D Kube1, B Milavetz. 1. Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine, Grand Forks 58202, USA.
Abstract
BACKGROUND: T-antigen binding site I has been shown previously to play a role in regulating the proportion of Simian Virus 40 (SV40) chromosomes containing a nuclease hypersensitive promoter region. In order to determine whether these changes in nuclease hypersensitivity were a result of changes in the proportion of SV40 chromosomes, which contained a nucleosome-free promoter region, SV40 chromosomes were visualized by electron microscopy. METHODS: SV40 chromosomes were prepared from cells infected with either wild-type or mutant virus lacking T-antigen binding site I, and the chromosomes were analyzed by electron microscopy for the presence of nucleosome-free regions. RESULTS: Both the wild-type and mutant chromatin were found to contain heterogeneous nucleosome-free promoter regions consisting of small (3-4 times the normal internucleosomal distance) and large (greater than four times the normal internucleosomal distance) regions. Quantification of the proportion of chromosomes containing each type of region indicated that deletion of site I resulted in a 25% increase in the proportion of chromosomes containing a large nucleosome-free region but had no effect on the proportion of chromosomes containing a small nucleosome-free region. CONCLUSIONS: The results indicate that there are two distinct classes of nucleosome-free promoter region in SV40 chromatin and that T-antigen binding to site I is specifically involved in the regulation of only the class that contains a large nucleosome-free region.
BACKGROUND: T-antigen binding site I has been shown previously to play a role in regulating the proportion of Simian Virus 40 (SV40) chromosomes containing a nuclease hypersensitive promoter region. In order to determine whether these changes in nuclease hypersensitivity were a result of changes in the proportion of SV40 chromosomes, which contained a nucleosome-free promoter region, SV40 chromosomes were visualized by electron microscopy. METHODS:SV40 chromosomes were prepared from cells infected with either wild-type or mutant virus lacking T-antigen binding site I, and the chromosomes were analyzed by electron microscopy for the presence of nucleosome-free regions. RESULTS: Both the wild-type and mutant chromatin were found to contain heterogeneous nucleosome-free promoter regions consisting of small (3-4 times the normal internucleosomal distance) and large (greater than four times the normal internucleosomal distance) regions. Quantification of the proportion of chromosomes containing each type of region indicated that deletion of site I resulted in a 25% increase in the proportion of chromosomes containing a large nucleosome-free region but had no effect on the proportion of chromosomes containing a small nucleosome-free region. CONCLUSIONS: The results indicate that there are two distinct classes of nucleosome-free promoter region in SV40 chromatin and that T-antigen binding to site I is specifically involved in the regulation of only the class that contains a large nucleosome-free region.