Our long-term objectives are to understand the mechanism of initiation at mammalian origins of replication, how the temporal order of activation of replicons in the genome is controlled, and how the structural organization of replicons in the nucleus facilitates the DNA synthetic process. As a model system, we have developed a methotrexate (MTX)-resistance CHO cell line (CHOC 400) that has amplified the dihydrofolate reductase (DHFR) gene and flanking sequences -1,000 times. The entire 240 kb repeating unit (amplicon) has been cloned in overlapping cosmids. We have shown that replication initiates downstream from the DHFR gene at two clustered sites (ori-beta and ori-gamma) separated by -20 kb that straddle a matrix attachment region (MAR). An additional initiation locus (ori-alpha) has been identified -200 kb upstream from ori-beta in the 550 kb amplicon of another MTX-resistant cell line. The specific aims of the proposed project are: 1) to compare the initiation reactions at ori-alpha, ori-beta, and ori-gamma by a two-dimensional gel electrophoretic method, in order to determine whether all chromosomal origins of replication contain two clusters of initiation sites flanking a MAR; 2) to determine whether cosmids containing ori-alpha, ori-beta and/or ori-gamma are capable of replicating autonomously or directing initiation from a new chromosomal location after transfection into CHO cells; if so, the cosmid inserts will be systematically mutagenized to determine which cis-acting elements are required for initiation; we will also determine whether the introduction of the MAR into clones containing ori-alpha, ori-beta, or ori-gamma alone converts these clones to autonomously-replicating sequences; 3) to develop strategies for identifying and purifying specific DNA binding proteins for subfragments from origin regions; appropriate fragments will be footprinted with crude cell extracts to delimit potential sequences of interest within the origin regions; hybridomas will be prepared against DNA binding proteins obtained by a preparative column method, and their antibodies will be screened with appropriate radiolabelled fragments saturated with specific DNA binding proteins.