The DNA of a strain of the pleuropneumonia-like organism Mycoplasma laidlawii B, adapted to grow in a semi-defined medium, was density-labeled for various periods of time by substituting 5-bromodeoxyuridine for thymidine in the growth medium. Analysis of extracted DNA fragments by CsCl equilibrium sedimentation demonstrated semi-conservative replication, proceeding sequentially along the chromosome. The kinetics of formation of density-labeled DNA showed that 5-bromodeoxyuridine probably effects a premature initiation of a round of replication. By lysing the cells directly in the centrifuge tube, the M. laidlawii B chromosome was isolated in one piece in the CsCl gradient. Comparison of the buoyant-density distribution of the intact chromosomes with that of very mildly sheared chromosomes (shear yielding pieces at least 1 8 to 1 4 the size of the total chromosome) eliminated nearly all dispersive replication mechanisms and suggested that replication probably proceeds from only a few growing points. The buoyant-density distributions of intact chromosomes isolated after different times of density labeling are shown to be consistent with theory. Some physicochemical properties of the hybrid (density-labeled) and normal M. laidlawii B DNA are presented. These properties, and similarities and differences of DNA replication in M. laidlawii B and in Escherichia coli, are discussed. © 1969.
