confidence: 97%

In vitro transcription of Moloney leukemia virus genes in infected cell nuclei and chromatin: elongation of chromatin associated ribonucleic acid by Escherichia coli ribonucleic acid polymerase

Shih¹,

Young²,

Parks³

et al. 1977

Biochemistry

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“…RESULTS Binding of Mercurated Nucleic Acids to SH-Agarose. In order to follow specific transcription in isolated nuclei, we distinguished in vitro synthesized from preexisting mRNA sequences either by incorporation of a mercurated nucleotide (9,(16)(17)(18)(19) and subsequent isolation of the mercurated RNA by affinity chromatography on SH-agarose or by incorporation of a radioactive nucleotide and isolation of the specific hybrids after hybridization to mercurated cDNA. The basis of both procedures is the affinity of mercurated polynucleotides for SH-agarose.…”

Section: Methodsmentioning

confidence: 99%

Preferential transcription of the ovalbumin gene in isolated hen oviduct nuclei by RNA polymerase B.

Nguyen-Huu¹,

Sippel²,

Hynes³

et al. 1978

Proc. Natl. Acad. Sci. U.S.A.

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The synthesis of ovalbumin mRNA sequences was studied in isolated nuclei from hen oviduct. Two different methods of analysis were used to distinguish in vitro synthesized from preexisting mRNA sequences: (i) Mercurated ribonucleotides were used for in vitro RNA synthesis, and the newly synthesized RNA was purified by chromatography on sulfhydrylagarose and hybridized to radioactive ovalbumin cDNA. (ii) (3HJUTP was used to label the in vitro synthesized RNA. Hybridization to unlabeled mercurated cDNA, RNase A digestion, and subsequent purification of the hybrids on SH-agarose allowed the quantitation of newly synthesized ovalbumin mRNA sequences. Approximately 0.1% of the newly synthesized RNA was identified as ovalbumin RNA by both methods. The synthesis of ovalbumin RNA progressed during the incubation of nuclei and was sensitive to actinomycin D and low concentrations of a-amanitin. The preferential in vitro transcription of the ovalbumin gene (1000-fold over random transcription of the chicken genome) by RNA polymerase B (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) suggests that the specificity of in vivo RNA synthesis is retained in isolated nuclei.The synthesis of the egg white proteins in the tubular gland cell of the chicken oviduct is primarily related to the cellular accumulation of the corresponding mRNAs (1-7). During stimulation with estradiol the amount of ovalbumin mRNA dramatically increases from a few copies to tens of thousands of copies per cell (2, 4, 5-7). Similar results have been found for the induction of ovomucoid and lysozyme mRNA (7). The steroid-mediated induction of specific mRNA molecules in the chicken oviduct may result from altered rates of synthesis and/or degradation. We therefore studied the synthesis rate of the egg white protein mRNAs. Because it was not possible to measure the rate of specific mRNA synthesis in the animals, we chose isolated oviduct nuclei as the cell-free system that might best reflect the transcriptional activity of the intact cell (8). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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“…According to Beebee and Butterworth and Zasloff and Felsenfeld inadequate hybridisation is obtained with RNA containing the level of mercuration chosen here, i.e. 25 % substitution in a single nucleotide [38,49]. However, the decrease in rate of hybridisation observed by those authors is explicable entirely in terms of the absence of a suitable thiol ligand for the hgRNA.…”

Section: Discussionmentioning

confidence: 93%

Transcription of Friend Virus Proviral Sequences in Isolated Nuclei

1980

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Transcription studies using isolated Friend nuclei and Escherichia coli polymerase are presented. Combination of the techniques of thiol-Sepharose chromatography and cDNA-Sepharose hybridisation has resulted in a system in which the transcription of the Friend virus proviral sequences with endogenous and E. coli polymerase can be examined. The results show that the percentage of Friend viral-specific sequences in RNA transcribed by E. coli RNA polymerase and by endogenous RNA polymerase in isolated nuclei are similar. The percentage of viral-specific sequences synthesized in isolated nuclei is similar to that found in Friend cell nuclear RNA.In vitro transcription systems have been developed to study the mechanisms of gene regulation in eukaryotes and in particular to study the role of proteins associated with the genomic DNA [1,2]. Two approaches have been widely used : (a) transcription from isolated chromatin by exogenous polymerases [3 -51; (b) transcription by endogenous polymerases in isolated nuclei [6-131. Both approaches have proved to have many problems.The assessment of fidelity of transcription by hybridisation analysis has recently concentrated mainly on the study of expression of single-copy genes, e.g. globin and ovalbumin. The sequences have usually been detected by hybridisation of RNA to radioactively labelled cDNA but this type of analysis is seriously compromised by the presence of endogenous RNA in isolated nuclei and chromatin [14]. In the present studies we have employed methods which circumvent this difficulty.Nuclear transcription systems in which transcription by endogenous polymerase is studied have consistently suffered from the drawback that, although important findings have been obtained about tranAbbreviations. Buffer A, 0.32 M sucrose, 10 mM Tris, 2 mM magnesium acetate, 3 mM CaC12, 0.1 % Triton X-100, 1 mM dithioerythritol pH 8.0. Buffer B, 2.0 M sucrose, 10 mM Tris, 5 mM magnesium acetate, 1 mM dithioerythritol pH 8.0. Buffer C, 25 % glycerol, 50 mM Tris, 5 mM magnesium acetate, 10 mM mercaptoethanol. Buffer D, 0.1 M NaC1, 10 mM Tris-C1, 1 mM EDTA, 0.1 % sodium dodecylsulphate pH 7.5. Buffer E, 0.75 M NaCl, 50 mM Tris-C1, 1 mM EDTA, 0.3 % sodium dodecylsulphate, 50% formamide, pH 7.5. NaCl/Cit ( x 2) 0.3 M NaC1, 0.03 M sodium citrate pH 7.0; hgUTP 5-mercuriuridine 5'-triphosphate hgCTP 5-mercuricytidine 5'-triphosphate.Enzymes (IUB Recommendations, 1978). Protease K (EC 3.4.21.14); RNase A (EC 3.1.27.5); RNase TI (EC 3.1.27.3); RNA polymerase (EC 2.7.7.6).scription by polymerase I11 [8,15 -171, the mass of RNA synthesised is low and the endogenous polymerases I and I1 do not appear to initiate to any great extent [18].Because of the failure of exogenous eukaryotic polymerase I1 to initiate efficiently and specifically on isolated chromatin 11 91 Escherichia coli RNA polymerase has been used in many studies as a putative probe for transcriptionally active regions of the genome but the utility of such a probe remains to be unequivocally demonstrated.In the present studies we have ...

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The Use of Mercurated Nucleoside Triphosphate as a Probe in Transcription Studies in vitro

Cited by 27 publications

References 22 publications

In vitro transcription of Moloney leukemia virus genes in infected cell nuclei and chromatin: elongation of chromatin associated ribonucleic acid by Escherichia coli ribonucleic acid polymerase

In vitro transcription of Moloney leukemia virus genes in infected cell nuclei and chromatin: elongation of chromatin associated ribonucleic acid by Escherichia coli ribonucleic acid polymerase

Preferential transcription of the ovalbumin gene in isolated hen oviduct nuclei by RNA polymerase B.

Transcription of Friend Virus Proviral Sequences in Isolated Nuclei

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