Separation of transcriptionally active from less active rat ventral prostate chromatin

Abstract

Rat ventral prostate chromatin, prepared by a method in which marked intentional shearing was avoided, and analysed by sucrose density gradient centrifugation. yielded two major components. 1. 1. L chromatin, the quantitatively minor, less dense and/or structurally less compact fraction remained near the top of the gradient. H chromatin, containing 85–90% of the chromatin DNA, migrated into the more dense regions of the gradient, and in some experiments, was resolved into two fractions, H 1 and H 2. 2. 2. The H fraction exhibited a typical chromatin absorption profile; the absorption maximum of L chromatin was shifted towards 270 nm. 3. 3. The Tm app of these fractions was: whole chromatin, 83 °C; H 1, 85 °C; H 2, 80.5 °C, and L, 62.5 °C compared with 69 °C for calf thymus DNA. 4. 4. The ratio of L chromatin protein to DNA was 5 to 1, compared with 1.7 for unfractionated chromatin. The ratio of RNA to DNA for H and L fractions was 0.055 and 0.075. 5. 5. L chromatin was associated with TCA-precipitable radioactivity after ( a) in vivo administration of 14C-uridine or orotic acid; ( b) after in vitro incubation with E. coli RNA polymerase and subsequent separation on the gradient, and ( c) exhibited 10–20-fold greater template activity for E. coli polymerase than H chromatin when it was first separated on a gradient and then incubated for RNA synthesis. 6. 6. Residual endogenous RNA polymerase activity (cpm/unit A 260, nucleoplasmic enzyme II, 0.2 M ammonium sulfate, Mn 2+) associated with L chromatin was 5 times that of H. 7. 7. Both H and L chromatin were associated with similar amounts of radioactivity derived from injected 3H-testosterone, but L chromatin had a specific radioactivity (per μg DNA) 10 times that of H chromatin. 8. 8. The fraction of ‘minimally-sheared’ chromatin excluded from Bio-Gel A50m represented 10–20% of the total A ̀ 260 , exhibited a melting profile and Tm app similar to L fractions isolated on sucrose gradients, included 80% of the remaining in vivo-labelled RNA and was a better template for E. coli RNA polymerase than unfractionated chromatin. L chromatin from a sucrose gradient was also excluded from Bio-Gel A50m. By these four criteria, the two fractions are similar, and may be virtually identical. 9. 9. ‘Minimally-sheared’ chromatin did not degrade 14C-labelled rat liver RNA or DNA, and nucleoside triphosphatase activity, although present, did not interfere with the assay for RNA polymerase. 10. 10. Total proteins and histones from ‘minimally-sheared’ chromatin prepared after swelling overnight, and from ‘rapidly prepared’ chromatin subjected to more shearing were compared by SDS polyacrylamide gel electrophoresis (pH 7.2, total proteins) and electrophoresis at pH 3.5 (histones). No differences in the binding of Coomassie blue by histones or by total proteins from these preparations were evident. 11. 11. SDS-polyacrylamide gel electrophoresis of total protein from unfractionated chromatin and from the fraction excluded from Bio-Gel A50m exhibited many similarities in the high molecular weight region of the gels, and some quantitative differences in the region from about 1.5–4.5 × 10 4 D. Properties 1–11 are consistent with the seperation of a transcriptionally more active L from of chromatin (euchromatin) from less active H hetrochromatin.