The overall objective of this proposal is to gain understanding of how genes are regulated in a temporal and tissue-specific manner during development. The CyI actin gene of the sea urchin, Strongylocentrotous purpuratus, exhibits a unique pattern of expression for each of several cell types of the embryo, hence it is an excellent model for the study of gene regulation. The DNA sequence elements required for the ontogenic regulation of the CyI actin gene will be identified. A series of deletions in the sequences surrounding the 5' cap site of the gene will be made by digestion with the exonuclease Bal 31 using a plasmid that contains the CyI actin gene 5' flanking sequences fused to the gene for the bacterial enzyme. Chloramphenicol acetyltransferase (CAT). Each of the different plasmid constructs will be tested for expression in the sea urchin transient expression system. The spatial pattern of expression for the various CyI-CAT fusion gene constructs will be determined by using the technique of in situ hybridization. Nuclear extracts will be prepared from 17 hr embryos and tested for the presence of specific DNA binding factors to the CyI actin regulatory sequences by the DNAse I footprinting assay. The binding of any detected factors will be correlated with transcriptional activation of the CyI gene by generating single-base pair mutations in the binding domain by oligonucleotide-directed mutagenesis and testing each mutation for expression and binding. Nuclear extracts will then be prepared from embryos prior to CyI gene activation and tested for the presence of CyI DNA binding factors using DNAase I footprinting. With this information it should be possible to formulate a general model for regulation of CyI expression. An attempt will be made to purify the specific DNA binding factors definitively shown to be involved in the ontogenic regulation of CyI expression using sequence-specific DNA affinity chromatography. knowledge of how genes are differentially activated is not only of fundamental biological importance but is important for understanding the process by which cells become transformed. In addition successful implementation of gene therapy will require an understanding of the DNA sequence elements needed for correct regulation and expression.
