Isolation, characterization, and structure of the folded interphase genome of Drosophila melanogaster.
The intact interphase genome of Drosophila melanogaster has been isolated by sucrose gradient centrifugation after gentle lysis of tissue culture cells in 0.9 M NaCl-0.4% nonidet P40. The non-viscous folded DNA sediments as a single broad 5000S peak in a complex with RNA (a fraction of the nuclear nascent RNA) and protein (all of the four intranuclesome histones: H2A, H2B, H3, and H4). The folded DNA is supercoiled and can be relaxed to slower sedimenting forms either by intercalating ethidium or by nicking with DNAase I. Incomplete DNAase treatment gives partially relaxed complexes, indicating that each nick relaxes only a stretch of DNA (defined as a supercoiled DNA loop) without affecting the superhelical content of the rest of the genome. The concentration of superhelices in the Drosophila folded DNA is the same as in the E. coli and SV40 closed circular DNAs-that is, about one negative turn every 200 base pairs (bp) in 0.15 M NaCl at 26 degrees C. The estimated average size of the supercoiled DNA loops, about 85,000 bp, equals the size of the larger Drosophila chromomeres. Ethidium intercalation in 0.9 M NaCl both removes the negative superhelical turns and dissociates the four histones from the DNA. The four histones are dissociated in equimolar concentrations, and the relative proportion of histones displaced from the DNA is a function of ethidium concentration. The histones are completely dissociated from the folded DNA at the ethidium concentration. The histones are completely dissociated from the folded DNA at the ethidium concentration which removes all of the negative superhelices. Thus the data strongly suggest that the rotation of the Watson Crick helix which accompanies ethidium intercalation causes the loss of nucleosomes from the DNA. The results are interpreted in terms of a model for the folded Drosophila genome which has the DNA constrained (by both protein-DNA and RNA- DNA interactions) into independent supercoiled loops containing on the average 400 nucleosomes per loop. Each nucleosome is composed of a histone core with the DNA wound around it in a 360 degrees left-handed toroidal supercoil; each nucleosome toroidal supercoil plus its relaxed internucleosome DNA contains, on the average, 200 bp.
D000818:Animals; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002868:Chromosomal Proteins, Non-Histone; D002875:Chromosomes / Q000032:analysis*; D004247:DNA / Q000302:isolation & purification; D003851:Deoxyribonucleases / Q000494:pharmacology; D004331:Drosophila melanogaster; D004996:Ethidium / Q000494:pharmacology; D006657:Histones; D008954:Models, Biological; D009690:Nucleic Acid Conformation / Q000187:drug effects; D011487:Protein Conformation / Q000187:drug effects; D012313:RNA
D002868:Chromosomal Proteins, Non-Histone; D006657:Histones; D012313:RNA; D004247:DNA; D003851:Deoxyribonucleases; D004996:Ethidium