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Changes in Structural Organization of the Nucleosome Fiber during Protooncogene Activation

A. A. Terent'ev, G. V. Kostyuk, and P. Ya. Boikov*

Institute of Chemical Physics, Russian Academy of Sciences, Chernogolovka, Moscow Region, 142432 Russia; fax: 8-(2) 515-5420; E-mail: boikov@icp.ac.ru

* To whom correspondence should be addressed.

Received November 28, 1996; Revision received August 13, 1997
Dynamics of structural changes in the chromatin of rat liver cells were studied during activation of the c-myc, c-fos, and c-jun protooncogenes. Nuclei were isolated at various stages of gene activation using cycloheximide and chromatin was subjected to partial nucleolysis by endogenous Ca2+/Mg2+-DNAse and extracted with low magnesium buffer; the nucleosome fragmentation was analyzed. DNA was isolated from the soluble chromatin and low-molecular-weight fragments were separated by electrophoresis through a composite gel system (3 and 1% agarose). This modification of DNA electrophoresis protocol improves the efficiency of separation of the low-molecular-weight DNA fragments. Increased protooncogene activity is associated with decreased content of DNAse-sensitive soluble chromatin. The fractional content of the c-fos gene is increased 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 protooncogene activation is associated with reorganization at the nucleosome level of the structural organization of the chromatin.
KEY WORDS: chromatin, nucleosome fiber, protooncogene, rat hepatocytes