Nucleic Acids Research, 1977, Vol. 4, No. 11 3943-3958
© 1977
Articles |
A RNA-dependent RNA polymerase activity: implications for chromatin transcription experiments
Max-Planck-Institut für Molekulare Genetik Berlin-Dahlem D-1000 Berlin 33, Ihnestrasse 63–73, GFR
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Received August 30, 1977. Mercurated nucleoside triphosphates have been used for transcription of chicken oviduct chromatin with E.coli RNA polymerase. The newly synthesized RNA was purified from preexisting RNA by SH-agarose chromatography and analyzed for the content of specific raRNA sequences. The apparent preferential production of ovalbumin mRNA sequences was not inhibited by actinomycin D, although total RNA synthesis was reduced by more than 90%. Furthermore, when globin mRNA alone, or added to oviduct chromatin, was incubated in the transcription assay, a significant fraction of this mRNA was retained on SH-agarose. The copurification of chromatin associated RNA with in vitro synthesized mercurated RNA was mainly due to a RNA-dependent synthesis of complementary sequences by the bacterial enzyme. Although denaturation of the transcripts prior to SH-agarose chromatography leads to a reduced contamination with endogenous ovalbumin specific RNA, we are unable to show that the messenger-specific RNA sequences purified with the newly mercurated RNA result from a DNA-dependent reaction.