Fushi tarazu (ftz) is one of several zygotically-active genes required to establish the proper segmental pattern in Drosophila. The expression of this gene is both temporally and spatially controlled. Between 2-4 hours after fertilization the transcripts begin to accumulate in a pattern of seven stripes. It is proposed that the cis-acting ftz transcripts in the developing embryo be mapped in detail. This will be accomplished be constructing sets of 5' and 3' deletion mutations in the element known to be essential for the striped pattern of expression. The ftz promoter deletions will be fused to the E. coli beta-galactosidase gene and introduced into Drosophila embryos by P-element mediated germ line transformation. The progeny of the transformed lines will be stained for beta-galactosidase activity to determine effects of the deletions on ftz expression. The reconstruction of the striped patten of ftz expression will be attempted by the combination of the appropriate activator and repressor binding sites. The potential interaction of known zygotically active segmentation genes with the ftz zebra element will be examined. The long term goal is to identify and purify the DNA binding proteins that specifically recognize the critical cis-acting promoter elements. Once a given binding site has been mapped in vivo by the deletion experiments, oligonucleotide resins that are specific for each binding protein will be prepared, and attempts will be made to purify these proteins to homogeneity. Once this has been accomplished, a partial amino acid sequence of tryptic peptides derived from the purified preparations will be obtained and the appropriate oligonucleotide probes synthesized. The oligonucleotides will be used to screen appropriately staged cDNA and genomic libraries in an effort to clone the genes for the regulatory proteins. Monoclonal antibodies will be raised against the purified proteins and used to localize the regulatory factors during embryogenesis. Various techniques will be used to demonstrate that the correct regulatory gene has been cloned. We also hope to be able to reconstruct some of the controlling steps (activation and repression) in vitro using the cloned regulatory factor and appropriately staged embryo extracts.
| Prakash, K; Fang, X D; Engelberg, D et al. (1992) dOct2, a Drosophila Oct transcription factor that functions in yeast. Proc Natl Acad Sci U S A 89:7080-4 |
| Topol, J; Dearolf, C R; Prakash, K et al. (1991) Synthetic oligonucleotides recreate Drosophila fushi tarazu zebra-stripe expression. Genes Dev 5:855-67 |
