ISOLATION OF THE GROWING POINT IN THE BACTERIAL CHROMOSOME

Abstract

A thymine-requiring bacterium was transferred to a medium containing the thymine analogue, 5-bromouracil, as a density label for newly replicated DNA. Following a gentle lysis procedure, the density distribution of the DNA molecules was examined in cesium chloride density gradients. A small fraction of the DNA molecules were isolated at densities intermediate between normal (unreplicated) and hybrid (5-bromouracil in one stand) and these were characterized as containing transition points (i.e., the replication point at instant of transfer to 5-bromouracil medium) or replication points (as caught at the instant growth was stopped). The isolation of replication points was specifically shown by P32 pulse-labeling during 5-bromouracil incorporation and the demonstration that the intermediate density P32 activity could be "chased" into the hybrid density region by subsequent growth with unlabeled phosphate. The extracted DNA was shown to have a mean molecular weight of 108, and mild shear which reduced this to the 3 to 6 x 107 range sheared all replication point containing molecules into hybrid and normal density fragments. The resistance to isolation and the sensitivity to shear of partially replicaed DNA molecules is discussed.