Two-dimensional hybridization mapping of nucleosomes: Comparison of DNA and protein patterns

Abstract

A new approach was developed for analysis of complex mixtures of staphylococcal nuclease-produced nucleosomes, consisting of two-dimensional electrophoretic fractionation of nucleosomes (low ionic strength electrophoresis of deoxyribo-nucleoproteins in the first dimension followed by DNA electrophoresis in the second dimension), with subsequent electrophoretic transfer of nucleosomal DNA from polyacrylamide gels to DBM paper for hybridization with specific DNA probes. Hybridization was accompanied by parallel two-dimensional analysis of protein composition of mononucleosomes from HeLa chromatin. The major results are: 1. (1) The H1-lacking mononucleosomes containing proteins HMG 14 and HMG 17 migrate as a broad band in the first dimension, and their DNA fragments form an identifiable subset of the total mononucleosomal DNA pattern. 2. (2) Hybridization of a highly repetitive human DNA fragment to fractionated HeLa mononucleosomal DNA lights up all major mononucleosomal DNA spots, suggesting that transcriptionally inactive regions of HeLa chromatin contain both A24 semihistone and HMG proteins. 3. (3) Hybridization of cloned ribosomal DNA to fractionated HeLa mononucleosomal DNA also lights up all major DNA spots. The regions corresponding to A24- and HMG-containing mononucleosomes, however, appear to be under-represented in the hybridization pattern. 4. (4) A fragment of highly repetitive rat DNA hybridizes to a subset of the total oligonucleosomal DNA pattern from rat 14B chromatin, strongly suggesting that the range of nucleosomal DNA repeat lengths within regions of chromatin homologous to this highly repetitive probe is much narrower than that in bulk chromatin. Our findings indicate that specific interactions of HMG proteins with transcriptionally active regions of chromatin (Weisbrod et al., 1980) are superimposed over a significant background of apparently non-specific HMG chromatin interactions. We suggest that the process of transcription itself is responsible for "saturation" of a chromatin region with HMG proteins in vitro.

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