The unique system of Sciara DNA puff amplification which occurs as a programmed developmental event in salivary gland giant chromosomes, offers an opportunity to investigate re-replication control, which is our long term goal. To approach this goal, we must first identify the origin of amplification, compare it the origin of the same locus that is activated just once per cell-cycle earlier in development, and study the cisacting regulatory elements. Recently we developed the method of Replication Initiation Point (RIP) mapping; application of this technique to yeast ARS1 and to Sciara DNA puff 11/9A, coupled with mapping the Origin Recognition Complex (ORC) binding site extended to eukaryotes the viral paradigm that the start site for DNA synthesis is adjacent to the binding site for the initiator protein, in this grant application we propose: (1) Identification of Cis-actinq Elements. We will extend our research to the unstudied right half of the Sciara 11/9A origin that appears to have another replication start site. In addition, we will map the origin and ORC binding site in DNA puff 11/2B,for sequence comparison with DNA puff 11/9Ato reveal conserved cis-acting sequences. (2) Functional Tests of Cis-acting Elements. We have shown that ORC binds in vitro to 80 bp in a 160 bp fragment from the 11/9Aorigin. Mutagenesis will identify sequences in the 160 bp fragment required for ORC binding, including a functional test of a 14 bp consensus sequence found both at the left and right replication start sites in the Sciara 11/9A origin. P-element transformation will provide an in vivo functional assay for putative cis-acting regulatory elements. Furthermore, mix-and-match experiments will give a functional comparison of cis-acting sequences in Sciara and Drosophila origins. A novel in vivo assay will use polyamides to block putative cis-acting elements, such as the ecdysone response element directly adjacent to the ORC binding site in the Sciara 11/9A origin, with analysis of the effect on re-replication. (3) Specification of the Initiation Zone. We have found that the Sciara 11/9A replication initiation zone (IZ) changes size during development. Chromatin ImmunoPrecipitation (CHIP) will indicate if proteins of the normal replication machinery are also present during DNA amplification, and will indicate their distribution in the initiation zone. Factors that may determine the IZ boundary will be studied by P-element transformation of an origin-containing clone with an insertion of the hsp26 promoter, where sequences important for binding RNA polymerase, a transcription factor (heat shock factor), or for nucleosome placement will be mutated in turn to analyze their effect on the IZ boundary. Information gained from the unique Sciara system should behelpful in understanding the mechanisms used by metazoa to specify an origin and to regulate its activation.