The labelling of DNA by pulse/chase experiments in has been investigated, analysing the products by alkaline sucrose velocity centrifugation. In NCTC 8325 a short (60 s) pulse of [H]thymidine labels both small (10 to 20 S) fragments and DNA that co-sediments with long-term label. In a thymidine-requiring derivative, 8325, most pulse label is incorporated into small fragments. In both bacterial strains small fragments can be chased into high molecular weight DNA. Thymidine starvation of 8325 prior to pulse labelling results in smaller fragments (4 to 10S) being labelled. In a subsequent chase with unlabelled thymidine this label is incorporated into high molecular weight DNA, although more slowly than in the absence of thymidine starvation. The fact that nalidixic acid, an antibiotic which specifically inhibits DNA replication in , does not inhibit the [H]thymidine incorporation immediately after thymidine starvation and that nalidixic acid slows down the increase in size of pulse-labelled fragments through inhibition of DNA synthesis suggests that thymidine starvation results in changes at the replication fork. The possible nature of these changes is discussed. It is proposed that one of the results of thymidine starvation is to cause a long-lived gap between DNA synthesized before starvation and DNA synthesized after starvation.


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