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J. Biochem, 1989, Vol. 106, No. 1 29-33
© 1989 Japanese Biochemical Society

research-article

Assembly of Transcriptionally Inactive Chromatin In Vitro1

Miya M. Shanahan and Eric B. Kmiec2

Department of Biochemistry and Biophysics, University of California Davis, Davis, California 95616, U.S.A

2To whom correspondence should be addressed

We have successfully uncoupled the previously interlocked activities of chromatin assembly and in vitro transcription promoted by the Xenopus oocyte S-150 cell-free extract. Our isolated fraction catalyzes extensive chromatin assembly measured both by changes in DNA topology and Micrococcal nuclease digestions. The assembly of chromatin is slowed by the exogenous addition of ATP. In the absence of exogenously added ATP, the fraction forms a chromatin template that is transcriptionally inert. Addition of small amounts of the HeLa cell extract (S-100) converts these templates into transcriptionally active ones without disrupting the chromatin structure. Our protocol defines a method for the isolation of a fraction from the Xenopus cell free extract that catalyzes the assembly of transcriptionally inactive chromatin. We characterize this reaction and establish conditions for the transcriptional activation of these inactive minichromosomes.

1This project is supported by a grant from the American Heart Association (California affiliate, 87-147) and the Cancer Research Coordinating Committee (3-504026).


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