Electron microscopic analysis of DNA replication in main band and satellite DNAs of Drosophila virilis

Abstract

DNA was isolated from pre-blastoderm embryos of Drosophila virilis and fractionated by CsCl centrifugation. Fractions containing main band or satellite DNAs were examined by electron microscopy for replicating structures. Replication of main band DNA proceeded from many sites on a chromosome. Two classes of presumptive replicating molecules with more than one site of replication were seen in main band DNA. The first class contained one or more clusters of small eyes (all eyes less than or equal to 900 bases in size), whereas the second class was comprised of molecules with eyes of larger sizes. Many cluster-to-cluster distances in molecules in the first class and many eye-to-eye distances in molecules in the second class were equal to about 4000 bases or multiples thereof. These data are consistent with the following model. Replication in main band DNA is initiated by activation of 2 to 12 closely spaced origins. These clusters of origins are spaced at regular intervals along the chromosome and eventually fuse to produce larger eyes which are also spaced in a regular manner. Regular spacing of initiation sites is true for at least short stretches of the chromosome. However, it is possible that these regularly spaced sites are themselves part of a larger pattern of chromosome organization. Like the replication of main band DNA, that of satellite DNA proceeded, at least in part, by a chromosomal fork mechanism. However, linear replicating molecules were about five times less common in satellite than in main band DNA. As in main band DNA, single-stranded regions at fork junctions were small in comparison to those described in prokaryotes. There was no evidence for either clustering of initiation sites or regular spacing of eyes in satellite DNA. Thus, the organization of initiation sites appeared different in the two classes of DNA. In addition to conventional linear replicating molecules, structures were seen in satellite DNA which could be involved in extrachromosomal replication of some satellite sequences. However, these structures could also be explained by breakage of replicating molecules followed by reassociation of single-stranded regions.