Nuclease sensitivity of chromatin containing active genes: kinetic analyses utilizing continuous elution of digestion products from an ultrafiltration cell.
Methods have been developed to analyze the kinetics of digestion of chromatin by nucleases. Radioactively labeled nuclei were incubated with enzyme in an ultrafiltration apparatus and digestion rates of different chromatin samples were computed employing a least-squares curve fitting technique to fit the data to zero-order and/or first-order kinetic models. These methods allow detailed kinetic analyses on small amounts of chromatin. Two biological systems were studied. 1) Tetrahymena thermophila macronuclei and micronuclei were compared; these nuclei differ in their transcriptional activities. 2) Ribosomal DNA (rDNA) of Tetrahymena pyriformis, approximately 60% of which codes for rRNA, can be preferentially labeled during starvation-refeeding; its digestion kinetics relative to bulk chromatin were studied. DNase I digested 20-40% of the macromolecular DNA about 3 times faster than bulk macronuclear or micronuclear DNA, and 60-80% of ribosomal gene-containing chromatin about 5 times faster than bulk chromatin. Filter hybridization studies of the DNAase I sensitivity of tRNA, 5S RNA, and ribosomal genes yielded similar results. These data are consistent with the observation that transcribed genes are especially sensitive to attach by DNase I and suggest that activated chromatin structure as probed by extensive DNase I digestion is the same in higher and lower eucaryotes for genes transcribed by all three RNA polymerases. Digestion kinetics of micrococcal nuclease were found to depend on the digestion conditions employed. These two biological systems and the methods we have developed should facilitate analyses of the factors responsible for maintaining an active chromatin structure.
D000818:Animals; D002467:Cell Nucleus / Q000648:ultrastructure; D002843:Chromatin / Q000648:ultrastructure*; D004247:DNA / Q000032:analysis*; D003850:Deoxyribonuclease I; D003851:Deoxyribonucleases / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D013769:Tetrahymena pyriformis / Q000032:analysis; D014462:Ultrafiltration
D002843:Chromatin; D004247:DNA; D003851:Deoxyribonucleases; D004720:Endonucleases; D003850:Deoxyribonuclease I