stocks carrying isolated naturally occurring inversions disrupting the ultraconserved region and none was found. Thus, we generated a disruption of the ultraconserved region CG15121– CG16894 by inducing the inversion In(2R)51F11-56E2 in D. melanogaster, a species in which nonallelic homologous recombina- tion (NAHR) events can be mediated between FLP recombination target-bearing transposable elements (FRT-bearing TEs hereafter) via activation of a heat-inducible flippase-recombinase [28,39–42] (Figure S3, S4, S5). For that, we used two TEs bearing FRT sites in opposite orientation: P{RS5}5-HA-1995, which is inside the ultraconserved region CG15121–CG16894; and P{RS3}CB-0236- 3, which is located 4.35 Mb upstream (Figure 1A). The outer element is virtually terminal within a collinear block of 15 genes and is inserted into a naturally occurring copy of the TE 1360{}835 [43]. We adopted several measures to avoid confounding effects that could overlay those of the intended disruption of the ultra- conserved region CG15121–CG16894. First, TEs were selected to avoid disrupting any known regulatory sequences of flanking genes and those presumably embedded in HCNE peaks, thus preventing the generation of artifactual position effects. The comparison of the size and sex ratio of the progeny of flies homozygous for each of the TEs confirmed the absence of any obvious detrimental effect Collinearity Conservation in Eukaryotes associated with particular TE insertions (Figure S6). Second, in addition to strains carrying the inversion In(2R)51F11-56E2 (INV1 and INV2), we generated several control strains to account for further mutations that could have been incidentally generated by our approach [44]. Specifically, three kinds of control strains were constructed: strains carrying two FRT-bearing TEs in cis (REC), i.e. just before inducing the NAHR event that mediates the inversion; strains carrying the standard arrangement as a result of failed induced NAHR events but that were exposed to the same experimental conditions as the INV strains -SIMultaneous controls- (SIM1, SIM2, and SIM3); and strains in which the inverted segment is reverted back to its original orientation - REVertant controls- (REV1 and REV2) (Figure 2A, Figure S3 and S4). The main molecular changes at the inversion breakpoints of all relevant strains, plus those carrying the original FRT-bearing TEs, are depicted in Figure S5. In the absence of any secondary effect of our procedure, SIM, REV, and REC should perform likewise as measured by the size and sex ratio in the progeny of low-density crosses with homozygous flies (Figure S7). INV strains for which no SIM and/or REV control lines could be generated were discarded. For the remaining strains, their putative karyotype was verified at the cytological level and the expected molecular organization at their breakpoint regions confirmed by PCR and Figure 2. Disruption of the ultraconserved region CG15121–CG16894. (A) Outline of the main steps used to obtain the strains carrying the ultraconserved region CG15121–CG16894 in its disrupted and intact form (Figures S3 and S4 for details). The strains 5-HA-1995 and CB-0236-3 carrying the FRT-bearing TEs [42] were used to induce the inversion In(2R)51F11-56E2, which disrupts the ultraconserved region (strains INV1 and INV2). Control strains carrying the ultraconserved region in its intact form are also shown (REC, SIM1-SIM3, REV1-REV2; Table S4). Black and red denote w2 and w+ phenotype, respectively; asterisk, heat-shock induced FLP expression. (B) Distinctive banding pattern for the PCR products of five different amplicons (D, F, G/G9, H) run simultaneously for each of the strains generated (Material and Methods, Figure S5, and Tables S5 and S6). Two ml from each individual PCR product were combined. Strains 5-HA-1995 and CB-0236-3 show the presence of only one of the FRT-bearing TEs in their genomes while the REC strain shows the presence of both. SIM and REV strains show the successful amplification of the amplicon G9, which denotes the presence of a derivative of P{RS3}CB-0236-3 at the outer breakpoint, but not of the amplicon H, indicating that they carry the 2R standard chromosomal arrangement. INV strains show the opposite pattern; the detection of amplicon H denotes the presence of the inverted chromosomal arrangement. (C) Representative polytene chromosome squashes. Top, an inversion loop is observed for the heterozygote progeny of INV1 and INV2 parentals when crossed with individuals from the strain w1118 thus confirming the presence of the inverted arrangement. Bottom, no inversion loop is observed in the progeny of similar crosses involving SIM3 and REV1. Red and blue lines connect the same polytene band inside and outside of the inverted fragment, respectively, between carriers for the standard arrangement (SIM3 and REV1) and homozygotes for the induced inversion (INV1; center). Apart from the inversion In(2R)51F11-56E2 in the strains INV1 and INV2, no other gross chromosome alteration was detected for the strains shown in (B). doi:10.1371/journal.pgen.1002475.g002 PLoS Genetics | www.plosgenetics.org 4 February 2012 | Volume 8 | Issue 2 | e1002475