SITE-SELECTED MUTAGENESIS OF THE DROSOPHILA 2ND CHROMOSOME VIA PLASMID RESCUE OF LETHAL P-ELEMENT INSERTIONS

This paper describes a fast and efficient approach to correlating clon ed genes with mutant phenotypes in Drosophila. We make use of a large collection D. melanogaster lines with recessive lethal insertions of a P[lacW] transposon on their second chromosome, Within this collection there clearly must be many insertions corresponding to Drosophila gen es that have been cloned and characterized, e.g., via homology with cl oned mammalian genes, but for which mutant phenotypes have yet to be i dentified. We have made use of the Fact that P[lacW] contains a plasmi d replicon to establish a collection of rescued plasmids containing ge nomic DNA flanking the sites of transposon insertion, Plasmids represe nting a total of 1836 lines were independently rescued and pooled in b atches of 10 and 100. Pools of 100 plasmids were screened by hybridiza tion with cDNAs corresponding to cloned second chromosome loci. Hybrid izing pools were then narrowed down to single plasmids by a process of subdivision and rehybridization, and corresponding mutant lines were obtained. The success rate was better than one in four. This rare woul d undoubtedly be improved by the use of genomic DNA probes.