Transferring DNA from electrophoretically resolved nucleosomes to diazobenzyloxymethyl cellulose: properties of nucleosomes along mouse satellite DNA

Abstract

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.

The active immunoglobulin kappa chain gene is packaged by non-ubiquitin-conjugated nucleosomes.

“…This has been possible because chromatin fractionation enriches transcribed sequences in the S1 fraction (6) much higher hybridization signals than does diazotized paper (18). Our results reveal that the nucleosomes along the active K locus have many features in common with bulk particles found in the S1 fraction, including the apparent association of accessory proteins HMG-14 or -17 and the absence ofhistone H1 (Fig.…”

“…Where indicated, linker DNA was trimmed by allowing residual micrococcal nuclease within the S1 fraction to digest samples at 4°C for about 1 hr. Samples adjusted to 10 mM EDTA were dialyzed against 2 mM EDTA, pH 7.4, concentrated to 1.5 mg of DNA per ml by using a Minicon-B15 concentrator at 4°C, and, when desired, extracted with 0.35 M NaCl (18) prior to repeated dialysis and concentration.…”

“…DNA of nucleoprotein gel strips (see above) was resolved on 5% polyacrylamide/0. 1% NaDodSO4 gels (9,18) and transferred to Zeta-Probe membrane (26). Membranes sprayed with EN3HANCE were fluorographed with preflashed film (25).…”

5-methylcytosine is localized in nucleosomes that contain histone H1.

“…Mouse mastocytoma (line P815) cells were grown and uniformly labeled as described elsewhere (13). Published techniques were used for nuclei isolation and micrococcal nuclease digestion (21), quantitation of digestion and DNA yields, and DNA purification (22). For preparation of chromatin fractions, digested nuclei were cooled rapidly and centrifuged, yielding the S1 supernatant; the nuclear pellet was then lysed by suspension in 2 mM EDTA and centrifuged, yielding the S2 supernatant and the P pellet fraction, as described in detail elsewhere (23).…”

“…Published procedures were employed for denaturing and electrophoretically transferring DNA from gels to diazobenzyloxymethyl-cellulose (DBM-paper) (ref. 21), except that a one-fifth power setting was used for the first hour to improve retention of 121-base-pair (bp) pBR322 fragments. Dot blots were performed essentially as described elsewhere (23).…”