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Molecular and Cellular Biology, January 1999, p. 547-555, Vol. 19, No. 1
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Specification of Regions of DNA Replication Initiation during Embryogenesis in the 65-Kilobase DNApolalpha -dE2F Locus of Drosophila melanogaster

Takayo Sasaki,1,dagger Tomoyuki Sawado,2,3 Masamitsu Yamaguchi,2 and Tomoyuki Shinomiya1,*

Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo 194-8511,1 Laboratory of Cell Biology, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya 464-8681,2 and Department of Applied Biological Science, Faculty of Science and Technology, Science University of Tokyo, Noda-shi, Chiba-ken 278-8510,3 Japan

Received 13 July 1998/Returned for modification 17 September 1998/Accepted 19 October 1998

In the early stage of Drosophila embryogenesis, DNA replication initiates at unspecified sites in the chromosome. In contrast, DNA replication initiates in specified regions in cultured cells. We investigated when and where the initiation regions are specified during embryogenesis and compared them with those observed in cultured cells by two-dimensional gel methods. In the DNA polymerase alpha  gene (DNApolalpha ) locus, where an initiation region, oriDalpha , had been identified in cultured Kc cells, repression of origin activity in the coding region was detected after formation of cellular blastoderms, and the range of the initiation region had become confined by 5 h after fertilization. During this work we identified other initiation regions between oriDalpha and the Drosophila E2F gene (dE2F) downstream of DNApolalpha . At least four initiation regions showing replication bubbles were identified in the 65-kb DNApolalpha -dE2F locus in 5-h embryos, but only two were observed in Kc cells. These results suggest that the specification levels of origin usage in 5-h embryos are in the intermediate state compared to those in more differentiated cells. Further, we found a spatial correlation between the active promoter regions for dE2F and the active initiation zones of replication. In 5-h embryos, two known transcripts differing in their first exons were expressed, and two regions close to the respective promoter regions for both transcripts functioned as replication origins. In Kc cells, only one transcript was expressed and functional replication origins were observed only in the region including the promoter region for this transcript.

* Corresponding author. Mailing address: Mitsubishi Kasei Institute of Life Sciences, 11 Minami-oya, Machida, Tokyo 194-8511, Japan. Phone: 81-427-24-6251. Fax: 81-427-24-6317. E-mail: tomo{at}libra.ls.m-kagaku.co.jp.

dagger Present address: Horikoshi Gene Selector Project, ERATO, JST, Tsukuba, Ibaraki 300-2635, Japan.

Molecular and Cellular Biology, January 1999, p. 547-555, Vol. 19, No. 1
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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