Mapping an initiation region of DNA replication at a single-copy chromosomal locus in Drosophila melanogaster cells by two-dimensional gel methods and PCR-mediated nascent-strand analysis: multiple replication origins in a broad zone.
We have mapped an initiation region of DNA replication at a single-copy chromosomal locus in exponentially proliferating Drosophila tissue culture cells, using two-dimensional (2D) gel replicon mapping methods and PCR-mediated analysis of nascent strands. The initiation region was first localized downstream of the DNA polymerase alpha gene by determining direction of replication forks with the neutral/alkaline 2D gel method. Distribution of replication origins in the initiation region was further analyzed by using two types of 2D gel methods (neutral/neutral and neutral/alkaline) and PCR-mediated nascent-strand analysis. Results obtained by three independent methods were essentially consistent with each other and indicated that multiple replication origins are distributed in a broad zone of approximately 10 kb. The nucleotide sequence of an approximately 20-kb region that encompasses the initiation region was determined and searched for sequence elements potentially related to function of replication origins.
Shinomiya
T
T
Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
Ina
S
S
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D004261:DNA Replication / Q000235:genetics*; D004331:Drosophila melanogaster / Q000235:genetics*; D015180:Electrophoresis, Gel, Two-Dimensional; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D018741:Replication Origin / Q000235:genetics*; D015183:Restriction Mapping
D004247:DNA