Possible chromosomal rearrangements following two DNA double-strand breaks (DSBs) and repair by single-strand annealing (SSA). Two transgenes (DE16 or DE18 and DE20) that share homologous sequences of the yellow (y) gene undergo homologous recombination (HR) upon cleavage by I-SceI. (A) DE20 is a P-element carrying a full-length y+ gene and a recognition site for the rare cutting endonuclease I-SceI in the intron. This y+ gene is functional and confers a dark body color to the fly. DE16 and DE18 carry the same sequences in reverse orientation (the 3′ part of y is placed upstream of the 5′ part), and the first exon of y is removed rendering this incomplete y gene nonfunctional. The recognition site for I-SceI is located at the junction of the two inverted sequences. In addition, DE16 and DE18 carry a white+ (w+) gene serving as a transformation marker. (B, C) Mechanism of SSA as described in the Introduction, leading to chromosomal inversion (B) or to chromosomal translocation (C). Sequences shared between the two recombining elements are reduced to single copy, and the y+ function within DE20 is thereby lost. (D) Recombination of DE20 with DE16 or DE18 results in two hybrid elements composed of DE16 or DE18 and DE20. (E) Possibilities of DSB repair leading to the loss of y gene function. Indicated are only elements undergoing a change in the respective case. (F) Cre-mediated excision from DE16 joins the 3xP3 promoter to the EGFP coding region, resulting in a transient EGFP expression from an episome. (Boxed key) An explanation of all symbols used in this and the other figures is as follows: The y gene sequences are shown as either two bars to represent the DNA double-strand or as a single box to illustrate the structure of the gene. Arrows indicate the arrangement of the homologous regions of the y gene shared between the P-elements. Sequences other than y are not drawn to scale. Numbers (one to seven) denote kilobase intervals on the y gene. In some figures, the P-elements DE16, DE18, and DE20 are simplified to emphasize the components involved in the depicted process.
