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

Tim L. Reudelhuber; Dorothy J. Ball; Alan H. Davis; William T. Garrard

doi:10.1093/nar/10.4.1311

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

Tim L. Reudelhuber, Dorothy J. Ball, Alan H. Davis, William T. Garrard

Molecular Biology

Research output: Contribution to journal › Article › peer-review

8 Scopus citations

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 DBN-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.

Original language	English (US)
Pages (from-to)	1311-1325
Number of pages	15
Journal	Nucleic acids research
Volume	10
Issue number	4
DOIs	https://doi.org/10.1093/nar/10.4.1311
State	Published - Feb 25 1982

ASJC Scopus subject areas

Genetics

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title = "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 DBN-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.",

author = "Reudelhuber, {Tim L.} and Ball, {Dorothy J.} and Davis, {Alan H.} and Garrard, {William T.}",

note = "Funding Information: ACKNOWLEDGEMENTS We thank Mr. J. M. Wiseman for expert technical assistance and Drs. Raymond J. MacDonald, Nancy C. Martin, and Stephen M. Rose for helpful suggestions. We are indebted to Drs. George R. Stark and Albert E. Dahlberg for providing us with preprints of their work prior to publication. We thank Dr. B. David Stollar and Ellen S. Vitetta for their generous gifts of monoclonal and polyclonal antibodies, respectively. This investigation was supported by Grant GM-22201 from the National Institutes of Health and by Grant 1-823 from the Robert A. Welch Foundation. A preliminary report describing a portion of this work has appeared (52).",

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AU - Reudelhuber, Tim L.

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AU - Davis, Alan H.

AU - Garrard, William T.

N1 - Funding Information: ACKNOWLEDGEMENTS We thank Mr. J. M. Wiseman for expert technical assistance and Drs. Raymond J. MacDonald, Nancy C. Martin, and Stephen M. Rose for helpful suggestions. We are indebted to Drs. George R. Stark and Albert E. Dahlberg for providing us with preprints of their work prior to publication. We thank Dr. B. David Stollar and Ellen S. Vitetta for their generous gifts of monoclonal and polyclonal antibodies, respectively. This investigation was supported by Grant GM-22201 from the National Institutes of Health and by Grant 1-823 from the Robert A. Welch Foundation. A preliminary report describing a portion of this work has appeared (52).

PY - 1982/2/25

Y1 - 1982/2/25

N2 - 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 DBN-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.

AB - 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 DBN-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.

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

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