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Nucleic Acids Research, 1978, Vol. 5, No. 4 1353-1370
© 1978

Articles

In vitro RNA synthesis and expression of vitellogenin gene in isolated chicken liver nuclei

Sakoi Panyim, Takeshi Ohno and Jean-Pierre Jost

Friedrich Miescher-Institut PO Box 273, CH-4002 Basel, Switzerland

Received February 9, 1978. Optimal conditions for prolonged in vitro synthesis of RNA in isolated chicken liver nuclei have been described. It is shown by incorporation of {gamma}32P-GTP into RNA, analysis of the product on sucrose density gradient, and digestion with alkaline phosphatase and ribonuclease A that there is reinitiation of RNA synthesis. Polynucleotide kinase activity has been ruled out as an explanation for the incorporation of {gamma}32P-GTP. {alpha}-Amanitin inhibits RNA synthesis by about 50%. Nuclei prepared from estradiol-treated chicks have twice the RNA synthesis activity as the controls. RNA is synthesized in the presence of Hg-UTP and the mercurated product separated by affinity chromatography on sulfhydryl-Sepharose column under stringent conditions. Vitellogenin mRNA sequences are measured by hybridization with DNA complementary to vitellogenin mRNA. Estradiol treatment leads to a 10-fold increase in vitellogenin mRNA sequences.


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