Molecular characterization of eag: A gene affecting potassium channels in Drosophila melanogaster

Abstract

Genes encoding proteins involved in the function of the nervous system can be identified via mutations causing behavioral abnormalities. An example is ether a go-go (eag) in Drosophila melanogaster, which was identified originally as an X-linked mutation that displayed ether-induced leg-shaking behavior. Electrophysiological and genetic evidence suggests that the product of the eag locus is intimately involved in the normal functioning of one or more types of voltage-gated potassium channels. To initiate a molecular analysis of eag we first generated a collection of deletions to pinpoint its cytological location. On the basis of this location, we identified an existing inversion, In(1)sc29, with one breakpoint at the eag locus and the other in the scute (sc) complex. A genomic library was prepared from In(1)sc29 and screened with a genomic DNA fragment that spanned the sc breakpoint to isolate DNA from the eag region. Beginning from this starting point over 85 kb of DNA were isolated by chromosome walking. Three additional eag alleles, including two dysgenesis-induced insertion mutations and a γ-ray-induced insertional translocation, were located on the molecular map of the eag locus by Southern blot analysis. The molecular defects associated with these alleles encompass a total of 27 kb within the chromosome walk. A 10-kb transcript derived from this region, which is expressed most abundantly in heads, was identified on Northern blots. Two different eag mutations separated by over 20 kb interrupt the same transcript identifying it as the likely eag message. cDNAs representing a portion of this transcript have been isolated. The genomic DNA sequences from which these cDNAs are derived extend over 37.5 kb, providing a minimum estimate of the size of the eag transcription unit. Ultimately, sequence analysis of these cDNAs should enable us to the identify the eag polypeptide and to elucidate its role in membrane excitability.