Studies on chromatin assembly in vitro and in transfected cells
Abstract
Chromatin structure is known to play an active role in regulation of gene expression. One of the features of chromatin in higher eukaryotic cells is that it consists of extensive, regular nucleosome arrays along the DNA sequence. The nucleosome spacing and the degree of regularity can strongly influence chromatin higher-order structure. However, the mechanisms which direct the physiological nucleosome spacing are not well understood. We have developed the first fully defined in vitro chromatin assembly system and have found that histone H5 (or H1) induces physiological nucleosome spacing and extensive ordering on some of the constructs derived from plasmid pBR327. A continuous approximately 800 bp region of the plasmid was required to nucleate histone H5-induced nucleosome alignment, which could then spread to adjacent chromatin. Supporting this idea, a positioned five-nucleosome array appears to originate in the required region. We have also investigated nucleosome structure in chromatin assembled on DNA transfected into cultured animal cells to obtain insight into the mechanisms by which a physiological nucleosome array is generated in vivo. We have observed that every replicating circular DNA does not assemble into a chromatin structure with a regular nucleosome spacing. Simian virus 40 (SV40) DNA transfected into COS-1 cells revealed a much more regular nucleosome spacing than other plasmids. The results suggest that DNA sequences in the SV40 early region are necessary for the regular nucleosome arrangement observed over the whole SV40 genome (5243 bp). Evidence is also provided that periodic nucleosome positioning signals, in two or more phasing frames with respect to DNA, exist throughout the SV40 early region. In addition, we also investigated chromatin assembled on non-replicating plasmid DNA. The data indicate that chromatin assembled on non-replicating plasmid DNA transiently transfected into cultured cells contains an unusual, altered nucleosome structure, and suggest that it might be associated with the nuclear matrix. These properties closely resemble those found for some active genes.
Degree
Ph.D.
Advisors
Stein, Purdue University.
Subject Area
Molecular biology
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