Modulation of the sensitivity of chromatin to exogenous nucleases: implications for the apparent increased sensitivity of transcriptionally active genes.
We have examined the effects of changing the ionic composition of the buffers in which nuclei are isolated on the sensitivity of chromatin to micrococcal nuclease and deoxyribonuclease I. Unless nuclei are isolated in buffers containing physiological levels of monovalent (150 mM KCl) and divalent (2-5 mM MgCl2) cations, there is a substantial loss of higher order structure. The ionic composition of the buffer in which the digestion is carried out also affects the amount of material digested both by modulating higher order structure and by determining the solubility of the released material. Magnesium ion concentrations greater than 2 mM and calcium ions at virtually any concentration precipitate substantial amounts of the released chromatin fragments. These observations can be interpreted in light of the known effects of the ions on 10- and 30-nm fiber structure and used as a basis for improvements in techniques for isolating chromatin and for studying its structure and function using exogenous nuclease probes. The apparent nuclease sensitivity of transcriptionally active chromatin was reexamined and shown to be more likely a reflection of differential solubility rather than an overall increase in nuclease sensitivity.
D000818:Animals; D002413:Cations, Divalent; D002414:Cations, Monovalent; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002843:Chromatin / Q000187:drug effects / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000494:pharmacology*; D004492:Edetic Acid / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D008297:Male; D008836:Micrococcal Nuclease / Q000494:pharmacology*; D051381:Rats; D011919:Rats, Inbred Strains; D014158:Transcription, Genetic / Q000187:drug effects*
D002413:Cations, Divalent; D002414:Cations, Monovalent; D002843:Chromatin; D004492:Edetic Acid; D003850:Deoxyribonuclease I; D008836:Micrococcal Nuclease