Author Summary Eukaryotic genomes have been reshaped by chromosomal rearrangements during evolution. However, the compari- son of distantly related species has uncovered unusually large genomic regions with conserved gene organization. A widely accepted explanation is that, in those regions, there exist genes with joint and/or intricate regulation, which, if altered, might affect the performance of the carriers. We used a system that allowed us to precisely disrupt one of the largest genomic regions that has been conserved in the fruit flies since ,63 million years ago and performed a variety of assays devised to detect differences in organismal performance and anomalous gene expres- sion. Despite the overrepresentation of genes with ex- pression profiles related to male fertility and detection of chemical stimuli in this genomic region, as well as the presence of genes expressed during development and putatively regulated by long distantly located sequences, we do not find evidence of diminished viability and fertility or of anomalous levels of expression. These results lead us to propose that regulatory constraints might not suffice to explain the maintenance of the integrity of some of the best candidate regions in one of the most dynamic eukaryotic genomes. enriched for genes associated with particular expression profiles, but the functional signature most prominently found in ultra- conserved regions are stretches of DNA with multiple HCNEs (14.5% of the 145 HCNE peaks mapped as compared to 6.7% expected). To our knowledge, only two empirical tests for the presence of functional constraints have been performed in eukaryotic genomes [20,28]. In both cases, engineered chromosomal inversions were induced to disrupt clusters of genes with shared expression attributes and, subsequently, the phenotypic consequences of such disruptions monitored. For example, the disruption of the mouse Hoxd cluster, which is conserved across vertebrates, helped determine the presence of two functional subdomains and two long-range enhancers on either side of the cluster. This functional organization of the Hoxd cluster was proposed as the underlying cause that kept the cluster intact during the evolution of vertebrate lineages. In Diptera, three gene neighborhoods expressed in testes and one gene neighborhood expressed during embryogenesis of D. melanogaster have been disrupted [28], but no modification of the expression of the genes included in the neighborhoods examined was detected. These neighborhoods are conserved within the D. melanogaster species subgroup but disrupted in some other Drosophila lineages (Table S1), which does not clarify the phylogenetic scope of the putative constraints tested. The phenotypic consequences of disrupting a collinear region conserved throughout the genus Drosophila, which is presumably maintained by constraints, are presently unknown. Here, we used chromosome engineering to disrupt an ultraconserved region located on Muller’s element C of the Drosophila genome (Figure 1A). This ultraconserved region is delimited by the genes CG15121 and CG16894 and is ,701 kb long in D. melanogaster [22]. Importantly, this ultraconserved region is, based on current information, the one with the largest number of functional signatures, suggestive of the presence of regulatory- based constraints [22]. After disrupting the ultraconserved region CG15121–CG16894, we examined the resulting phenotypic consequences both by performing a variety of genetic and competition experiments that tested for differences in viability, Collinearity Conservation in Eukaryotes fertility, and relevant parameters of general homeostasis and by characterizing levels of mRNA abundance in both sexes. Our results indicate that, contrary to the prevalent view, the extraordinary conservation of some of the largest collinear blocks in eukaryotic genomes might not necessarily derive only from functional constraints that result in severe detrimental effects. Results Functional and comparative organization of the ultraconserved region CG15121–CG16894 The ultraconserved region CG15121–CG16894 ranked first in length and eleventh in the number of genes encompassed among 2,683 regions of conserved collinearity across nine Drosophila species [22]. Data from another comparative analysis on gene organization in the genus Drosophila [29] are consistent with the overall maintenance of the collinearity in this genomic region. In addition, this region shows statistically significant enrichment for genes encoding proteins involved in chemosensory perception and for genes preferentially expressed in males, many of them showing this same trend across multiple Drosophila species (Figure 1B and Table S2). This higher-than-expected local density of genes with coherent patterns of expression supports the presence of a male- biased gene expression neighborhood, which is intertwined with a smaller chemosensory perception gene neighborhood. This ultraconserved region is spanned by four HCNE peaks [12] (Figure 1C), more than any other collinear block. Genes responsive to HCNEs have been postulated to be preferentially associated with a particular kind of core promoters. Specifically, using promoter predictions for 42% of the protein-coding genes of D. melanogaster, a significant overrepresentation of genes with some kind of Inr-motif (Inr only, Inr/DPE, or Inr/TATA) was found in HCNE-dense regions [12]. We screened 500 nt upstream of each protein-coding gene in the region under study using McPromoter and obtained reliable predictions for eight genes. Six of these genes were predicted to have a core promoter responsive to HCNEs. These genes are found scattered along the region (Figure 1B and Figure S1) and encode proteins involved in the regulation of gene expression, from mRNA binding proteins (sm), to signal transduc- ers (Toll-7, 18-w), to ubiquitins (Isopeptidase-T-3), and also to others that we did not predict a priori (Obp56a and CG8654). Given the large number of genes for which a prediction was not provided by McPromoter, we checked for the presence of genes that are expressed during key developmental stages using the expression profile characterization generated by modENCODE [2]. Fifteen out of 36 protein-coding genes show a preferential pattern of expression during embryogenesis; among them, there is a cluster of eight genes mostly displaying high levels of expression during the first 16 h of development and moderate expression during the larva-pupa transition (Figure S1). Six of these eight genes are predicted to have core promoters of the Inr-motif type and four are associated with lethal phenotypes, the latter underscoring their functional relevance prior to imago emergence (Figure S1) [30,31]. Together, these functional features associated with some of the genes in the region are suggestive of regulation by HCNEs. Examination of the organization of the ultraconserved region CG15121–CG16894 in Anopheles gambiae revealed the presence of orthologues in six different locations (Figure S2A and Table S3). This degree of dispersion is not surprising given the fast differentiation of the Drosophila and Anopheles genomes via the accumulation of chromosomal rearrangements [32–34] and the lack of common constraints reflected in the pronounced differences in development, morphology, and ecology of these two Diptera [33,35,36]. Nevertheless, we detected a conserved PLoS Genetics | www.plosgenetics.org 2 February 2012 | Volume 8 | Issue 2 | e1002475