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. 2001 Nov 1;15(21):2837–2851. doi: 10.1101/gad.937401

Figure 5.

Figure 5

Transcription on RSF-assembled chromatin. (A) Southern blot of micrococcal nuclease digestions of RSF-assembled and remodeled chromatin. Chromatin was assembled with RSF in the presence and absence of the activator Gal4VP16, added during the assembly or after the assembly as indicated. The Southern blot was probed with a promoter probe (left), followed by stripping off the probe and reprobing with a distal probe (right). The illustration (bottom) shows schematically the location of the probes (for details, see Orphanides et al. 1998). Each lane number, at the bottom of the panel, denotes two different concentrations of micrococcal nuclease used in the assays. (B) Transcription on RSF-assembled chromatin. (Top) A scheme of the procedure used to deposit chromatin and its analysis on transcription. Transcription was performed with nuclear extract in the presence and absence of the activator Gal4VP16. As a control, transcription from naked DNA is shown (lanes 1–3). The same amount of DNA was used in all lanes. Products of the transcription reactions were separated by electrophoresis on a denaturing polyacrylamide gel. (Right) Full-length transcripts (390 nt). (C) Reconstituted transcription on RSF-assembled chromatin. Transcription was performed using a reconstituted system, in the presence and absence of FACT. All reactions contained the activator Gal4VP16. Short products are observed as a result of transcription without FACT. Products of the transcription reactions were separated by electrophoresis on a denaturing polyacrylamide gel. (Right) Full-length transcripts obtained in the presence of FACT (390 nt). (D) Transcription on nuclear extract using chromatin templates reconstituted with hypo- and hyperacetylated histones isolated from HeLa cells. Increasing amounts of hypo- and hyperacetylated chromatin (equal molar amounts) were added to nuclear extract in the presence and absence of the activator Gal4VP16. Products of the transcription reactions were separated by electrophoresis on a denaturing polyacrylamide gel. (Right) Full-length transcripts (390 nt). (E) Analysis of the chromatin reconstituted with hypo- and hyperacetylated histones using a reconstituted transcription system. Transcription reactions were performed in the presence of Gal4–VP16, but with and without FACT, as described in Materials and Methods. Products of the transcription reactions were separated by electrophoresis on a denaturing polyacrylamide gel. (Right) Full-length transcripts (390 nt).