Fig. 1. Two-step construction of the Ubx^QA allele. (A) An X chromosome insertion of the P element construct containing the desired changes to Ubx and the necessary sequences for allelic replacement (Rong et al., 2002) was used to manipulate the endogenous Ubx locus on the 3rd chromosome. Only the 3' end of Ubx, which is also the edge of the BX-C, is shown. The scale is approximate; the targeting construct contained 10,893 bp between the vector cloning sites. At each step, the expressed polypeptide sequence of Ubx following the homeodomain is shown. Each step was catalyzed by crossing to flies containing the appropriate heat-shock transgenes and heat-shocking the progeny (see Materials and methods) (Rong et al., 2002). (B) The first step created a targeted duplication of the 3' end of the Ubx locus that contained one complete and expressed Ubx⁺ allele and a partial unexpressed Ubx^QA allele. (C) The second step catalyzed the reduction of this duplication to a single copy, creating both full-length Ubx^QA alleles and control Ubx⁺ alleles in independent reduction events. Only the recovery of an Ubx^QA allele is shown. Black indicates sequences with homology to Ubx; gray indicates vector or unknown sequences; red indicates marker w^hs gene for screening; blue indicates site-specific recombinase Flp and FRT targets (triangles); purple indicates homing endonuclease I-SceI and I-SceI target (bar); pink indicates homing endonuclease I-CreI and I-CreI targets (bars); green indicates PCR primers (half arrows) and restriction sites (bars) used in preliminary screens of putative targeted duplication and reduction events. Ultimately, the entire region was sequenced from alleles selected for study.