In vitro transcription of eukaryotic genes is affected differently by the degree of DNA supercoiling.

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RESUMO

In a posterior silk gland extract, covalently closed circular (ccc) DNA is in a superhelical state that supports more transcription of fibroin gene than does linear DNA. A HeLa cell extract showed neither the supercoiling activity nor the preference for the transcription of ccc DNA over linear DNA. These activities could be added to the HeLa cell extract. Phosphocellulose fractionation of the posterior silk gland extract yielded a flow-through fraction and a 0.6 M KCl eluate fraction that were required for the supercoiling and for the efficient transcription of the ccc template in the acceptor HeLa cell extract. The 0.6 M KCl fraction had a DNA topoisomerase II activity, and the flow-through fraction contained a supercoiling factor that, with the aid of topoisomerase II, introduced negative supercoils into ccc DNA. When both fractions were added to the posterior silk gland extract, more supercoiling occurred than with the extract alone. Several genes were optimally transcribed under various extents of supercoiling. The fibroin gene and adenovirus 2 major late promoter were fully transcribed as partially supercoiled templates. The sericin gene required more supercoiling for full transcription, whereas no preference for supercoiling was seen with the transcription of hsp70. These results suggest that DNA topology plays a role in the regulation of gene expression.

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