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PMID: 634792 Published · ppublish English Journal Article Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

Chromatin assembly in isolated mammalian nuclei.

Nucleic acids research ·Vol. 5 ·No. 2 ·1978-02-00 ·Pages 349-62

Shelton ER, Kang J, Wassarman PM, DePamphilis ML

Abstract

Cellular DNA replication was stimulated in confluent monolayers of CV-1 monkey kidney cells following infection with SV40. Nuclei were isolated from CV-1 cells labeled with [3H]thymidine and then incubated in the presence of [alpha-32P]deoxyribonucleoside triphosphates under conditions that support DNA replication. To determine whether or not the cellular DNA synthesized in vitro was assembled into nucleosomes the DNA was digested in situ with either micrococcal nuclease or pancreatic DNase I, and the products were examined by electrophoretic and sedimentation analysis. The distribution of DNA fragment lengths on agarose gels following micrococcal nuclease digestion was more heterogeneous for newly replicated than for the bulk of the DNA. Nonetheless, the state of cellular DNA synthesized in vitro (32P-labeled) was found to be identical with that of the DNA in the bulk of the chromatin (3H-labeled) by the following criteria: (i) The extent of protection against digestion by micrococcal nuclease of DNase I. (ii) The size of the nucleosomes (180 base pairs) and core particles (145 base pairs). (iii) The number and sizes of DNA fragments produced by micrococcal nuclease in a limit digest. (iv) The sedimentation behavior on neutral sucrose gradients of nucleoprotein particles released by micrococcal nuclease. (v) The number and sizes of DNA fragments produced by DNase I digestion. These results demonstrate that cellular DNA replicated in isolated nuclei is organized into typical nucleosomes. Consequently, subcellular systems can be used to study the relationship between DNA replication and the assembly of chromatin under physiological conditions.

MeSH Terms
Cell Line Cell Nucleus/metabolism,ultrastructure Cell-Free System Chromatin/metabolism,ultrastructure Cytosol/metabolism DNA Replication Deoxyribonucleases/metabolism Micrococcal Nuclease/metabolism Time Factors
Chemicals
Chromatin Deoxyribonucleases Micrococcal Nuclease
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Shelton E R
Kang J
Wassarman P M
DePamphilis M L
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36 references, click to expand
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/5.2.349
Published
1978-02-00
Pages
349-62
Language
English
Region
England
NLM ID
0411011
PMCID
PMC341988
Subset
IM
External Links
PubMed source DOI: 10.1093/nar/5.2.349 PMC Full Text View journal details
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