Figure 5

Levels of in vitro transcription from chromatin assembled IgH enhancer templates correlate with levels of endogenous or recombinant-complemented Bright. Transcription reactions and templates utilizing the Eμ core without (Eμ) or with flanking MARs (Eμ+MARs) were measured by RNase protection as described in legends to Figs. 1, 3, and Materials and Methods. Transcription reactions were performed on N, naked DNA; R, reconstituted chromatin (Step 2 reaction order conditions); or D, prebound chromatin (Step 1 conditions). Protein sources: Upper panel (lanes 1–8): heparin agarose purified nuclear extracts prepared from Namalwa (5 μg/reaction) that contain low levels of endogenous Bright (Fig. 4A, lane 8); Lower panel (lanes 9–16): Nalm6 nuclear extract (5 μg/reaction) containing high levels of endogenous Bright (Fig. 4A, lane 4). Extracts were supplemented in the indicated lanes (+) with ~20 ng of purified Bright (177–601; Fig 4B, lane 5). Shown are phosphoimages of transcription reactions following protein removal and fractionation on 6% acrylamide/5 M urea gels.