Title

An analysis of transcription factor binding sites within histone non-coding sequences in Drosophila

Date of Completion

January 2003

Keywords

Biology, Genetics

Degree

Ph.D.

Abstract

A global objective in the study of Drosophila histone genes is to decipher the mechanism by which they are regulated and evolve. Yet before regulatory mechanisms can be investigated, the identity of transcription factors involved in the regulation of these genes must be uncovered. Therefore an immediate goal is to identify transcription factor binding sites within the non-coding region of H2A and H2B genes as an initial step in deciphering the mechanism by which all Drosophila histone genes are regulated. ^ The approach undertaken to identify transcription factor binding sites within the H2A-H2B intergenic spacer was in silico analysis, experimentation that utilizes bioinformatics or the computer based analysis of biological data. The intergenic spacer for H2A and H2B genes was subjected to various bioinformatic search tools that are capable of sequence comparisons and identification of transcription factor consensus binding sites. Through resulting data an organizational model of transcription factor binding sites was generated for the H2A-H2B spacer. Empirical investigation of the model was carried out using both phylogenetic and biochemical analyses. The evolutionary conservation of the putative transcription factor binding sites within the organizational model for the H2A-H2B spacer was analyzed using organizational models that were created for fourteen additional, widely diverged Drosophila species. Common to all organizational models across long evolutionary distances was a centrally located putative binding site for the yeast transcription factor Repressor Activator Protein-1 (Rap1p). ^ Investigation of the functionality of the putative binding site for Rap1p entailed a search for a Drosophila counterpart to the yeast transcription factor. A PCR based strategy was used in this search, which resulted in the identification of the Drosophila mismatch repair protein, Spellchecker1. Further investigations are required to establish the precise association between Spellchecker1 and histone genes and to determine whether Spellchecker1 can be positively identified as the Drosophila counterpart to yeast Rap1p. ^