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Published June 1977 | Published
Journal Article Open

The problems of eukaryotic and prokaryotic DNA packaging and in vivo conformation posed by superhelix density heterogeneity


Systems for gel electrophoresis in the presence of one of the inter-calative unwinding ligands, ethidium or chloroquine, have been developed which permit the resolution of highly supercoiled closed circular DNA molecules differing by unit values of the topological winding number, . All native closed circular DNAs examined, including the viral and intra-cellular forms of SV40 and polyoma DNA, bacterial plasmid DNAs, and the double stranded closed circular DNA genome of the marine bacterio-phage, PM2, are more heterogeneous with respect to the number of super-helical turns present than are the thermal distributions observed in the limit products of the action of nicking-closing (N-C) enzyme on the respective DNAs. In the cases of SV40 and polyoma, where it has been shown that the supercoiling is a combined consequence of the binding of the four nucleosomal histones, H2a, H2b, H3 and H4, and the action of N-C enzyme, the breadth of the distributions within the form I DNAs poses specific problems since the work of other laboratories indicates that the number of nucleosomes on the respective minichromosomes falls within a narrow distribution of 21. If it is assumed that all nucleosomes have identical structures, and that the DNA within a nucleosome is not free to rotate, the native DNA would be anticipated to be less heterogeneous than the thermal equilibrium mixtures present in N-C enzyme relaxed SV40 and polyoma DNAs. The absolute number of superhelical turns (at 37°C in 0. 2 M NaCl) in virion polyoma DNA has been determined to be 26 ± 1, which is the same value obtained for virion SV40 DNA. This is consistent with the observations that polyoma DNA has a higher molecular weight, a lower superhelix density, but the same number of nucleosomes as SV40 DNA. In addition, the distributions within the virion and intracellular form I DNAs of both SV40 and polyoma were found to be indistinguishable.

Additional Information

© 1977 Oxford University Press. Received December 24, 1976. We are grateful to all of those who gave us samples of DNA, virus, and bacterial strains, to M. Kiernan for assistance with tissue culture and virus propagation, and to D. Agard for help with the fitting procedures used. This work was supported in part by grants from the National Cancer Institute and the National Institute of General Medical Sciences. M. S. is a recipient of an NSF graduate fellowship. D. E. P. is a recipient of a Medical Research Council of Canada fellowship. This is contribution no. 5442 from the Division of Chemistry and Chemical Engineering.

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