Functional analysis of a mosquito gamma-aminobutyric acid receptor gene promoter.
A single point mutation in the insect gamma-aminobutyric acid receptor (GABAR)-encoding gene (Rdl) confers high levels of resistance to cyclodienes in Drosophila and other insects. We were interested in studying the promoter of this gene for two reasons. Firstly, to define the elements underlying Rdl expression. Secondly, to identify the minimum set of regulatory elements necessary for construction of a functional Rdl minigene. Such an insecticide-resistance-associated minigene should form a strong selectable marker for use in the genetic transformation of non-drosophilid pest insects, such as mosquitoes. Here, we report the identification of the region containing the rdl promoter, via transient expression of a luc reporter gene following micro-injection into embryos of the mosquito Aedes aegypti. Promoter activity is contained within a 2.53-kb fragment immediately upstream from the rdl start codon. Primer extension shows three closely linked sites for transcript initiation within this region and sequence analysis reveals anumber of putative consensus regulatory sequences shared by other genes expressed in the nervous system. The implications for construction of a functional minigene and the identification of cis-acting control elements underlying ion-channel gene regulation are discussed.
Shotkoski
F
F
Department of Neurobiology, University of Wisconsin-Madison 53706, USA.
Morris
A C
AC
James
A A
AA
ffrench-Constant
R H
RH
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D009033:Culicidae / Q000235:genetics*; D017931:DNA Primers; D004330:Drosophila / Q000235:genetics; D004351:Drug Resistance; D004625:Embryo, Nonmammalian; D017344:Genes, Insect; D008156:Luciferases / Q000032:analysis / Q000096:biosynthesis; D008969:Molecular Sequence Data; D009424:Nervous System Physiological Phenomena; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D018079:Receptors, GABA / Q000235:genetics*; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D017384:Sequence Deletion; D016385:TATA Box
D017931:DNA Primers; D018079:Receptors, GABA; D011994:Recombinant Proteins; D008156:Luciferases