CHANGES IN STRUCTURAL ORGANIZATION OF THE NUCLEOSOME FIBER DURING PROTOONCOGENE ACTIVATION

Dynamics of structural changes in the chromatin of rat liver cells wer e studied during activation of the c-myc, c-fos, and c-jun protooncoge nes. Nuclei were isolated at various stages of gene activation using c ycloheximide and chromatin was subjected to partial nucleolysis by end ogenous Ca2+/Mg2+-DNAse and extracted with low magnesium buffer; the n ucleosome fragmentation was analyzed. DNA was isolated from the solubl e chromatin and low-molecular-weight fragments were separated by elect rophoresis through a composite gel system (3 and 1% agarose). This mod ification of DNA electrophoresis protocol improves the efficiency of s eparation of the low-molecular-weight DNA fragments. Increased protoon cogene activity is associated with decreased content of DNAse-sensitiv e soluble chromatin. The fractional content of the c-fos gene is incre ased in the soluble chromatin. In control nuclei, endogenous Ca2+/Mg2-DNAse cleaves mononucleosomes containing DNA fragments with variable length. Upon protooncogene activation, mono-and oligonucleosomes with elongated DNA fragments are predominantly formed. The changes involve about 40% of the nuclear chromatin. The data suggest that protooncogen e activation is associated with reorganization at the nucleosome level of the structural organization of the chromatin.