UBC Theses and Dissertations
Analysis of protein products encoded by the polyhomeotic locus, a member of the polycomb-group of genes in drosophila melanogaster DeCamillis, Mark Andrew
Polyhomeotic (ph) belongs to the Polycomb-Group (PcG) of genes in Drosophila melanogaster. 12 PcG genes have so far been described, originally identified as trans-regulatory repressors of homeotic gene expression. PcG genes interact in multiple heterozygous combinations to mutually enhance gain of function homeotic phenotypes. In addition to homeotic effects, many PcG genes including ph exhibit pleiotropic phenotypes. Two models have been proposed to explain PcG interactions and function: 1) PcG genes form a hierarchical regulatory network which functions to elaborate the correct expression of a subset of members required at specific regulatory targets; 2) PcG members constitute single components of a multimeric protein complex that functions at specific regulatory targets. To further the molecular characterization of ph protein products and better understand its role in the PcG, I have produced a mono-specific polyclonal antiserum to ph proteins. Using this reagent I show that at least 4 distinct ph protein products are produced that are differentially expressed during development. Several correlations between ph phenotypes and ph protein expression are made. As expected from conceptual translation of a ph cDNA, I show that ph proteins are localized in the nucleus. Further analysis shows that ph is a chromatin binding protein that recognizes 80 specific sites on the polytene chromosome including locations of the homeotic gene complexes and several PcG genes. Comparison of ph and. Polycomb binding sites show a striking degree of overlap in which 71 of 80 ph sites are indistinguishable from Pc sites. I show that ph recognizes ectopically located bxd sequences (an upstream regulatory element in the Ubx gene of the bithorax complex) also recognized by Pc, indicating that ph localization is sequence dependent. Co-localization of ph and Pc throughout the genome and at bxd sequences suggest that these proteins either interact directly or function in close proximity at similar target genes. Co-immunoprecipitation experiments done on nuclear extracts show that ph and Pc proteins are present in the same irnmunoprecipitates, providing the first evidence that they associate in a multimeric complex.
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