The long-term objective of the proposed research is to discover how the functions of specific genes of Drosophila melanogaster are integrated into the overall program of imaginal disc determination. The normal determined state of an imaginal disc can be transformed to that of some other imaginal disc either by transdetermination or by homeotic mutations. These transformations reveal an underlying regulatory network which connects the determined states of different imaginal discs. Three sets of genes give rise to homeotic mutations; the segment-specific genes, the trithorax set of genes and the polycomb set of genes. The segment-specific genes include those of the bithorax and Antennapedia complexes. Mutations in these genes cause transformations of specific segments and/or specific imaginal discs. Most of the segment-specific genes have been cloned and, at least, partially sequenced. The polypeptide products of each of those genes are expressed predominantly in different segments. Mutations in the trithorax set and in the polycomb set of genes cause abnormal patterns of expression of the segment-specific homeotic genes. Normal determination requires the expression of these two sets of genes in all segment and/or imaginal discs. There are two aspect of the trithorax and polycomb sets of genes that are especially significant. First, there are many genes in each set which implies that the products of many genes must be integrated to perform their function. Second, the phenotype caused by a trithorax mutation can be suppressed by a polycomb mutation and vice versa implying that normal determination requires a balance between the products of the trithorax set and the polycomb set of genes. Two genes of the trithorax set (ash-1 and ash-2) and one gent of the polycomb set (polycombeotic) have been studied in this lab. The goal of this proposal is to understand at the molecular level how these genes regulate segment-specific homeotic genes. There are four specific aims. (1) Clone the ash-1, ash-2, and polycombeotic genes by transposon tagging. Examine the temporal and tissue-specific patterns of transcription of the genes, isolate cDNAs for the transcript(s) of the genes and determine the nucleotide sequences of those cDNAs; (2) Use antibodies raised against fusion polypeptides to identify the polypeptide product(s) of the genes and determine the sub-cellular localization of those products: (3) Use the antibodies to examine the interactions of these three genes with each other and with other genes of the trithorax and polycomb sets; (4) Analyze the consequences of mutations in the ash-1, ash- 2, and polycombeotic genes on the expression of segment-specific homeotic genes using the molecular probes for those genes that are available. Transdetermination leads to the same transformations as those caused by homeotic mutations; it occurs when the proliferative phase of mature imaginal discs is extended by wounding and subsequent culture. An understanding of the regulator network which maintains the determined state should help to explain how such perturbations of normal cells can cause alterations of the determined state.
