It is well known that DNA replication decreases significantly during senescence in higher organisms. The mechanism(s) underlying this decrease are unknown. Bacterial plasmids provide an easily manipulated experimental system that is suitable for studying the control of DNA replication. Prokaryotic systems have consistently provided crucial insights that are directly applicable to the functioning of eukaryotic cells. I believe that the proposed study of plasmid replication will greatly further our understanding of the mechanisms by which replication is altered during aging. I propose to analyze the region of the resistance plasmid pSC101 that is essential for plasmid replication. My goals are (1) the identification of specific genes and sites that are essential for control of plasmid DNA replication and (2) the delineation of the mechanisms by which this control of replication is effected. The proposed research combines analysis of DNA, RNA, and polypeptides with the isolation and characterization of mutant plasmids. The combination of these techniques will allow me to examine the structure of regions of DNA critical for the control of replication. For example, I have isolated several mutant pSC101 plasmids, each with a characteristic level of increased copy number. I will use these mutant plasmids to identify alterations in DNA sequence and will correlate these alternations in sequence to changes in structure and function of polypeptides and RNA's. In this way, I will characterize the mechanisms by which copy number is altered and maintained at different levels. Similar procedures will be used to probe the molecular basis of aspects of pSC101 plasmid replication in addition to copy number.