Combinatorial mechanisms of repression involving the gap protein giant. L..P.M. Andrioli , X. Wu , T. Schinecker , S. Small. Biology, New York University, New York, NY.

   The repressor activity of the gap gene giant (gt) is required in anterior regions of the developing embryo. At the pair-rule level, gt is involved in the formation of the anterior border of even-skipped stripe 2 (eve st2). Target misexpression of gt suggests however, that its activity is not sufficient for efficient repression of the stripe 2 response. Since gt is a leucine zipper protein, we are testing the hypothesis that a cofactor heterodimerizes with gt in order to create an effective repressor complex. For this, we used gt and 2 fragments of gt that contain the leucine zipper as baits to screen an embryonic cDNA library in a two-hybrid assay. We isolated 28 positive clones that are now being characterized. We are also focusing on a 44bp complex binding site in the stripe 2 enhancer that is critical for formation at the anterior border. This site binds gt protein weakly, but may contain sites for a putative repressor. To test this, we have generated transgenic lines with gt2-lacZ reporter gene that contain small deletions within the 44bp site. Patterning changes caused by these deletions will be discussed. If any of these constructs show an expansion of the anterior border, the deleted regions will be used in in vitro assays to identify proteins that bind these sites. These studies will increase our understanding of combinatorial repression mechanisms involved in early development.