Molecular genetic analysis of glucose repression in Drosophila.

Abstract

The research described in this thesis focuses on glucose repression of gene expression in a higher eukaryotic organism, the fruit fly Drosophila melanogaster. The results show that DNA sequences which are located upstream of the transcribed region of the amylase gene (Amy) control glucose repression in D. melanogaster larvae. Hybrid constructs, in which the upstream flanking sequences of the amylase gene were fused with the transcribed region of the alcohol dehydrogenase gene (Adh), were expressed in transgenic $Adh\sp{\rm null}$ larvae. The expression of ADH from the hybrid gene was found to be subject to glucose repression. The function of potential regulatory cis-acting elements within the glucose responsive upstream region of the amylase gene was examined by deletion analysis and site-directed mutagenesis, coupled with expression assays in somatically transformed $Amy\sp{\rm null}$ larvae. The upstream deletion analysis showed that essential elements, both for overall activity and glucose repression of the amylase gene, are located close to the transcription start site (within 109 base pairs). In vitro site-directed mutagenesis of upstream sequences revealed that the TATA motif, at position $-$28, and a novel 35 base pair element, at position $-$109 to $-$74 with respect to the transcription start site, were necessary for overall activity of the amylase promoter. None of the introduced mutations, which scanned the upstream regulatory region of the amylase gene, resulted in the loss of glucose responsiveness. These results suggest that glucose repression, in Drosophila, is mediated by transcriptional mechanisms which involve multiple functionally redundant DNA elements. Variation in the degree of glucose repression of the amylase genes was examined at a molecular level in D. melanogaster. Gene-specific elements located within a short upstream DNA region were shown to regulate the difference in response to dietary glucose between duplicated amylase genes. In addition, in order to assess the effect of interspecific variation of regulatory DNA sequences on glucose repression, the function of glucose repressible amylase promoters was examined in distantly related species. The D. virilis amylase gene was shown to be repressed by dietary glucose in somatically transformed D. melanogaster larvae. (Abstract shortened by UMI.)