among HCNE peaks should result in certain regionalization of the expression profiles, i.e. physically closer HCNE targets should exhibit more similar expression profiles. In fact, we find putative targets of HCNEs located at both sides of the disruption, such as CG9854 and CG8896, showing very similar expression profiles (Figure 1 and Figure S1). If this similarity denotes the existence of long-range interactions, the latter are not associated with detrimental effects that explain the collinearity conservation. Alternatively, some degree of regulatory redun- dancy could exist at both sides of the disruption, which would explain the lack of phenotypic effect but not the collinearity conservation. A different view of collinearity conservation entails factors other than functional constraints. DNA stretches with sufficient sequence identity can mediate NAHR events that give rise to chromosomal rearrangements [53]. Large collinear regions could be depleted of this type of sequences thus explaining why this ultraconserved region has maintained its integrity. A search for well-annotated sequences with the potential to mediate NAHR events [54] in the studied region across Drosophila species revealed that these sequences are present (Table S20). These sequences include ncRNA genes such as tRNAs [55,56], rRNAs genes [57,58], and snoRNAs [59]. Equivalent comparative analysis focusing on TEs, once properly annotated, will enable to test whether the region under study is particularly depleted for these sequences, which would decrease its propensity of being rearranged. Further, recent findings indicate that orthologous landmarks harboring genes that bind to the nuclear periphery are significantly larger than those that do not harbor any suggesting that particular intranuclear localizations might provide molecular environments associated with higher levels of genome stability [60]. The ultraconserved region CG15121–CG16894 is known to establish some contact with the nuclear periphery [61]. Specifi- cally, it contains at least five protein-coding genes (CG16716, CG13872, CG10822, CG8654, CG16898) that exhibit statistically significant association with the B-type Lamin protein, a key component of the inner nuclear membrane [60,62]. This pattern leaves open the possibility that some regulatory-based constraints had evolved under the enhanced evolutionary stability enjoyed by genomic regions associated with the nuclear periphery, and therefore functional constraints would not be the only mechanism contributing to their collinearity conservation. Regardless how extensive the characterization of individuals carrying engineered genomic regions could be, our ability to detect phenotypic effects will always be contingent to the experimental setting used and the timescale in which the assays are performed. Nevertheless, our results raise the possibility that, at least in Diptera genomes, the mechanistic basis of collinearity conserva- tion might be much more subtle and diverse than previously thought and that regulatory-based interactions might not suffice to account for the patterns of extensive conservation seen in some genomic regions [22]. Only further empirical tests for this and other ultraconserved regions can shed light on the scope of our observations. Materials and Methods Fly husbandry Table S4 describes the strains used. Fly cultures were grown and maintained on dextrose-cornmeal-yeast medium at room temper- ature. Unless otherwise stated, all phenotypic assays were performed at 25uC in a temperature-controlled chamber and fly manipulation, sorting, and scoring were carried out under CO2 anesthesia. Strains generated are available upon request. Collinearity Conservation in Eukaryotes Inversion generation Strains carrying the original FRT-bearing TE insertions (5-HA- 1996 and CB-0236-3) were selected from the DrosDel collection and examined at different levels prior to their use in the generation of the chromosomal inversion In(2R)51F11-56E2. Sanger se- quencing confirmed the insertion point of the FRT-bearing TEs used. Low-density crosses using homozygous flies were also performed to test for potential detrimental effects associated with the TE insertions. Briefly, six sexually mature individuals (three four-day-old females and three two-day-old males) were used per cross and strain; each cross was replicated five times. Males were discarded after 24 hours while females were transferred twice every three days until discarded on the ninth day. To generate the inversion In(2R)51F11-56E2, we followed essentially [42] with slight modifications (John Roote, pers. comm.; Figure S3A–S3B). Three types of strains are generated under this procedure (REC, INV, and SIM); a fourth type of strain (REV; Figure S3C) was generated by reverting the inversion In(2R)51F11-56E2 [40]. The strains generated were scrutinized for evidence of side effect associated with our procedure (see below). Individuals from strains deemed as valid were crossed with those from the strain w1118, which possesses the standard arrangement for all chromosomes, and the third-instar salivary gland polytene chromosomes of the progeny examined. Chromo- some squashes were stained with orcein and inspected with a Zeiss AX10 Imager M1 microscope. Cytological analysis was performed using the photographic polytene maps of D. melanogaster as a reference [63]. Diagnostic DNA stretches at the breakpoint regions were PCR amplified and their identity verified by Sanger sequencing; sequences were deposited at GenBank (http://www. ncbi.nlm.nih.gov/genbank/; JN805541–JN805602). Amplicons C, D, E, and F confirmed the presence of FRT-bearing TEs and their derivatives at the breakpoint regions. Amplicon G, or G9 after excision of one of the exons of the reporter gene, and H correlate with the absence and presence of the inversion, respectively (Tables S5 and S6 for further details on primers used and amplicons). Genomic DNA used in PCR genotyping was extracted from 50 individuals of each strain as described [39] and quantified using a NanoDrop 8000 Spectrophotometer. Takara Taq and Takara Ex Taq, depending on the size of the DNA fragment to be amplified, were used according to manufacturer conditions. PCR products were resolved on 1% agarose gels and visualized in an AlphaImager HP system. Amplicon sequences were examined for point mutations and indels that could have been generated incidentally during the course of our procedure. Viability tests Low-density crosses using homozygous flies were set up for each strain, as described above for the evaluation of the TE insertions, to confirm absence of differences among control strains and to test for differences among the latter and the strains carrying the disrupted ultraconserved region. Additional crosses, five per strain, evaluated the effects of the disruption in heterozygous condition with different chromosomes carrying the standard arrangement (SIM1, REV1, REV2); 10 sexually mature individuals of each sex were used per cross. Further, adapted frequency-dependent experiments [64] evaluated differences in viability of mixtures of embryos for two different genotypes. Two types of mixtures were prepared. In both, w+ individuals (INV1, INV2, or REC; the tested chromosome) compete with w2 individuals carrying the ultraconserved region in its intact form. The only difference between the mixtures was the condition of the tested chromosome (heterozygosis, e.g. INV1/SIM1 versus SIM1/SIM1; homozygosis, e.g. INV1/INV1 versus SIM1/SIM1). For both types of mixtures, PLoS Genetics | www.plosgenetics.org 7 February 2012 | Volume 8 | Issue 2 | e1002475