The evolution of cis regulatory elements (enhancers) of developmentally regulated genes plays a large role in the evolution of animal morphology. However, the mutational path of enhancer evolution-the number, origin, effect, and order of mutations that alter enhancer function-has not been elucidated. Here, we localized a suite of substitutions in a modular enhancer of the ebony locus responsible for adaptive melanism in a Ugandan Drosophila population. We show that at least five mutations with varied effects arose recently from a combination of standing variation and new mutations and combined to create an allele of large phenotypic effect. We underscore how enhancers are distinct macromolecular entities, subject to fundamentally different, and generally more relaxed, functional constraints relative to protein sequences.Three major challenges for understanding the genetic and molecular bases of morphological evolution are to identify loci underlying trait divergence, to pinpoint functional changes within these loci, and to trace the origin of functional variation in populations. The evolution of animal morphological diversity is generally associated with changes in the spatial expression of genes that govern development (1,2). The divergence of particular morphological traits has been linked to changes in specific enhancers of individual loci (3-9). Mutations in individual, modular enhancers are thought to circumvent the potentially pleiotropic effects of mutations in coding sequences of genes that participate in many developmental processes (10-12).Nonetheless, there is relatively little detailed knowledge of how enhancer sequences evolve, of the genetic path of enhancer evolution. In most instances, functional mutations have not been identified, so their individual effects and origins have not been traced. In contrast, the evolutionary paths of several proteins have been traced and revealed that many trajectories, including reversals, are not allowed because of structural constraints (13-15). To decipher the mode and tempo of regulatory sequence evolution, we must determine the following:

Understanding the mechanisms underlying the morphological divergence of species is one of the central goals of evolutionary biology. Here, we analyze the genetic and molecular bases of the divergence of body pigmentation patterns between Drosophila yakuba and its sister species Drosophila santomea. We found that loss of pigmentation in D. santomea involved the selective loss of expression of the tan and yellow pigmentation genes. We demonstrate that tan gene expression was eliminated through the mutational inactivation of one specific tan cis-regulatory element (CRE) whereas the Tan protein sequence remained unchanged. Surprisingly, we identify three independent loss-of-function alleles of the tan CRE in the young D. santomea lineage. We submit that there is sufficient empirical evidence to support the general prediction that functional evolutionary changes at pleiotropic loci will most often involve mutations in their discrete, modular cis-regulatory elements.

A cattle-human whole-genome comparative map was constructed using parallel radiation hybrid (RH) mapping in conjunction with EST sequencing, database mining for unmapped cattle genes, and a predictive bioinformatics approach (COMPASS) for targeting specific homologous regions. A total of 768 genes were placed on the RH map in addition to 319 microsatellites used as anchor markers. Of these, 638 had human orthologs with mapping data, thus permitting construction of an ordered comparative map. The large number of ordered loci revealed ը 105 conserved segments between the two genomes. The comparative map suggests that 41 translocation events, a minimum of 54 internal rearrangements, and repositioning of all but one centromere can account for the observed organizations of the cattle and human genomes. In addition, the COMPASS in silico mapping tool was shown to be 95% accurate in its ability to predict cattle chromosome location from random sequence data, demonstrating this tool to be valuable for efficient targeting of specific regions for detailed mapping. The comparative map generated will be a cornerstone for elucidating mammalian chromosome phylogeny and the identification of genes of agricultural importance. "Ought we, for instance, to begin by discussing each separate species-in virtue of some common element of their nature, and proceed from this as a basis for the consideration of them separately?" from Aristotle, On the Parts of Animals, 350 B.C.E.

The origination and diversification of morphological characteristics represents a key problem in understanding the evolution of development. Morphological traits result from gene regulatory networks (GRNs) that form a web of transcription factors, which regulate multiple cis-regulatory element (CRE) sequences to control the coordinated expression of differentiation genes. The formation and modification of GRNs must ultimately be understood at the level of individual regulatory linkages (i.e., transcription factor binding sites within CREs) that constitute the network. Here, we investigate how elements within a network originated and diversified to generate a broad range of abdominal pigmentation phenotypes among Sophophora fruit flies. Our data indicates that the coordinated expression of two melanin synthesis enzymes, Yellow and Tan, recently evolved through novel CRE activities that respond to the spatial patterning inputs of Hox proteins and the sex-specific input of Bric-à-brac transcription factors. Once established, it seems that these newly evolved activities were repeatedly modified by evolutionary changes in the network’s trans-regulators to generate large-scale changes in pigment pattern. By elucidating how yellow and tan are connected to the web of abdominal trans-regulators, we discovered that the yellow and tan abdominal CREs are composed of distinct regulatory inputs that exhibit contrasting responses to the same Hox proteins and Hox cofactors. These results provide an example in which CRE origination underlies a recently evolved novel trait, and highlights how coordinated expression patterns can evolve in parallel through the generation of unique regulatory linkages.

SUMMARY The evolutionary origins of complex morphological structures such as the vertebrate eye or insect wing remain one of the greatest mysteries of biology. Recent comparative studies of gene expression imply that new structures are not built from scratch, but rather form by co-opting preexisting gene networks. A key prediction of this model is that upstream factors within the network will activate their preexisting targets (i.e. enhancers) to form novel anatomies. Here, we show how a recently derived morphological novelty present in the genitalia of D. melanogaster employs an ancestral Hox-regulated network deployed in the embryo to generate the larval posterior spiracle. We demonstrate how transcriptional enhancers and constituent transcription factor binding sites are used in both ancestral and novel contexts. These results illustrate network co-option at the level of individual connections between regulatory genes, and highlight how morphological novelty may originate through the co-option of networks controlling seemingly unrelated structures.

A second-generation 5000 rad radiation hybrid (RH) map of the cattle genome was constructed primarily using cattle ESTs that were targeted to gaps in the existing cattle–human comparative map, as well as to sparsely populated map intervals. A total of 870 targeted markers were added, bringing the number of markers mapped on the RH5000 panel to 1913. Of these, 1463 have significant BLASTN hits (E < e–5) against the human genome sequence. A cattle–human comparative map was created using human genome sequence coordinates of the paired orthologs. One-hundred and ninety-five conserved segments (defined by two or more genes) were identified between the cattle and human genomes, of which 31 are newly discovered and 34 were extended singletons on the first-generation map. The new map represents an improvement of 20% genome-wide comparative coverage compared with the first-generation map. Analysis of gene content within human genome regions where there are gaps in the comparative map revealed gaps with both significantly greater and significantly lower gene content. The new, more detailed cattle–human comparative map provides an improved resource for the analysis of mammalian chromosome evolution, the identification of candidate genes for economically important traits, and for proper alignment of sequence contigs on cattle chromosomes.

Spatiotemporal changes in gene expression underlie many evolutionary novelties in nature. However, the evolutionary origins of novel expression patterns, and the transcriptional control elements ("enhancers") that govern them, remain unclear. Here, we sought to explore the molecular genetic mechanisms by which new enhancers arise. We undertook a survey of closely related Drosophila species to identify recently evolved novel gene expression patterns and traced their evolutionary history. Analyses of gene expression in a variety of developing tissues of the Drosophila melanogaster species subgroup revealed high rates of expression pattern divergence, including numerous evolutionary losses, heterochronic shifts, and expansions or contractions of expression domains. However, gains of novel expression patterns were much less frequent. One gain was observed for the Neprilysin-1 (Nep1) gene, which has evolved a unique expression pattern in optic lobe neuroblasts of Drosophila santomea. Dissection of the Nep1 cis-regulatory region localized a newly derived optic lobe enhancer activity to a region of an intron that has accumulated a small number of mutations. The Nep1 optic lobe enhancer overlaps with other enhancer activities, from which the novel activity was co-opted. We suggest that the novel optic lobe enhancer evolved by exploiting the cryptic activity of extant regulatory sequences, and this may reflect a general mechanism whereby new enhancers evolve.cis regulation | enhancer evolution | gene regulation | novelty Evolution does not produce novelties from scratch. It works on what already exists, either transforming a system to give it new functions or combining several systems to produce a more elaborate one.

A large fraction of the information content of metazoan genomes resides in the transcriptional and posttranscriptional cis-regulatory elements that collectively provide the blueprint for using the proteincoding capacity of the DNA, thus guiding the development and physiology of the entire organism. As successive whole-genome sequencing projects--including those of mice and humans-are completed, we have full access to the regulatory genome of yet another species. But our ability to decipher the cis-regulatory code, and hence to link genes into regulatory networks on a global scale, is currently very limited. Here we describe SCORE (Site Clustering Over Random Expectation), a computational method for identifying transcriptional cis-regulatory modules based on the fact that they often contain, in statistically improbable concentrations, multiple binding sites for the same transcription factor. We have carried out a Drosophila genomewide inventory of predicted binding sites for the Notch-regulated transcription factor Suppressor of Hairless [Su(H)] and found that the fly genome contains highly nonrandom clusterings of Su(H) sites over a broad range of sequence intervals. We found that the most statistically significant clusters are very heavily enriched in both known and logical targets of Su(H) binding and regulation. The utility of the SCORE approach was validated by in vivo experiments showing that proper expression of the novel gene Him in adult muscle precursor cells depends both on Su(H) gene activity and sequences that include a previously unstudied cluster of four Su(H) sites, indicating that Him is a likely direct target of Su(H).R ealizing the full promise of whole-genome sequencing projects depends on our ability to read and understand the tremendous informational richness contained therein. Computational methods for predicting novel protein coding genes in whole-genome sequence data are quite advanced, and various strategies for recognizing transcription units that generate noncoding RNAs are also available. However, these techniques address only part of the informational content of the genome. The complex blueprint that controls the utilization of the coding information in DNA is contained in the huge number of cis-regulatory elements, both transcriptional and posttranscriptional, that surround and invade the transcribed part of the genome. But it is clear that we are in our infancy in learning how to read the regulatory genome and thus decipher the blueprint.Here we describe SCORE (Site Clustering Over Random Expectation), a computational method for identifying potential cisregulatory modules and the target genes they serve. Transcriptional enhancer elements are generally quite compact, and they frequently include closely spaced binding sites for the same or multiple transcription factors (1). SCORE is designed to detect and statistically evaluate these structural features in whole-genome sequence data, and thus to reveal previously unrecognized enhancers. A conceptually similar method has been describ...