IDENTIFYING SITES OF REPLICATION INITIATION IN YEAST CHROMOSOMES - LOOKING FOR ORIGINS IN ALL THE RIGHT PLACES

DNA fragments that contain an active origin of replication generate bu bble-shaped replication intermediates with diverging forks. We describ e two methods that use two-dimensional (2-D) agarose gel electrophores is along with DNA sequence information to identify replication origins in natural and artificial Saccharomyces cerevisiae chromosomes. The f irst method uses 2-D gels of overlapping DNA fragments to locate an ac tive chromosomal replication origin within a region known to confer au tonomous replication on a plasmid. A variant form of 2-D gels can be u sed to determine the direction of fork movement, and the second method uses this technique to find restriction fragments that are replicated by diverging forks, indicating that a bidirectional replication origi n is located between the two fragments. Either of these two methods ca n be applied to the analysis of any genomic region for which there is DNA sequence information or an adequate restriction map.