RESUMO
Simian virus 40 [SV40] replicating chromosomes were labelled in vivo with 3H - thymidine [3H - dThd] and were extracted from SV40 infected green monkey kidney cells [CV1] under very low ionic strength buffer. The chromosomes were partially purified by centrifugation into through neutral glycerol gradient. Sub - fractions of chromosmes were pooled consecutively five batches. Each batch of chromosomes was used for in vitro DNA synthetic capability separately and the reactions were carried out in the presence of alpha 32 P - deoxythymidine 5' triphosphate [alpha 32 P - dTTP]. After termination of the reactions, the chromosomes of these sub - fractions were further analzed by resedimentation on the second cycle of glycerol gradients. Resedimentation of SV40 chromosomes have indicated that the sedimentation values of replicating or non - replicating chromosomes were not appreciably changed though some proteins might have been removed during the above procedures. The values ranged between 92 S and 55 S. Both the peaks of 3H radioactivity [incorporated in vivo] and the 32p radioactivity [incorporated in vitro] coincided with each other in these gradients. The chromosomes from these gradients were pooled separately, deproteinized and the products were further analyzed by sedimentation through alkaline sucrose gradients. The data suggest that the 32p label was observed in the Okazaki pieces [nascent DNA strands] of various lengths according to the extent of replication. The maximum efficiency of vitro DNA synthesis [as revealed by 32p/3H ratio] of SV40 sub - fractions from neutral glycerol gradient occurred in the replicating chromosomes which were sedimenting in the lagging side of the peak