Specification of regions of DNA replication initiation during embryogenesis in the 65-kilobase DNApol alpha-dE2F locus of Drosophila melanogaster

In the early stage of Drosophila embryogenesis, DNA replication initiates a t unspecified sites in the chromosome. In contrast, DNA replication initiat es in specified regions in cultured cells. We investigated when and where t he initiation regions are specified during embryogenesis and compared them with those observed in cultured cells by two-dimensional gel methods. In th e DNA polymerase alpha gene (DNApol alpha) locus, where an initiation regio n, oriD alpha, had been identified in cultured Kc cells, repression of orig in activity in the coding region was detected after formation of cellular b lastoderms, and the range of the initiation region had become confined by 5 h after fertilization. During this work we identified other initiation reg ions between oriD alpha and the Drosophila E2F gene (dE2F) downstream of DN Apol alpha. At least four initiation regions showing replication bubbles we re identified in the 65-kb DNApol alpha-dE2F locus in 5-h embryos, but only two were observed in Kc cells. These results suggest that the specificatio n levels of origin usage in 5-h embryos are in the intermediate state compa red to those in more differentiated cells. Further, we found a spatial corr elation between the active promoter regions for dE2F and the active initiat ion zones of replication. In 5-h embryos, two known transcripts differing i n their first exons were expressed, and two regions close to the respective promoter regions for both transcripts functioned as replication origins. I n Kc cells, only one transcript was expressed and functional replication or igins were observed only in the region including the promoter region for th is transcript.