AN INVERSE PCR SCREEN FOR THE DETECTION OF P-ELEMENT INSERTIONS IN CLONED GENOMIC INTERVALS IN DROSOPHILA-MELANOGASTER

We developed a screening approach that utilizes an inverse polymerase chain reaction (PCR) to detect P element insertions in or near previou sly cloned genes in Drosophila melanogaster. We used this approach in a large scale genetic screen in which P elements were mobilized from s ites on the X chromosome to new autosomal locations. Mutagenized flies were combined in pools, and our screening approach was used to genera te probes corresponding to the sequences flanking each site of inserti on. These probes then were used for hybridization to cloned genomic in tervals, allowing individuals carrying insertions in them to be detect ed. We used the same approach to perform repeated rounds of sib-select ion to generate stable insertion lines. We screened 16,100 insert bear ing individuals and recovered 11 insertions in five intervals containi ng genes encoding members of the kinesin superfamily in Drosophila mel anogater. In addition, we recovered an insertion in the region includi ng the Larval Serum Protein-2 gene. Examination by Southern hybridizat ion confirms that the lines we recovered represent genuine insertions in the corresponding genomic intervals. Our data indicates that this a pproach will be very efficient both for P element mutagenesis of new g enomic regions and for detection and recovery of ''local'' P element t ransposition events. In addition, our data constitutes a survey of pre ferred P element insertion sites in the Drosophila genome and suggests that insertion sites that are mutable at a rate of similar to 10(-4) are distributed every 40-50 kb.