Analysis of high-molecular-weight free nuclear DNAs revealed in mammalian cells by pulsed-field gel electrophoresis

Pulsed-field electrophoresis revealed minor free DNA (fDNA) fractions a and b in mammalian cells. The fractions differed in some properties from chrom osomal DNA (chrDNA). Electron microscopy revealed rosette-like structures r esistant: to drastic deproteinization in fDNAs from Namalwa cells. Selectiv e labeling and immunochemical visualization located both fractions to the n ucleus in terminally differentiated N1-E115 cells. Alu oligonucleotide-dire cted PCR showed that the distribution of unique and repetitive sequences in fDNAs differed from that in chrDNA. Representational difference analysis r evealed four sequences probably located in b fDNA. Of these, one proved hom ologous to the genes for human interleukin enhancer-binding factors (ILFs) and one, to the transcribed region neighboring the second internal transcri bed spacer in the rDNA cluster. The b fDNA was cleaved with BamHI, BglII, a nd BclI and cloned. The inserts ranging 120-7000 nt were completely or part ly sequenced in 60 clones. The fDNAs were assumed to result from selective excision of some chromatin regions or to be extrachromosomal elements of un known nature.