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

M. C. Nguyen-Huu, A. A. Sippel, N. E. Hynes, B. Groner, G. Schutz
1978 Proceedings of the National Academy of Sciences of the United States of America  
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
more » ... 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 analysis of specific mRNA synthesis in isolated nuclei and chromatin is difficult for two reasons. First, a specific mRNA is expected to represent only a small fraction of the total RNA synthesized. Thus, hybridization of in vitro synthesized, radiolabeled RNA to unlabeled cDNA and digestion of the nonhybridized RNA does not allow the detection of specific mRNA sequences over the RNase digestion background. Second, large amounts of the investigated sequences are usually present in the RNA endogenous to isolated nuclei and chromatin. Hybridization to radiolabeled cDNA would not be a suitable detection method because it does not distinguish in vitro synthesized from preexisting mRNA molecules. To overcome these difficulties, we used two different approaches, both exploiting the affinity of mercurated polynucleotides for sulfhydrylagarose (SH-agarose) (9, 10). Using either method we could show that ovalbumin mRNA sequences have been synthesized by RNA polymerase B in a highly selective manner in isolated hen oviduct nuclei. MATERIALS AND METHODS Preparation of Mercurated Nucleotides and SH-Agarose. Hg-CTP and Hg-dUTP were prepared and characterized as described by Dale et al. (9) . SH-agarose (2 ,umol of sulfhydryl per ml) was prepared essentially as described by Cuatrecasas (11) from aminoethylagarose (Servachrom A-Al). Isolation of Ovalbumin mRNA and Preparation of cDNAs. Ovalbumin mRNA (mRNAes) was purified and [3H]cDNA was prepared as described (7, 12 ). Hg-cDNA was synthesized essentially under the same conditions with the following changes: the deoxynucleotides were used at 1 mM, dTTP was substituted by a mixture of 87.5% dTTP and 12.5% Hg-dUTP, 5 mM 2mercaptoethanol replaced dithiothreitol, and pyrophosphate was omitted. [3H]dCTP (22 Ci/mmol) or [32P]dCTP (2 mCi/ mmol) was used to label the Hg-cDNAs. Approximately 80% of [3H]cDNAoa and Hg-[3H]cDNAoa synthesized with a 12.5% substitution of dTTP by Hg-dUTP represented full-length transcripts. Hg-[3H]cDNA and Hg-[32P]cDNA were characterized by hybridization to mRNAes and 125I-labeled mRNAes (125I-mRNAes). Substitutions above 12.5% Hg-dUTP in the cDNA led to a lower rate of hybridization with mRNAs,. Hg-cDNAoa synthesized with 100% Hg-dUTP substitution could not hybridize to mRNA~a (A. E. Sippel, unpublished observation). 125I-mRNAes was prepared according to Curtis and Weissmann (13). In Vitro Synthesis and Purification of Hg-RNA and Hybridization to [3HJcDNA. Oviduct nuclei were prepared from HNL laying hens according to Ernest et al. (8) with the buffers used by Marshall and Burgoyne (14) . The conditions for RNA synthesis were as reported previously (8) except that 14 mM 2-mercaptoethanol replaced 2.5 mM dithiothreitol, Tris-HCl at pH 8.0 replaced sodium N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Na Hepes) at pH 8.0, MgCl2 replaced magnesium acetate, bovine serum albumin was omitted, and CTP or Hg-CTP was used at 0.5 mM. Nuclei were incubated at 25°at a DNA concentration of 1.5 mg/ml. [32P]UTP (2 mCi/mmol) was used to label the RNA to low specific activity. The reaction mixture-was then diluted 2-fold with a buffer containing 100 mM Tris-HCI at pH 7.5, 0.3 M NaCl, 15 mM Abbreviations: mRNAa, ovalbumin mRNA; cDNAoa, DNA complementary to ovalbumin mRNA; Hg-CTP, 5-mercuricytidine triphosphate; Hg-dUTP, 5-mercurideoxyuridine triphosphate; Hg-RNA, RNA containing Hg-CMP; Hg-cDNA, cDNA containing Hg-dUMP; SHagarose, sulfhydrylagarose; R-SH eluate, the fraction eluted with 2mercaptoethanol from the SH-agarose column; TN buffer, Tris/NaCl buffer; Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; DNAfd, phage fd DNA. 686 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.
doi:10.1073/pnas.75.2.686 pmid:273231 pmcid:PMC411321 fatcat:u5h6jd3feffhzlzftebrrel75e