The nuclear location and chromatin organization of active chorion amplification origins

It remains unclear how certain regions on metazoan chromosomes are selected to initiate DNA replication. In recent years a number of origins of DNA re plication have been mapped, but there is still no DNA consensus for predict ing, where replication will initiate. Evidence suggests that the higher ord er structure of the nucleus and chromosome influences origin activity. Chro mosomal DNA replication is proposed to occur in special compartments in the nucleus called replication foci. Foci in different regions of the nucleus initiate replication at different times of S-phase, suggesting nuclear posi tion may contribute to where and when replication begins. Here we test the contribution of nuclear compartments for well-defined origins, those involv ed in amplification of the chorion (eggshell) genes during Drosophila oogen esis. The results of three-dimensional confocal microscopy indicate that ch orion DNA replication origins are highly active in diverse positions within the nucleus. We also find that chorion replication origins inserted at ect opic chromosomal sites can amplify highly in diverse nuclear locations dist inct from the endogenous loci, including when they are buffered against gen omic position effects. We used fluorescence in situ hybridization to analyz e chromosome structure during amplification. Contrary to the replication fa ctory model, we find no evidence for spooling of DNA toward a replication c enter. We discuss the implications of these results for understanding the r ole of higher order structure in amplification and chromosome duplication.