The regulation of DNA replication in animal cells is of utmost importance for understanding cell growth and transformation. In order to study this process, the investigator has developed a novel assay which measures replication directions and allows one to define, for the first time, chromosomal replicons by identifying both origins and termination sites. Since the origin element appears to remain functional after integration into the genome, reverse genetics can be used in conjunction with transfection to characterize the cis acting elements required for DNA synthesis. The human beta-globin domain will serve as a model system to detect origins and determine any possible relationship between replication and gene expression in different cell types. This transfection approach is also suitable for examining the mechanism of action of chromosomal terminators and their function will be verified on specially constructed replicating SV-40 vectors in vivo. Initial studies on individual gene regions suggest a relationship between the replicon unit and the chromatin boundaries of the DNase I sensitive domain. In these cases, termination appears to take place at or near the matrix attachment region which serves as anchors for chromosomal loops. DNA replication is also temporally regulated in animal cells and some specific genes are late replicating and transcriptionally inactive in a tissue specific manner. In order to learn more about this process, the investigator has designed a new approach for measuring replication timing of individual genes which involves in situ hybridization to nuclei. This method is extremely simple to perform and can be used on any cell type in culture and even on dividing cells in vivo, thus making it possible to determine how replication is developmentally regulated. The cystic fibrosis chromosomal domain will be used as a model system for evaluating the extent of the genomic unit which undergoes replication timing control.
