title: A broad replication origin of Drosophila melanogaster, oriDalpha, consists of AT-rich multiple discrete initiation sites.
abstract: This work shows that the replication origin of Drosophila melanogaster, oriDalpha, consists of multiple discrete initiation sites. We attempted to map at high resolution the initiation sites in oriDalpha with a quantitative nascent DNA abundance assay using a competitive polymerase chain reaction (PCR) method. Nascent DNA was prepared from either cells blocked in very early S-phase and then labeled with 5-bromo-2'-deoxyuridine (BrdU), or asynchronously growing cells labeled briefly with BrdU. Denatured DNA was size-fractionated in alkaline sucrose gradients. BrdU-labeled nascent DNA was immuno-affinity purified using anti-BrdU antibodies. DNA was quantified with a competitive PCR method before and after immuno-purification. The results indicated that oriDalpha, whose size was presumed to be about 10 kb from two-dimensional gel electrophoretic analysis, contained four major initiation sites in its central 2.8 kb region, and six to approximately eight sites in 8.4 kb. All initiation sites corresponded with AT-rich sequences. Detailed analysis of one major initiation site indicated that its range was restricted to 700 bp.
authors: Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo, Japan. sawako@libra.ls.m-kagaku.co.jp
authors: S
authors: S
authors: Ina
authors: T
authors: T
authors: Sasaki
authors: Y
authors: Y
authors: Yokota
authors: T
authors: T
authors: Shinomiya
mesh_terms: D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004331:Drosophila melanogaster / Q000235:genetics*; D016133:Polymerase Chain Reaction; D018741:Replication Origin; D016196:S Phase
chemical_list: D017931:DNA Primers