ISOLATION AND CHARACTERIZATION OF SPERMIDINE NUCLEOIDS FROM ESCHERICHIA-COLI

Nucleoids isolated from Escherichia coli at low salt concentrations in the presence of spermidine (Kornberg et al., Proc. Natl. Acad. Sci. U SA, 71, 3189-3193 (1974)) retain large amounts of protein and RNA and are, thus, potentially useful in structural and other studies, However , these preparations have neither been visualized nor extensively char acterized with regard to their protein and other components. We have i nvestigated this type of nucleoid preparation and here supply both Lig ht and electron microscope appearances and a description of the DNA-as sociated proteins. Light microscopy is used to follow the stages of nu cleoid release and to demonstrate characteristically rounded nucleoids after chloramphenicol treatment of the cells from which the nucleoids were isolated. The nucleoids are ''envelope-associated'' particles. E lectron microscopy shows an irregular central core that is partially c overed with small, membranous vesicles. A significant fraction of the nucleoids have a characteristic doublet/dumbbell-shaped appearance by light microscopy, The nucleoids contain large amounts of protein and R NA in addition to DNA, The DNA and RNA are rendered acid-soluble by ve ry low levels of nucleases, indicating an open structure. A small grou p of proteins, including H-NS, FIS, HU, and RNA polymerase, is release d from the particles upon enzymatic digestion of the DNA. (C) 1997 Aca demic Press.