Functional analysis of dMed6 in development of drosophila melanogaster

Abstract

A multi-protein Mediator complex consisting RNA polymerase II holoenzyme is required for both general and regulated transcription in Saccharomyces cerevisiae and higher eukaryotes. A Mediator of higher Eukaryotes like Drosophila and human is consisted of a subset of proteins conserved within metazoans and the other subset of proteins that are conserved throughout development. The MED6 is one of the evolutionary conserved subunit of Mediator complex and required for the regulation of about 10% of whole yeast genome (DNAchip data from yeast, Holstege et al., 1998). To identify the function of the mediator in higher eukaryotic developmental regulation, we clone dMed6, a Drosophila homolog of yeast and human Med6. dMED6 protein was localized in most cell nuclei of Drosophila embryo and ovary. The dMed6 proteins were associated with high-molecular weight complex as yeast and human counterparts and coimmunoprecipitated with other Mediator component. dMed6 mutants were isolated from F2 lethal screening from random mutagenized lethal mutation in dMed6 locus. $dMed6^{26}$ had splicing junction mutation result in truncation of protein and $dMed6^{64}$ had 5 bp deletion in exon 2 also result in truncation. The $dMed6^{26/26}$ could develop to larva like wild type. However, their development were delayed and most of them died as a $3_rd$ instar larva whereas wild type isogenic line could develop to pupa and adult. In fact, the dMED6 protein that was maternally provided was totally disappeared 3 days after egg laying (AEL). That indicated $dMed6^{26}$ is a null mutation. Yeast flipase and recognition sequence (FLP-FRT) mediated clonal analysis on eye and wing imaginal disc showed that $dMed6^{26/26}$ clone could not undergo proliferation. Clonal analysis on ovary shows inappropriate follicle and germ cell division. $dMed6^{26/26}$ expression profile of 192 genes showed that genes required for cell proliferation (cdc2, son of sevenless, and rolled), metabolism (glutamine syn...