Transcription of chromatin from mouse fibroblasts.
Chromatin purified from mouse fibroblasts can be fractionated after shearing by sedimentation in a sucrose gradient into an extended "light" and a compact "heavy" component. Further purification of these classically described components can be achieved by a second cycle of centrifugation of the light and heavy components through an equilibrium density gradient of metrizamide. The light component purified from sucrose gradient sediments faster (peak I) on metrizamide than its heavy counterpart (peak II). Template activity for DNA directed RNA synthesis in the presence of E. coli RNA polymerase is negligible in peak II but very pronounced in the peak I fraction. The difference in template activity appears to be connected with differences in propagation rather than initiation rates. Comparison of gel electrophoresis patterns of proteins indicate that the active subcomponent includes high molecular weight components not present in the inactive one, but that its histone content is somewhat lower. Using a very highly sensitive automatic recording apparatus for the measurement of melting profiles, no clear cut difference has been observed in the behaviour of active and inactive chromatin subcomponents nor in that of their total DNA.
D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002843:Chromatin / Q000302:isolation & purification / Q000378:metabolism*; D004247:DNA / Q000032:analysis; D012321:DNA-Directed RNA Polymerases; D005347:Fibroblasts / Q000378:metabolism*; D009691:Nucleic Acid Denaturation; D012313:RNA / Q000096:biosynthesis; D013698:Templates, Genetic; D014158:Transcription, Genetic
D002843:Chromatin; D012313:RNA; D004247:DNA; D012321:DNA-Directed RNA Polymerases