Studies on the compaction of isolated nucleoids from Escherichia coli.
The genomic DNA of Escherichia coli is contained in one or two compact bodies known as nucleoids. Isolation of typically shaped nucleoids requires control of DNA expansion, accomplished here by a modification of the polylysine-spermidine procedure. The ability to control expansion of in vitro nucleoids has application in nucleoid purification and in preparation of samples for high-resolution imaging, and may allow an increased resolution in gene localization studies. Polylysine of relatively low average molecular weight (approximately 3 kDa) is used to produce lysates containing nucleoids that are several-fold expanded relative to the sizes of in vivo nucleoids. These expanded forms can be converted to compact forms similar in dimensions to the cellular nucleoids by either a further addition of polylysine or by incubation of diluted lysates at 37 degrees C. The incubation at 37 degrees C is accompanied by autolytic degradation of most ribosomal RNA. Hyperchromism and circular dichroism spectra indicate that polylysine-DNA complexes are modified during the incubation. Compact forms of the nucleoid can be progressively reexpanded by exposure to salt solutions. Nucleoid compaction was similar in lysates made from rapidly or slowly growing cells or from cells that had been briefly treated with chloramphenicol to reduce linkages between DNA and cell envelope.
Zimmerman
Steven B
SB
Laboratory of Molecular Biology, National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0560, USA. stevenz@bdg5.niddk.nih.gov
D002701:Chloramphenicol / Q000494:pharmacology; D002876:Chromosomes, Bacterial / Q000737:chemistry* / Q000187:drug effects / Q000648:ultrastructure*; D004269:DNA, Bacterial / Q000032:analysis / Q000737:chemistry; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D011107:Polylysine / Q000737:chemistry
D004269:DNA, Bacterial; D011107:Polylysine; D002701:Chloramphenicol