Collinearity Conservation in Eukaryotes Figure 1. Schematic representation of the inversion engineered to disrupt the ultraconserved region CG15121–CG16894 and its salient features. (A) Schematic representation showing the surrounding regions and location of the FRT-bearing TEs P{RS5}5-HA-1995 and P{RS3}CB- 0236-3 (green double arrowhead lines) used to generate the inversion In(2R)51F11-56E2 (white double arrowhead line), which disrupts the ultraconserved region CG15121–CG16894 (blue box). The element P{RS3}CB-0236-3 is inserted in a naturally occurring copy of the TE 1360{}835 [43]. Distances between the FRT-bearing TEs and the flanking genes are indicated in nucleotides. Notice that flanking transcription units at the immediate vicinity of the inner breakpoint are two non-protein-coding genes: CR30451, which codes for tRNA:E4:56Fc; and CR33930, which codes for snoRNA:185. (B) Annotation of the ultraconserved region CG15121–CG16894 using D. melanogaster as a reference. From top to bottom: 36 protein-coding genes; 122 non-protein-coding genes (8 miRNAs, 13 tRNAs, 1 snoRNA, and 100 5S rRNAs); 29 protein-coding genes whose collinearity is maintained across nine species of the genus Drosophila; 20 protein-coding genes with male-biased gene expression using mRNA levels as a proxy; 10 protein-coding genes related to odor-guided behavior; and 8 putative targets of Highly Conserved Non-coding Elements (HCNEs) based on their expression profile, core promoter predictions, and mutant phenotypes. Details on the protein-coding genes in this region and their annotation are provided in Table S2. (C) Ancora [82] snapshot (http://ancora.genereg.net) of the distribution of HCNEs when genome sequences of D. melanogaster and D. virilis are compared. Green, orange, and red denote 96%, 98%, and 100% nucleotide identity, respectively. doi:10.1371/journal.pgen.1002475.g001 gene arrangement including the gene Toll-7 and the Obp genes, an association that is also present in A. aegypti (Figure S2B). Phylogenetic analyses [37] unambiguously support the close phylogenetic relationship among several Obp genes in the ultraconserved region under study (Obp56a, Obp56d, and Obp56e) and some that are adjacent to Toll-7 in A. gambiae (OBP23, OBP25, OBP26, OBP28), which in turn are closely related to those that cluster nearby Toll-7 in A. aegypti ([38] and this work; Figure S2C). Interestingly, the genes Obp56a, Obp56d and Toll-7 are found within the same expression cluster in D. melanogaster and two of them have core promoter types presumably responsive to HCNE- mediated regulation (Figure S1). To sum up, the existence of two intertwined gene neighborhoods associated with very marked expression profiles, the enrichment for HCNE peaks and presence of their putative targets, and the detection of a region conserved across Diptera reinforces the possibility that one or more regulatory-based constraints might exist in the ultraconserved region CG15121–CG16894. Disruption of the ultraconserved region CG15121–CG16894 To assess the importance of the integrity of the ultraconserved region CG15121–CG16894, we aimed to disrupt it and character- ize the resulting phenotypic effects. We examined the existence of PLoS Genetics | www.plosgenetics.org 3 February 2012 | Volume 8 | Issue 2 | e1002475