A nuclear extract of Xenopus laevis oocytes that accurately transcribes 5S RNA genes

Abstract

Xenopus 5S RNA genes in recombinant form with the plasmid pMB9 are transcribed accurately when added to a supernatant fraction obtained from disrupted nuclei of Xenopus laevis oocytes. After an initial 30 min lag period, the rate of synthesis of 5S RNA is constant for at least an hour and synthesis is still detected after 18 hr. As much as 40% of the total RNA synthesized from the recombinant DNA used in these experiments can be 5S RNA. The coding strand of the 5S RNA genes is transcribed at a rate 10 to 15 times greater than the noncoding strand. Plasmid and spacer DNA, however, are also transcribed. What fraction of total RNA synthesized is 5S RNA is strongly affected by DNA concentration, ionic strength and MgCl2 concentration. Inhibition of transcription by intermediate concentrations of alpha-amanitin demonstrates that RNA polymerase III transcribes at least 90% of all RNA synthesized. Adenovirus 2 DNA is also transcribed in the nuclear supernatant by RNA polymerase III. Approximately 15% of the total RNA synthesized migrates in an acrylamide gel as a band of 5.5S RNA and has been identified as virus-associated RNA1 by its oligonucleotide fingerprint.