Chromosomal Digests Research Articles

Replication of DNA in the chromosomes of eukaryotes.

The evidence that each chromatid of a eukaryotic organism contains only one DNA double helix comes from a variety of observations. It begins with the autoradiographic demonstration by J. H. Taylor that tritiated thymidine, incorporated into chromosomes during one round of DNA synthesis, is present in both chromatids at the first division after labelling, but in only one chromatid after a further round of DNA synthesis accomplished in the absence of label. Further evidence comes from those experiments which demonstrate that when two sister chromatids break and fuse one with the other, each chromatid behaves as though it contained two chains of opposite polarity, fusion between chains being restricted to those of like polarity. J. G. Gall’s study of the kinetics of digestion of lampbrush chromosomes by pancreatic DNase also supports the view that each chromatid contains only two polynucleotide chains which are cleaved by this enzyme independently of one another; while O. L. Miller’s observations on the dimensions of the fibres remaining after lampbrush chromosomes have been digested by trypsin only allow for there being two polynucleotide chains per chromatid. By means of the technique of DNA fibre autoradiography devised by J. A. Huberman and A. D. Riggs, the units involved in replicating the chromosomal DNA of somatic cells ofXenopushave been compared with those ofTriturus. Both these organisms have initiation points for DNA replication that are arranged in tandem, and from each initiation point replication proceeds in opposite directions at divergent forks. The intervals between initiation points inXenopusrange from about 20 to 125µm apart, whereas those ofTriturusare much more widely separated. At 25 °C replication of DNA inXenopussomatic cells proceeds at 9µm per hour one-way at each fork, whereas the corresponding rate inTriturusis 20µm per hour.Triturussomatic cells take about 4 times longer than comparable cells ofXenopusto replicate their DNA. TheTriturusgenome contains about 10 times as much DNA as theXenopusgenome, and comparison of the replication process in these two organisms indirectly adds weight to the view that theTriturusgenome is 10 timeslongerthan that ofXenopus, rather than that it contains 10 times as many DNA double helices per chromatid. DNA fibre autoradiography has also been used to study replication inTriturusspermato-cytes. The round of DNA synthesis just before meiosis inTriturusis an exceptionally long-drawn-out process, taking 9 to 10 days for completion at 16 °C. This lengthy S-phase is not occasioned by abnormally slow replication, the rate being 12µm per hour one-way at 18 °C, nor is it the result of an exceptional staggering of replication starts. Instead it appears to be correlated with a gross reduction in the number of initiation points for replication. i.e. with an increase in the lengths of the replicating units. A rough calculation suggests that each meiotic chromomere may correspond to a unit of replication during the pre-meiotic S-phase.