MICROCOCCAL NUCLEASE DIGESTION OF NUCLEI REVEALS EXTENDED NUCLEOSOME LADDERS HAVING ANOMALOUS DNA LENGTHS FOR CHROMATIN ASSEMBLED ON NONREPLICATING PLASMIDS IN TRANSFECTED CELLS

The chromatin structures of a variety of plasmids and plasmid construc tions, transiently transfected into mouse Ltk(-) cells using the DEAE- dextran procedure, were studied by micrococcal nuclease digestion of n uclei and Southern hybridization. Although regularly arranged nucleoso me-like particles clearly were formed on the transfected DNA, the nucl eosome ladders, in some cases with 13 - 14 bands, were anomalous. Most often, a ladder of DNA fragments with lengths of approximately 300, 5 00, 700, 900 bp, etc. was generated. In contrast, typical 180 - 190 bp multiples were generated from bulk cellular or endogenous beta-actin gene chromatin. Very similar results were obtained with all DNA's tran sfected, and in a variety of cell lines, provided that plasmid replica tion did not occur. Additionally, after digestion of nuclei, about 90% of the chromatin fragments that contained transfected DNA sequences c ould not be solubilized at low ionic strength, in contrast with bulk c ellular chromatin, suggesting association with nuclear structures or n uclear matrix. The remaining 10% of transfected DNA sequences, arising from soluble chromatin fragments, generated a typical nucleosome ladd er. These results are consistent with the idea that assembly of atypic al chromatin structures might be induced by proximity to elements of t he nuclear pore complex or by nuclear compartmentalization.