Construction and Characterization of Deletions with Defined Endpoints in Drosophila using P-elements in Trans

We used P-element transposase mediated "male recombination" between two P-elements in trans to create genetic deletions that removed a number of loci, including the gene encoding the neuropeptide Crustacean Cardioactive Peptide (CCAP). Two classes of recombinant chromosomes were produced. Approximately one quarter were viable when homozygous or hemizygous, whereas the remaining lines caused homozygous and hemizygous lethality. Preliminary analyses using PCR and CCAP immunohistochemistry suggested that, whereas the DNA of the viable lines was largely intact, most lethal lines contained chromosomal deletions that were roughly bounded by the insertion sites of the two P-elements used. Southern blot analyses of select lethal lines showed that the DNA flanking the deletion was indeed grossly intact whereas the intervening DNA could not be detected. Sequencing across the deletion in three of these lethal lines identified a single line bearing intact genomic DNA on either side of the deletion separated by 30 base pairs of P-element DNA. The method described here suggests a simple procedure for creating deletions with defined end-points. Importantly, it can use pre-existing P-element insertion strains and does not rely on the use of transposable elements that are engineered to cause specific DNA rearrangements.

Key Words: CCAP, excision, male recombination, mobile genetic element, neuropeptide

Online ISS 1091-6490
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