FIG. 1.

Effect of nucleosome reconstitution on 5S rRNA gene transcription. (A) Micrococcal nuclease digestion of nucleosomes reconstituted on full-length X. laevis oocyte and somatic genes. Digestions were carried out at a nucleosome concentration of 0.1 mg/ml (DNA weight) and an enzyme concentration of 10 U/ml for the times (minutes) indicated above each lane. The resulting DNA fragments were deproteinized and electrophoresed on a 4% nondenaturing gel. Lanes M, HhaI-cut pBR322. (B) Approximately 250 ng of the oocyte (lanes 3 and 4) or somatic (lanes 5 and 6) 5S rRNA genes, either uncomplexed (lanes 3 and 5) or reconstituted with histones isolated from chicken erythrocytes (lanes 4 and 6), was transcribed in HeLa cell nuclear extracts supplemented with 150 nM recombinant Xenopus TFIIIA and 2 mM MgCl₂. Each reaction mixture contained 50 ng of CMV DNA as an internal transcription control, which is visible only after longer exposures. Transcripts were analyzed by denaturing polyacrylamide gel electrophoresis (8% acrylamide and 8.3 M urea in 1× TBE). Lane 1, Klenow fragment-end-labeled HinfI-cut φX174 DNA (sizes of marker fragments are shown as numbers of nucleotides); lane 2, transcribed CMV DNA alone.