Transcription of Friend Virus Proviral Sequences in Isolated Nuclei

Abstract

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 ...