Ruhr-Universität Bochum, Bochum,FRG , # Universidad de Oviedo, Spain,
We are aiming to understand perception of odour quality and -concentration as well as odour coding in the brain. To approach this goal we study the molecular components involved in olfactory signal transduction and -coding as well as the modulation of this information by other inputs and the developmental processes that lead to the underlying neuronal network. We use the model system Drosophila because of its relatively simple architecture consisting of only 1300 receptor neurons which connect to about 35 glomeruli in the olfactory lobe of the deuterocerebrum. The experimental approach is to genetically decipher genes which are involved in these processes and to study their function. This is done by the introduction of enhancer trap transposons at random into the genome of Drosophila and by the selection of those lines which report on genes activated in cells of the olfactory organs. Lines that fulfil these criteria serve as the basis of our experimental work. During the last three years we have generated 2500 lines. 120 lines have been selected from this collection because they reveal specific gene activity in antenna or brain. Our experimental approach can be divided into three parts. First, the lines selected by the disclosure of gene activity of the reporter gene lacZ are tested for an olfactory mutant phenotype by measuring the behavioural response to six different chemicals. By applying direct electrophysiological measurements at the third antennal segment we confirm the mutant phenotype found in the behavioural test paradigm. The application of different chemicals allows us to distinguish effects on the general transduction pathway from those on specific olfactory perception and coding. From 30 lines tested 10 behaved as mutant against single chemicals. Second, the transposon insertion leads us to the mutant genes which are in the wildtype involved in olfactory perception. We analyse the insertion site of the genome using molecular cloning techniques. The affected transcript will be determined by cDNA cloning from an antennal library and comparison of the corresponding RNA expression pattern observed from Northern and in situ hybridization data with the X-gal staining pattern in the enhancer trap line. After having confirmed that the cloned cDNA is identical with the transcript regulated by the detected enhancer an extensive molecular characterization of the corresponding gene will be performed. Third, at the same time we concentrate on functional studies of the identified genes. This is approached by generation of additional alleles either as deletions, reinsertions at new chromosomal sites closeby or by in vitro mutagenesis followed by transformation into null mutant background.