We propose to analyze initiation of DNA replication in DNA puff II/9A of Sciara salivary gland polytene chromosomes because it is the only non-viral, metazoan eukaryotic origin unambiguously mapped to a small, well-defined region (less than 1 kb). We will address the following questions that have never been answered for any eukaryotic cellular origin: (1) What activates an origin to fire? (2) What control mechanism exists to ensure that an origin will fire not more than once per S phase? We will complete our studies on the structure of the II/9A origin: (a) definition of the boundaries of the II/9A origin and how it changes during development, (b) identification of replication initiation points (mapped to the nucleotide level), and (c) identification of cis-acting elements that regulate II/9A origin activity (by chromatin studies and P-element transformation). Our major thrust will be to identify proteins that associate with the II/9A origin and are important for control of initiation of DNA replication and prevention of rereplication. We will define the binding sites of the origin recognition complex (ORC) with the II/9a origin, having isolated the ORC2 polypeptide and raised antibodies against it. This will be the first case where ORC is demonstrated to bind to a specific DNA sequence in any metazoan. Also, we will investigate if ORC binds to a consensus sequence in the genome, which is currently unknown for any metazoan. Whether all ORC binding sites function as active ORIs in Sciara will be explored by PCR analysis of nascent strands and by double immunofluorescence of polytene chromosomes. Finally, we will identify by yeast one hybrid screens other proteins that bind the II/9A origin in a stage specific manner, differing between pre-amplification to amplification stages, suggesting a role for such proteins in rereplication control. Yeast two hybrid screens will identify interactions of these proteins with others, such as ORC. Cdc6 is an important component associated with ORC, and it appears to transmit cell cycle controls to ORC. We will clone Sciara Cdc6 and use it for a yeast two hybrid screen to identify interacting proteins that change from pre- amplification to amplification stage.