Both the inhibitory effect of aphidicolin on the replicative alpha-polymerase and the reversibility of its action in vivo (Pedrali-Noy & Spadari, 1979, Biochem. Biophys. Res. Commun. 88, 1194-2002) allow the synchronization of cells in culture. Aphidicolin prevents G1 cells from entering the DNA synthetic period, blocks cells in "S" phase, allows G2, M and G1 cells to continue the cell cycle and to accumulate at the G1/S border. Aphidicolin is a more useful reagent than hydroxyurea and thymidine because it does not affect cell viability or "S" phase duration and does not interfere with the synthesis of dNTPs or DNA polymerases. In fact cells exposed to the drug continue to synthesize all three DNA polymerases alpha, beta and gamma as well as all dNTPs which, when the block is removed, are present at levels optimal for DNA initiation and replication. The technique is simple and can be applied to cells growing in suspension or monolayers and allows one to harvest large quantities of synchronized cells.

No abstract

HeLa cells were labelled for 10 or 30 minutes with [3H]thymidine and the DNA fractionated with chloroform-isoamylalcohol ; almost goo/, of the labelled DNA remained in the interphase whilst about 60°/, of total DNA (bulk) was extracted in the aqueous phase.When added to a column of methylated serum albumin adsorbed on kieselguhr the extracted DNA was eluted as a major labelled peak with 0.8 M NaCl but a small fraction could only be eluted at higher NaCl concentrations. This "tail" contained both bulk and recently labelled DNA.After a short pulse the specific activity of the DNA eluted at higher salt concentrations increased ; this was confirmed when pooled fractions were concentrated on a caesium sulphate gradient.Electron microscopy and sucrose gradient centrifugation of fractions recovered on a Cs,SO, gradient shows that the eluted DNA was polydisperse (peak at about 18s) with molecular dimensions of a few to almost 20 p.When [3H]uridine was used as a label, both labelled DNA and rapidly labelled RNA could be studied simultaneously in the same way: these experiments indicated that in the fractions analysed, t8he radioactivity due to RNA and to DNA was not carried on the same molecules, suggesting the mutual exclusion of replication and transcription.Although the proposed mechanism of semiconservative DNA replication [I] has been demonstrated in bacteria [ 2 ] and also in higher cells [3-51, information concerning the mechanism of this replication i n vivo is still rather scanty. In bacteriophage and in bacteria, the single "chromosome" contains one molecule of double helical DNA which is often circular and it replicates from one point of origin in one definite direction. This has now been established both from genetical studies [6,7] and by electron microscopy and radioautography [S, 91. The term replicon has been suggested to define the unit of replication and regulation of DNA synthesis [lo] but the mechanism of replication a t the molecular level is still subject to discussion [l I , 121.In higher cells, the mode of organization of DNA in the chromosomes is still little known and open to speculation [4,13-171. The great fragility of the DNA molecules makes it difficult to estimate their number per chromosome, which is probably the requisite step to understanding their replication.Another approach is a search for unique properties of replicating DNA which would allow it to be isolated. It has been characterized as a "metastable" structure [IS-201 which is liable to appear as native Non-Standard Abbreviations. Tritiated uridine, 3H-Urd; tritiated thymidine, 3H-Thd; methylated bovine serum albumin adsorbed on kieselguhr, MAK; sodium dodecyl sulfate, SDS; ribosomal RNA, rRNA; rapidly labelled RNA, rlRNA. or denatured according to the method of preparation. The replicating structure is probably most susceptible to breakage by the action of shearing forces in bacteria [21] as well as in higher organisms [22]. I n addition, the replicating portion of DNA, perhaps because it is tightly bound with specific pr...

No abstract

The technique of laser flow cytofluorometry has been used to monitor the arrival in G1 and the subsequent progression through the cell cycle of HTC cells accumulated in metaphase with colcemid alone or after treatment with hydroxyurea and Nocodazole. Under the experimental conditions used in this study, the latter procedure gives much better results, avoiding in particular the extensive formation of micronucleated cells. Aphidicolin, an inhibitor of DNA polymerase, in combination with Nocodazole, provides a useful method to tightly synchronize these cells at the G1/S border.