Transferring DNA from electrophoretically resolved nucleosomes to diazobenzyloxymethyl cellulose: properties of nucleosomes along mouse satellite DNA.
AUTOR(ES)
Reudelhuber, T L
RESUMO
Electrophoresis fractionates nucleosomes which possess different protein compositions. We report here a procedure for transferring the DNA components of electrophoretically resolved nucleosomes to diazobenzyloxymethyl cellulose (DBM-paper). Histones are first removed from nucleosome components by electrophoresis in the presence of cetyltrimethylammonium bromide (CTAB), leaving DNA fragments fixed within the original gel as the CTAB salts. The DNA is then converted to the sodium salt, denatured, and electrophoretically transferred to DBM-paper. The overall pattern of DNA on the resulting blot is visualized either by fluorography or by immunoautoradiography. This DNA pattern is then compared with autoradiograms obtained after hybridizing the same blot with specific 32P-labeled probes. Using mouse satellite DNA as a hybridization probe, we illustrate the above techniques and demonstrate that nucleosomes carrying satellite sequences are compositionally heterogeneous. The procedures described here should also be useful in the analysis of the nucleic acid components associated with other nucleoprotein complexes.
