The bithorax complex in Drosophila melanogaster is a cluster of genes controlling segmental differentiation in the fly. Mutations in the complex are "homeotic"; they cause transformations of one segment into another. Our studies of the locus aim to discover the molecular mechanisms of such switches in development. The locus is quite large; mutant lesions are spread over 300 kb along the chromosome. Surprisingly few protein products have been found encoded within the 300 kb. We are looking for additional products, and working to understand the functions of those in hand. The protein coding regions are apparently flanked by cis-acting DNA elements which regulate the cell specificity of protein expression. We hope to separate out these elements, and to understand the higher order arrangement of these elements on the chromosome. We have worked together with E. B. Lewis to define mutant lesions on the DNA, and to correlate lesion with phenotype. This collaboration will continue, but the lab has adopted a variety of new approaches. Some techniques focus on the protein products. New cDNA clones are being sequenced; they show that the initial RNA transcripts can be spliced in different ways. Point mutations are being mapped in and around the known mRNA exons, using gradient denaturing gels. One protein product is being synthesized in-vitro; it will be tested for binding activity to specific DNA sites within the complex. Other experiments address the spatial regulation of expression. We are using antibodies to describe protein expression cell by cell in early embryos. The description will be repeated in mutant embryos, where the mutations remove putative regulatory elements. Potential regulatory elements can be tested by P element transformation. Fragments of bithorax DNA will be cloned into a P element next to a small marker gene. Vectors and techniques have also been developed to build P elements with very large insertions of Drosophila DNA. These P elements could carry intact the large transcription domains of the complex.