The normal development and maintenance of the differentiated state of complex eucaryotic organisms results from a precisely orchestrated, differential expression of specific genes throughout ontogeny. Progress toward a detailed description of gene control in complex organisms has been impeded largely because of an inability to generate specific gene modifications and then monitor the effects of the altered gene within the temporal and spatial milieu of the developing intact organism. These problems have been overcome by recent advances in the genetic and molecular biology of Droscophila which allow the return of in vitro modified genes to the germ line of the animal for subsequent functional analysis. The urate oxidase gene-enzyme system is an ideal experimental model for identifying the nucleotide sequences responsible for controlling timing and tissue specific gene expression. Urate oxidase is detected only in the Malpighian tubules at two noncontiguous periods of development. The appearances of activity in the adult is controlled by an autonomous timing mechanism in the tubules. The level of urate oxidase activity is induced by a diffusable factor in the hemolymph and is repressed by ecdysterone, a steroid hormone, in the late third instar larva. Changes in the level of urate oxidase activity are due to differences in the quantity of urate oxidase mRNA. That the urate oxidase locus contains specific nucleotide sequences responsible for controlling the above mentioned complex pattern of gene expression is the hypothesis to be tested.
The specific aims of this project are to determine the sequences of these transcription control sites by two approaches: (1) in vitro site directed mutagenesis of the cloned urate oxidase gene and (2) the construction of hybrid genes incorporating regulatory sequences of the urate oxidase gene and the transcriptional unit of a Drosophila enzyme responsible for a basic biochemical process, adenine phosphoribosyltransferase gene (UO5'-APRT). The in vitro mutagenized urate oxidase gene and the U05'-APRT hybrid genes, using P-element vectors, will be returned to the germ line of intact animals for functional testing.
| Friedman, T B; Burnett, J B; Lootens, S et al. (1992) The urate oxidase gene of Drosophila pseudoobscura and Drosophila melanogaster: evolutionary changes of sequence and regulation. J Mol Evol 34:62-77 |
| Wallrath, L L; Friedman, T B (1992) Combinative oligonucleotide-directed large deletions as a strategy for surveying the regulatory region of a gene. Biotechniques 12:214-6 |
| Friedman, T B; Owens, K N; Burnett, J B et al. (1991) The faint band/interband region 28C2 to 28C4-5(-) of the Drosophila melanogaster salivary gland polytene chromosomes is rich in transcripts. Mol Gen Genet 226:81-7 |
| Wallrath, L L; Friedman, T B (1991) Species differences in the temporal pattern of Drosophila urate oxidase gene expression are attributed to trans-acting regulatory changes. Proc Natl Acad Sci U S A 88:5489-93 |
