DNAase I and cellular factors that affect chromatin structure.
The use of DNAase I as a probe of chromatin structure is frequently fraught with problems of irreproducibility. We have recently evaluated this procedure, documented the sources of the problems, and standardized the method for reproducible results (Prentice and Gurley (1983) Biochim. Biophys. Acta 740, 134-144). We have now used this probe to detect differences in chromatin structure between cells blocked (1) in G1 phase by isoleucine deprivation, or (2) in early S phase by sequential use of isoleucine deprivation followed by release into the presence of hydroxyurea. The cells blocked in G1 phase have easily-digestible chromatin, while cells blocked in early S phase have chromatin which is much more resistant to DNAase I. These differences were found to be the result of diffusible factors found in the cytoplasm and nuclei of G1- and S-phase cells, respectively. The G1 cells contained a cytoplasmic factor which modulates the chromatin structure of S-phase nuclei to a more easily digestible state, while cells blocked in S phase contain a nuclear factor which modulates the chromatin structure of G1 nuclei to a state more resistant to digestion. DNAase I is much more sensitive to these cell cycle-specific chromatin changes than is micrococcal nuclease. The results indicate that, under controlled conditions, DNAase I should be a valuable probe for detecting chromatin structural changes associated with cell cycle traverse, differentiation, development, hormone action and chemical toxicity.
D000818:Animals; D002453:Cell Cycle / Q000187:drug effects; D002460:Cell Line; D002843:Chromatin / Q000187:drug effects / Q000648:ultrastructure*; D006224:Cricetinae; D003412:Cricetulus; D003850:Deoxyribonuclease I; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry; D006918:Hydroxyurea / Q000633:toxicity; D007532:Isoleucine / Q000378:metabolism; D007700:Kinetics; D008836:Micrococcal Nuclease / Q000378:metabolism; D010053:Ovary
D002843:Chromatin; D007532:Isoleucine; D004706:Endodeoxyribonucleases; D003850:Deoxyribonuclease I; D008836:Micrococcal Nuclease; D006918:Hydroxyurea