Distribution of proviral sequences in chromatin of embryonic fibroblasts infected by Rous sarcoma virus.

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RESUMO

Sheared chromatin prepared from chicken embryo fibroblasts and fibroblasts transformed by exogenous Rous sarcoma virus (Schmidt--Ruppin strain D) was separated by rate sedimentation on glycerol gradients into two components: fast-migrating (heavy chromatin fraction) and slow-migrating (light chromatin fraction). DNAs extracted from these fractions were assayed for proviral sequences by molecular hybridization using DNA complementary to the viral sequences. In uninfected cells, the endogenous complementary sequences were found to be equally distributed between heavy and light fractions. However, the newly integrated exogenous proviral sequences were found mostly in the light chromatin fraction in the transformed cells. Additionally, the light fraction was more sensitive to DNase I digestion and contains more material melting at low temperatures when compared with the heavy fraction. The results show that (i) distribution of endogenous proviral sequences is independent of chromatin conformation, and (ii) most of the newly acquired exogenous sequences are integrated within the host's chromatin fraction that exhibits properties of euchromatin. Because chromatin fragmentation and fractionation is accomplished without digestion with degrading enzymes, the chromatin fractions enriched in exogenous sequences remain intact and thus are suitable for further studies.

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