The replication of chromosomal DNA in mammalian cells is subject to several levels of temporal and spatial control. It has been estimated that there are 10-4 or more origins of replication in the genomes of higher eucaryotes, which are activated at specific times during S-phase, and it has been proposed that the control of replication timing could play an important role in the control of genetic activity. One of the most obvious changes in replication patterns is associated with X-chromosome inactivation where, early in embryogenesis, one of the X chromosomes in female cells becomes transcriptionally inactive and simultaneously becomes late-replicating relative to the remaining active copy. The overall goal of this work is to evaluate the significance of this change in replication timing and to examine potential mechanisms for control of the event. This will first involve structural and functional characterization of a potential replication origin from within the X-linked human hypoxanthine phosphoribosyltransferase (HPRT) gene that has been found to function as an antonomously replicating sequence (ars) in yeast cells. Activity in promoting replication of recombinant plasmids in transfected mammalian cells will be examined, as will other potentially important properties, including nuclear matrix association and specific interactions with nuclear proteins. If these assays fail to support the authenticity of the ars element as an origin of replication, similar studies will be conducted with an extensive collection of subcloned fragments from the HPRT locus to identify a bone fide replication origin. Two independent approaches to localize such a sequence by direct analysis of properties of the HPRT locus in its normal chromosomal context will also be sued. These will involve examination of nucleosome segregation patterns under conditions of protein synthesis inhibition and determination of replication polarity by in vitro replication studies with isolated nuclei. In addition, a set of hamster/human somatic cell hybrids (containing either an active or an inactive human X chromosome or derivatives of the latter where the human HPRT gene on the inactivated chromosome has been reactivated by 5azacytidine treatment) will be used to examine the correlation between gene expression and replication timing and also to compare properties of putative replication origins on the active and inactive chromosomes to try to identify factors involve in their differential replication control.
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