Mapping of replication initiation sites in mammalian genomes by two-dimensional gel analysis: stabilization and enrichment of replication intermediates by isolation on the nuclear matrix.

摘要: Two complementary two-dimensional gel electrophoretic techniques have recently been developed that allow initiation sites to be mapped with relative precision in eukaryotic genomes at least as complex those of yeast and Drosophila melanogaster. We reported the first application these mapping methods a mammalian genome study on amplified dihydrofolate reductase (DHFR) domain methotrexate-resistant CHO cell line CHOC 400 (J.P. Vaughn, P.A. Dijkwel, J.L. Hamlin, Cell 61:1075-1087, 1990). Our results suggested this 240-kb domain, nascent DNA strands occurs many within 30- 35-kb zone immediately downstream from DHFR gene. In course studies, it was necessary develop stabilize replication intermediates against branch migration shear. This report describes stabilization detail presents new enrichment protocol extends neutral/neutral method single-copy loci cells. Preliminary analysis purified cells by suggests synthesis may initiate broad locus well.