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.
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 as those of yeast and Drosophila melanogaster. We reported the first application of these mapping methods to a mammalian genome in a study on the amplified dihydrofolate reductase (DHFR) domain of the methotrexate-resistant CHO cell line CHOC 400 (J.P. Vaughn, P.A. Dijkwel, and J.L. Hamlin, Cell 61:1075-1087, 1990). Our results suggested that in this 240-kb domain, initiation of nascent DNA strands occurs at many sites within a 30- to 35-kb zone mapping immediately downstream from the DHFR gene. In the course of these studies, it was necessary to develop methods to stabilize replication intermediates against branch migration and shear. This report describes these stabilization methods in detail and presents a new enrichment protocol that extends the neutral/neutral two-dimensional gel mapping method to single-copy loci in mammalian cells. Preliminary analysis of replication intermediates purified from CHO cells by this method suggests that DNA synthesis may initiate at many sites within a broad zone in the single-copy DHFR locus as well.
Dijkwel
P A
PA
Department of Biochemistry, University of Virginia School of Medicine, Charlottesville 22908.
Vaughn
J P
JP
Hamlin
J L
JL
D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D002846:Chromatography, Affinity; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification / Q000648:ultrastructure; D004261:DNA Replication; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D005796:Genes; D007700:Kinetics; D015530:Nuclear Matrix / Q000502:physiology* / Q000648:ultrastructure; D010053:Ovary; D015183:Restriction Mapping; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*
D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase