HOMOLOGY REQUIREMENTS FOR TARGETING HETEROLOGOUS SEQUENCES DURING P-INDUCED GAP REPAIR IN DROSOPHILA-MELANOGASTER

The effect of homology on gene targeting was studied in the context of P-element-induced double-strand breaks at the white locus of Drosophi la melanogaster. Double-strand breaks were made by excision of P-w(hd) , a P-element insertion in the white gene. A nested set of repair temp lates was generated that contained the 8 kilobase (kb) Bellow gene emb edded within varying amounts of white gene sequence. Repair with unlim ited homology was also analyzed. Flies were scored phenotypically for conversion of the yellow gene to the white locus. Targeting of the yel low gene was abolished when all of the 3' homology was removed. Increa ses in template homology up to 51 base pairs (bp) did not significantl y promote targeting. Maximum conversion was observed with a construct containing 493 bp of homology, without a significant increase in frequ ency when homology extended to the tips of the chromosome. These resul ts demonstrate that the homology requirements for targeting a large he terologous insertion are quite different than those for a point mutati on. Furthermore, heterologous insertions strongly affect the homology requirements for the conversion of distal point mutations. Several abe rrant conversion tracts, which arose from templates that contained red uced homology, also were examined and characterized.