ABSTRACT
The Resistance to dieldrin (Rdl) gene encodes a novel subunit of a gamma-aminobutyric acid (GABA)-gated chloride ion channel in Drosophila. We were interested in defining the spatial and temporal expression pattern of this gene and in understanding the basis of its regulation. Rdl is expressed in both the embryonic central and peripheral nervous system. Here, we describe the complete Rdl transcription unit (approximately 50 kb) via localization of the flanking transcripts. The Rdl transcript itself is large (8.8 kb) and is composed of a short open reading frame (2 kb) with exceptionally long 5' (1.8-kb) and 3' (5-kb) untranslated regions (UTRs). The correct spatial and temporal expression of Rdl can be rescued by transformation constructs containing only 3.5 kb of DNA, a region which encompasses the transcription start point (tsp). This region also contains sequences strikingly similar to those found in other ion channel gene promoters. Failure of minigene constructs lacking the long 3' UTR to fully rescue both the lethal and resistance phenotypes associated with the Rdl locus might arise owing to the role of these sequences in message stability or trafficking.
Subject(s)
Dieldrin/pharmacology , Insecticide Resistance/genetics , Insecticides/pharmacology , Receptors, GABA/drug effects , Receptors, GABA/genetics , Animals , Base Sequence , Blotting, Northern , Cloning, Molecular , Drosophila melanogaster , Genes, Reporter , In Situ Hybridization , Molecular Sequence Data , RNA, Messenger/biosynthesis , Regulatory Sequences, Nucleic Acid , Transcription, GeneticABSTRACT
A single point mutation within the GABA receptor gene Resistance to dieldrin (Rdl) confers a high level of resistance to cyclodiene insecticides in a wide range of insects. Previous studies have shown partial rescue of the susceptible phenotype via germline transformation of a 36 kb cosmid coding (or all four alternative Rdl splice forms. Here, we describe the construction of two Rdl promoter/cDNA minigenes, each coding for one of the splice forms alone. Single splice forms rescued both the insecticide susceptible and resistant phenotypes associated with the locus as effectively as the complete cosmid. The minigenes also rescue the lethality associated with homozygous re-arrangements disrupting the Rdl gene, and the level of rescue observed is not increased by the addition of more than one splice form. This demonstrates that only a single Rdl splice form is necessary both to confer insecticide sensitivity and also to rescue lethality. Methods by which phenotype rescue could be enhanced and the potential advantages of using Rdl as a selectable marker are discussed.
