Enrichment of transcribed and newly replicated DNA in soluble chromatin released from nuclei by mild micrococcal nuclease digestion.
A chromatin fraction solubilized from mouse myeloma nuclei under near-physiological ionic conditions by very mild micrococcal nuclease digestion at 0 degrees C is enriched at least 7-fold in DNA complementary to total myeloma polyadenylated mRNA, and 15-fold in DNA originating near the replication fork (labeled within 30 s). Newly replicated DNA recovered in solubilized chromatin after brief labeling was incorporated mainly into particles sedimenting with, or faster than, mononucleosomes. A rapid decrease in enrichment of newly replicated DNA in readily released, soluble chromatin with increasing labeling times indicated that newly replicated chromatin matured within 90 s to a form that was partitioned similarly to bulk chromatin by this fractionation method. Previous studies showed that chromatin readily solubilized from myeloma nuclei is enriched in high-mobility-group (HMG) and other non-histone proteins, RNA and single-stranded DNA; and depleted in H1 and 5-methylcytosine, relative to bulk chromatin (Jackson, J.B., Pollock , J.M., Jr., and Rill , R.L. (1979) Biochemistry 18, 3739-3748). Mild digestion of chicken erythrocyte nuclei with micrococcal nuclease yielded a soluble chromatin fraction (1-2% of the total DNA) with similar properties. This fraction was enriched at least 6-fold in DNA complementary to chicken globin mRNA, relative to total erythrocyte DNA.
D000818:Animals; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000648:ultrastructure; D002645:Chickens; D002843:Chromatin / Q000378:metabolism* / Q000648:ultrastructure; D004247:DNA / Q000032:analysis; D004261:DNA Replication; D004912:Erythrocytes / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008836:Micrococcal Nuclease / Q000378:metabolism*; D009693:Nucleic Acid Hybridization; D010954:Plasmacytoma; D014158:Transcription, Genetic
D002843:Chromatin; D004247:DNA; D008836:Micrococcal Nuclease