This is a request for four years of support to examine replication of mammalian chromosomes with a focus upon X-inactivation and genomic imprinting. Two broad specific aims are listed. The first is to map replication timing domains utilizing FISH to interphase nuclei. Replication can be observed by the appearance of two adjacent signals per chromosome and the synchrony of homologous regions determined by whether or not the second signal appears jointly or at different times. The probes will be YAC and cosmid clones from two regions of the X chromosome (the ZFX-POLA region and the XIST region) and a portion of chromosome 15 involved in the imprinting disorders of Prader-Willi and Angelman syndromes. YACs of the region will be converted to cosmid contigs and used to achieve a high resolution analysis to demarcate replication domain boundaries. The origin would be thought to lie between such boundaries and it is hoped this can be reduced to a region of several kb.
The second aim will be to characterize and compare these interdomain boundaries. Several approaches are proposed. Scanning for nascent strand template switching will be accomplished by inhibiting Okazaki fragment synthesis while pulsing with BrdU. The leading strand can be isolated and characterized by single-strand probes of the region. Another approach is 2D gel electrophoresis to isolated replication intermediates for characterization. Finally, characterization of interdomain boundaries in autonomously replicating constructs.
