Assembly of transcriptionally inactive chromatin in vitro.
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.
Shanahan
M M
MM
Department of Biochemistry and Biophysics, University of California, Davis 95616.
Kmiec
E B
EB
D000818:Animals; D002474:Cell-Free System; D005591:Chemical Fractionation; D002843:Chromatin / Q000096:biosynthesis / Q000235:genetics*; D004270:DNA, Circular / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D005786:Gene Expression Regulation; D012343:RNA, Transfer / Q000235:genetics; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014982:Xenopus laevis
D002843:Chromatin; D004270:DNA, Circular; D004278:DNA, Superhelical; D012343:RNA, Transfer