Structure of eukaryotic chromatin. Evaluation of periodicity using endogenous and exogenous nucleases.
DNA isolated from (a) liver chromatin digested in situ with endogenous Ca2+, Mg2+-dependent endonuclease, (b) prostate chromatin digested in situ with micrococcal nuclease or pancreatic DNAase I, and (c) isolated liver chromatin digested with micrococcal nuclease or pancreatic DNAase I has been analyzed electrophoretically on polyacrylamide gels. The electrophoretic patterns of DNA prepared from chromatin digested in situ with either endogenous endonuclease (liver nuclei) or micrococcal nuclease (prostate nuclei) are virtually identical. Each pattern consists of a series of discrete bands representing multiples of the smallest fragment of DNA 200 +/- 20 base pairs in length. The smallest DNA fragment (monomer) accumulates during prolonged digestion of chromatin in situ until it accounts for nearly all of the DNA on the gel; approx. 20% of the DNA of chromatin is rendered acid soluble during this period. Digestion of liver chromatin in situ in the presence of micrococcal nuclease results initially in the reduction of the size of the monomer from 200 to 170 base pairs of DNA and subsequently results in its conversion to as many as eight smaller fragments. The electrophoretic pattern obtained with DNA prepared from micrococcal nuclease digests of isolated liver chromatin is similar, but not identical, to that obtained with liver chromatin in situ. These preparations are more heterogeneous and contain DNA fragments smaller than 200 base pairs in length. These results suggest that not all of the chromatin isolated from liver nuclei retains its native structure. In contrast to endogenous endonuclease and micrococcal nuclease digests of chromatin, pancreatic DNAase I digests of isolated chromatin and of chromatin in situ consist of an extremely heterogeneous population of DNA fragments which migrates as a continuum on gels. A similar electrophoretic pattern is obtained with purified DNA digested by micrococcal nuclease. The presence of spermine (0.15 mM) and spermidine (0.5 mM) in preparative and incubation buffers decreases the rate of digestion of chromatin by endogenous endonuclease in situ approx. 10-fold, without affecting the size of the resulting DNA fragments. The rates of production of the smallest DNA fragments, monomer, dimer, and trimer, are nearly identical when high molecular weight DNA is present in excess, indicating that all of the chromatin multimers are equally susceptible to endogenous endonuclease. These observations points out the effects of various experimental conditions on the digestion of chromatin by nucleases.
D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D002467:Cell Nucleus / Q000032:analysis*; D002843:Chromatin / Q000032:analysis; D004247:DNA / Q000032:analysis; D003851:Deoxyribonucleases / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005092:Exonucleases / Q000378:metabolism; D008099:Liver / Q000032:analysis*; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008837:Micrococcus / Q000201:enzymology; D009928:Organ Specificity; D010179:Pancreas / Q000201:enzymology; D011073:Polyamines / Q000494:pharmacology; D011467:Prostate / Q000032:analysis*; D051381:Rats
D002843:Chromatin; D046911:Macromolecular Substances; D011073:Polyamines; D004247:DNA; D003851:Deoxyribonucleases; D005092:Exonucleases; D008274:Magnesium; D002118:Calcium