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Problems involved in using the Hg-nucleotide technique for in vitro chromatin transcription are 2-fold. First, Escherichia coli RNA polymerase can utilize endogenous RNA as template and synthesize complementary sequences which remain base-paired to the template, thereby allowing it to bind to the SH-Sepharose column and copurify with the newly synthesized Hg-RNA. Second, non-mercurated endogenous RNA can bind to the SH-Sepharose through aggregation with Hg-RNA and thus be retained in the final RNA preparation. These two problems associated with the Hg-nucleotide technique can be minimized by modifying the conditions for RNA synthesis and SH-Sepharose chromatography. Using the modified procedure, the Hg-nucleotide and SH-Sepharose technique can remove more than 90% of endogenous RNA contaminants. In order to directly demonstrate that the mRNA ov sequences detected in vitro result from de novo transcription of oviduct chromatin, experiments were carried out which show that the hybridizable RNA sequences contain the Hg element and that the synthesis of these RNA sequences is sensitive to low concentrations of actinomycin D. These combined results strongly suggest that the majority of mRNA ov sequences detected by hybridization to cDNA ov is indeed due to DNA-dependent RNA synthesis by E. coli RNA polymerase and not due to an artifact of endogenous RNA contamination. This observation was further supported by data obtained using a filter hybridization method which measures directly the mRNA ov sequences present in [ 3 H]RNA synthesized from chromatin. The 3 H-labeled ovalbumin messenger RNA was assayed by hybridization to cloned pOV230 DNA containing the ovalbumin structural gene sequences. With this modified Hg-nucleotide-SH-Sepharose technique and filter hybridization technique, we have restudied the in vitro transcription of the ovalbumin gene from chromatins isolated at different stages of hormone-induced oviduct development. The results are in agreement with our previous findings which suggest that the primary regulation of ovalbumin synthesis by steroid hormones occurs at the transcriptional level.