Analysis by Isopycnic centrifugation of isolated nucleoids of Escherichia coli

Abstract

The isolated, formaldehyde-fixed nucleoid of E. coli has been analyzed by isopycnic centrifugation in CsCl density gradients. The membrane-free nucleoid bands at a density of 1.69 ± 0.02 g/cm3. The membrane-associated nucleoid bands at a density of 1.46 ± 0.02 g/cm3. Both species sediment to equilibrium as nearly monodisperse bands in CsCl, suggesting that the nucleoid components of DNA, RNA and protein are present in relatively constant ratios. These ratios are constant regardless of the position of the nucleoids in the heterogeneous sedimentation profile of a preparative sucrose gradient. The fixed nucleoids remain condensed during isopycnic centrifugation and there is no detectable loss of RNA from the nucleoid.

Organization of supercoil domains and their reorganization by transcription

“…A partial solution to the packing problem is negative supercoiling, which causes the double helix to adopt a branched and plectonemic or interwound structure. Cells treated with lysozyme and mild ionic detergent release non‐viscous ‘nucleoids’ 1 that can be analysed by sedimentation through sucrose gradients and by electron microscopy (Worcel and Burgi, 1972; Giorno et al ., 1975; Kavenoff and Ryder, 1976). The unfolded structure reveals an interwound DNA conformation.…”

Ionizing radiation damage to the folded chromosome of Escherichia coli K-12: sedimentation properties of irradiated nucleoids and chromosomal deoxyribonucleic acid

“…Much of the early work on nucleoids focused on the membrane-associated form, largely due to difficulties in obtaining quantitative yields of the membrane-free chromosomes. An observation by Worcel and Burgi (37) that Sarkosyl could release the nucleoid from the membrane in vitro has led to the development of isolation procedures specific for membrane-free chromosomes (19)(20)(21). We have devised a similar protocol (K. M. Ulner, Ph.D. thesis, Massachusetts Institute of Technology, Cambridge, 1978) for isolation of t Present address: Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, MA…”