Sequences In Drosophila Research Articles

Divergent evolution and purifying selection of the Type 2 diabetes gene sequences in Drosophila: a phylogenomic study.

The recently developed phylogenomic approach provides a unique way to identify disease risk or protective allele in any organism. While risk alleles evolve mostly under purifying selection, protective alleles are evolving either under balancing or positive selection. Owing to insufficient information, authors employed the phylogenomic approach to detect the nature of selection acting on type 2 diabetes (T2D) genes in Drosophila genus using various models of CODEML utility of PAML. The obtained result revealed that T2D gene sequences are evolving under purifying selection. However, only a few sites in membrane proteins encoded via CG8051, ZnT35C, and kar, are significantly evolving under positive selection under specific scenarios, which might be because of positive or adaptive evolution in response to changing niche, diet or other factors. In the near future, this information will be highly useful in the field of evolutionary medicine and the drug discovery process.

O-GlcNAcylation Dampens Dpp/BMP Signaling to Ensure Proper Drosophila Embryonic Development.

BMP (bone morphogenetic protein) signaling activity is precisely controlled by both pathway agonists andantagonists. Here, we identify a previously unrecognized BMP signaling antagonist. We demonstrate that the Drosophila BMP type I receptor Sax(Saxophone) functions as a Dpp (Decapentaplegic) receptor in Drosophila embryos, but that its activity is normally inhibited by the O-linked glycosyltransferase Sxc (Super sex combs). In wild-type embryos, Sax activity is inhibited, and the BMP type I receptor Tkv (Thickveins) is the sole conduit for Dpp. In contrast, in sxc mutants, the Dpp signal istransduced by both Tkv and Sax, and elevated Dpp signaling results in embryonic lethality. We also demonstrate that Sxc O-glycosylates Sax andobserve elevated Dpp signaling in response to maternal restriction of dietary sugar. These findings link fertility to nutritive environment and point to Sax signaling as the nutrient-sensitive branch of BMP signaling.

"What You Need, Baby, I Got It": Transposable Elements as Suppliers of Cis-Operating Sequences in Drosophila.

Transposable elements (TEs) are constitutive components of both eukaryotic and prokaryotic genomes. The role of TEs in the evolution of genes and genomes has been widely assessed over the past years in a variety of model and non-model organisms. Drosophila is undoubtedly among the most powerful model organisms used for the purpose of studying the role of transposons and their effects on the stability and evolution of genes and genomes. Besides their most intuitive role as insertional mutagens, TEs can modify the transcriptional pattern of host genes by juxtaposing new cis-regulatory sequences. A key element of TE biology is that they carry transcriptional control elements that fine-tune the transcription of their own genes, but that can also perturb the transcriptional activity of neighboring host genes. From this perspective, the transposition-mediated modulation of gene expression is an important issue for the short-term adaptation of physiological functions to the environmental changes, and for long-term evolutionary changes. Here, we review the current literature concerning the regulatory and structural elements operating in cis provided by TEs in Drosophila. Furthermore, we highlight that, besides their influence on both TEs and host genes expression, they can affect the chromatin structure and epigenetic status as well as both the chromosome’s structure and stability. It emerges that Drosophila is a good model organism to study the effect of TE-linked regulatory sequences, and it could help future studies on TE–host interactions in any complex eukaryotic genome.

Transcription elongation factor AFF2/FMR2 regulates expression of expanded GGGGCC repeat-containing C9ORF72 allele in ALS/FTD

Expanded GGGGCC (G4C2) repeats in C9ORF72 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How RNAs containing expanded G4C2 repeats are transcribed in human neurons is largely unknown. Here we describe a Drosophila model in which poly(GR) expression in adult neurons causes axonal and locomotor defects and premature death without apparent TDP-43 pathology. In an unbiased genetic screen, partial loss of Lilliputian (Lilli) activity strongly suppresses poly(GR) toxicity by specifically downregulating the transcription of GC-rich sequences in Drosophila. Knockout of AFF2/FMR2 (one of four mammalian homologues of Lilli) with CRISPR-Cas9 decreases the expression of the mutant C9ORF72 allele containing expanded G4C2 repeats and the levels of repeat RNA foci and dipeptide repeat proteins in cortical neurons derived from induced pluripotent stem cells of C9ORF72 patients, resulting in rescue of axonal degeneration and TDP-43 pathology. Thus, AFF2/FMR2 regulates the transcription and toxicity of expanded G4C2 repeats in human C9ORF72-ALS/FTD neurons.

Influence of epistasis on response to genomic selection using complete sequence data

BackgroundThe effect of epistasis on response to selection is a highly debated topic. Here, we investigated the impact of epistasis on response to sequence-based selection via genomic best linear prediction (GBLUP) in a regime of strong non-symmetrical epistasis under divergent selection, using real Drosophila sequence data. We also explored the possible advantage of including epistasis in the evaluation model and/or of knowing the causal mutations.ResultsResponse to selection was almost exclusively due to changes in allele frequency at a few loci with a large effect. Response was highly asymmetric (about four phenotypic standard deviations higher for upward than downward selection) due to the highly skewed site frequency spectrum. Epistasis accentuated this asymmetry and affected response to selection by modulating the additive genetic variance, which was sustained for longer under upward selection whereas it eroded rapidly under downward selection. Response to selection was quite insensitive to the evaluation model, especially under an additive scenario. Nevertheless, including epistasis in the model when there was none eventually led to lower accuracies as selection proceeded. Accounting for epistasis in the model, if it existed, was beneficial but only in the medium term. There was not much gain in response if causal mutations were known, compared to using sequence data, which is likely due to strong linkage disequilibrium, high heritability and availability of phenotypes on candidates.ConclusionsEpistatic interactions affect the response to genomic selection by modulating the additive genetic variance used for selection. Epistasis releases additive variance that may increase response to selection compared to a pure additive genetic action. Furthermore, genomic evaluation models and, in particular, GBLUP are robust, i.e. adding complexity to the model did not modify substantially the response (for a given architecture).

Competitive Disinhibition Mediates Behavioral Choice and Sequences in Drosophila

Even a simple sensory stimulus can elicit distinct innate behaviors and sequences. During sensorimotor decisions, competitive interactions among neurons that promote distinct behaviors must ensure the selection and maintenance of one behavior, while suppressing others. The circuit implementation of these competitive interactions is still an open question. By combining comprehensive electron microscopy reconstruction of inhibitory interneuron networks, modeling, electrophysiology, and behavioral studies, we determined the circuit mechanisms thatcontribute to the Drosophila larval sensorimotor decision to startle, explore, or perform a sequence of the two in response to a mechanosensory stimulus. Together, these studies reveal that, early in sensory processing, (1) reciprocally connected feedforward inhibitory interneurons implement behavioral choice, (2) local feedback disinhibition provides positive feedback that consolidates and maintains the chosen behavior, and (3) lateral disinhibition promotes sequence transitions. The combination of these interconnected circuit motifs can implement both behavior selection and the serial organization of behaviors into a sequence.

Redeployment of a conserved gene regulatory network during Aedes aegypti development

Changes in gene regulatory networks (GRNs) underlie the evolution of morphological novelty and developmental system drift. The fruitfly Drosophila melanogaster and the dengue and Zika vector mosquito Aedes aegypti have substantially similar nervous system morphology. Nevertheless, they show significant divergence in a set of genes co-expressed in the midline of the Drosophila central nervous system, including the master regulator single minded and downstream genes including short gastrulation, Star, and NetrinA. In contrast to Drosophila, we find that midline expression of these genes is either absent or severely diminished in A. aegypti. Instead, they are co-expressed in the lateral nervous system. This suggests that in A. aegypti this “midline GRN” has been redeployed to a new location while lost from its previous site of activity. In order to characterize the relevant GRNs, we employed the SCRMshaw method we previously developed to identify transcriptional cis-regulatory modules in both species. Analysis of these regulatory sequences in transgenic Drosophila suggests that the altered gene expression observed in A. aegypti is the result of trans-dependent redeployment of the GRN, potentially stemming from cis-mediated changes in the expression of sim and other as-yet unidentified regulators. Our results illustrate a novel “repeal, replace, and redeploy” mode of evolution in which a conserved GRN acquires a different function at a new site while its original function is co-opted by a different GRN. This represents a striking example of developmental system drift in which the dramatic shift in gene expression does not result in gross morphological changes, but in more subtle differences in development and function of the late embryonic nervous system.

LedPred: an R/bioconductor package to predict regulatory sequences using support vector machines.

Supervised classification based on support vector machines (SVMs) has successfully been used for the prediction of cis-regulatory modules (CRMs). However, no integrated tool using such heterogeneous data as position-specific scoring matrices, ChIP-seq data or conservation scores is currently available. Here, we present LedPred, a flexible SVM workflow that predicts new regulatory sequences based on the annotation of known CRMs, which are associated to a large variety of feature types. LedPred is provided as an R/Bioconductor package connected to an online server to avoid installation of non-R software. Due to the heterogeneous CRM feature integration, LedPred excels at the prediction of regulatory sequences in Drosophila and mouse datasets compared with similar SVM-based software. LedPred is available on GitHub: https://github.com/aitgon/LedPred and Bioconductor: http://bioconductor.org/packages/release/bioc/html/LedPred.html under the MIT license. aitor.gonzalez@univ-amu.fr Supplementary data are available at Bioinformatics online.

The achaete-scute complex in Diptera: patterns of noncoding sequence evolution.

The achaete‐scute complex (AS‐C) has been a useful paradigm for the study of pattern formation and its evolution. achaete‐scute genes have duplicated and evolved distinct expression patterns during the evolution of cyclorraphous Diptera. Are the expression patterns in different species driven by conserved regulatory elements? If so, when did such regulatory elements arise? Here, we have sequenced most of the AS‐C of the fly Calliphora vicina (including the genes achaete, scute and lethal of scute) to compare noncoding sequences with known cis‐regulatory sequences in Drosophila. The organization of the complex is conserved with respect to Drosophila species. There are numerous small stretches of conserved noncoding sequence that, in spite of high sequence turnover, display binding sites for known transcription factors. Synteny of the blocks of conserved noncoding sequences is maintained suggesting not only conservation of the position of regulatory elements but also an origin prior to the divergence between these two species. We propose that some of these enhancers originated by duplication with their target genes.

Parallel faster-X evolution of gene expression and protein sequences in Drosophila: beyond differences in expression properties and protein interactions.

Population genetics models predict that the X (or Z) chromosome will evolve at faster rates than the autosomes in XY (or ZW) systems. Studies of molecular evolution using large datasets in multiple species have provided evidence supporting this faster-X effect in protein-coding sequences and, more recently, in transcriptomes. However, X-linked and autosomal genes differ significantly in important properties besides hemizygosity in males, including gene expression levels, tissue specificity in gene expression, and the number of interactions in which they are involved (i.e., protein-protein or DNA-protein interactions). Most important, these properties are known to correlate with rates of evolution, which raises the question of whether differences between the X chromosome and autosomes in gene properties, rather than hemizygosity, are sufficient to explain faster-X evolution. Here I investigate this possibility using whole genome sequences and transcriptomes of Drosophila yakuba and D. santomea and show that this is not the case. Additional factors are needed to account for faster-X evolution of both gene expression and protein-coding sequences beyond differences in gene properties, likely a higher incidence of positive selection in combination with the accumulation of weakly deleterious mutations.

Co-regulation of invected and engrailed by a complex array of regulatory sequences in Drosophila

invected (inv) and engrailed (en) form a gene complex that extends about 115kb. These two genes encode highly related homeodomain proteins that are co-regulated in a complex manner throughout development. Our dissection of inv/en regulatory DNA shows that most enhancers are spread throughout a 62kb region. We used two types of constructs to analyze the function of this DNA: P-element based reporter constructs with small pieces of DNA fused to the en promoter driving lacZ expression and large constructs with HA-tagged en and inv inserted in the genome with the phiC31 system. In addition, we generated deletions of inv and en DNA in situ and assayed their effects on inv/en expression. Our results support and extend our knowledge of inv/en regulation. First, inv and en share regulatory DNA, most of which is flanking the en transcription unit. In support of this, a 79-kb HA-en transgene can rescue inv en double mutants to viable, fertile adults. In contrast, an 84-kb HA-inv transgene lacks most of the enhancers for inv/en expression. Second, there are multiple enhancers for inv/en stripes in embryos; some of these may be redundant but others play discrete roles at different stages of embryonic development. Finally, no small reporter construct gave expression in the posterior compartment of imaginal discs, a hallmark of inv/en expression. Robust expression of HA-en in the posterior compartment of imaginal discs is evident from the 79-kb HA-en transgene, while a 45-kb HA-en transgene gives weaker, variable imaginal disc expression. We suggest that the activity of the imaginal disc enhancer(s) is dependent on the chromatin structure of the inv/en domain.

Evolution and function of the extended miR-2 microRNA family

MicroRNAs are essential post-transcriptional regulators. Many animal microRNAs are clustered in the genome, and it has been shown that clustered microRNAs may be transcribed as a single transcript. Polycistronic microRNAs are often members of the same family, suggesting a role of tandem duplication in the emergence of clusters. The mir-2 microRNA family is the largest in Drosophila melanogaster, with 8 members that are mostly clustered in the genome. Previous studies suggest that the copy number and genomic distribution of mir-2 family members has been subject to significant change during evolution. The effects of such changes on their function are still unknown. Here we study the evolution of function in the mir-2 family. Our analyses show that, in spite of the change in number and organization among invertebrates, most mir-2 loci produce very similar mature microRNA products. Multiple mature miR-2 sequences are predicted to target genes involved in neural development in Drosophila. These targeting properties are conserved in the distant species Caenorhabditis elegans. Duplication followed by functional diversification is frequent during protein-coding gene evolution. However, our results suggest that the production of microRNA clusters by gene duplication rarely involves functional changes. This pattern of functional redundancy among clustered paralogous microRNAs reflects birth-and-death evolutionary dynamics. However, we identified a small number of mir-2 sequences in Drosophila that may have undergone functional shifts associated with genomic rearrangements. Therefore, redundancy in microRNA families may facilitate the acquisition of novel functional features.

Optimization of SVM parameters for recognition of regulatory DNA sequences

Identification and recognition of specific functionally-important DNA sequence fragments such as regulatory sequences are considered the most important problems in bioinformatics. One type of such fragments are promoters, i.e., short regulatory DNA sequences located upstream of a gene. Detection of regulatory DNA sequences is important for successful gene prediction and gene expression studies. In this paper, Support Vector Machine (SVM) is used for classification of DNA sequences and recognition of the regulatory sequences. For optimal classification, various SVM learning and kernel parameters (hyperparameters) and their optimization methods are analyzed. In a case study, optimization of the SVM hyperparameters for linear, polynomial and power series kernels is performed using a modification of the Nelder–Mead (downhill simplex) algorithm. The method allows for improving the precision of identification of the regulatory DNA sequences. The results of promoter recognition for the drosophila sequence datasets are presented.

Hyper-expansion of large DNA segments in the genome of kuruma shrimp, Marsupenaeus japonicus

BackgroundHigher crustaceans (class Malacostraca) represent the most species-rich and morphologically diverse group of non-insect arthropods and many of its members are commercially important. Although the crustacean DNA sequence information is growing exponentially, little is known about the genome organization of Malacostraca. Here, we constructed a bacterial artificial chromosome (BAC) library and performed BAC-end sequencing to provide genomic information for kuruma shrimp (Marsupenaeus japonicus), one of the most widely cultured species among crustaceans, and found the presence of a redundant sequence in the BAC library. We examined the BAC clone that includes the redundant sequence to further analyze its length, copy number and location in the kuruma shrimp genome.ResultsMj024A04 BAC clone, which includes one redundant sequence, contained 27 putative genes and seemed to display a normal genomic DNA structure. Notably, of the putative genes, 3 genes encode homologous proteins to the inhibitor of apoptosis protein and 7 genes encode homologous proteins to white spot syndrome virus, a virulent pathogen known to affect crustaceans. Colony hybridization and PCR analysis of 381 BAC clones showed that almost half of the BAC clones maintain DNA segments whose sequences are homologous to the representative BAC clone Mj024A04. The Mj024A04 partial sequence was detected multiple times in the kuruma shrimp nuclear genome with a calculated copy number of at least 100. Microsatellites based BAC genotyping clearly showed that Mj024A04 homologous sequences were cloned from at least 48 different chromosomal loci. The absence of micro-syntenic relationships with the available genomic sequences of Daphnia and Drosophila suggests the uniqueness of these fragments in kuruma shrimp from current arthropod genome sequences.ConclusionsOur results demonstrate that hyper-expansion of large DNA segments took place in the kuruma shrimp genome. Although we analyzed only a part of the duplicated DNA segments, our result suggested that it is difficult to analyze the shrimp genome following normal analytical methodology. Hence, it is necessary to avoid repetitive sequence (such as segmental duplications) when studying the other unique structures in the shrimp genome.

DISCOVER: a feature-based discriminative method for motif search in complex genomes.

Motivation: Identifying transcription factor binding sites (TFBSs) encoding complex regulatory signals in metazoan genomes remains a challenging problem in computational genomics. Due to degeneracy of nucleotide content among binding site instances or motifs, and intricate ‘grammatical organization’ of motifs within cis-regulatory modules (CRMs), extant pattern matching-based in silico motif search methods often suffer from impractically high false positive rates, especially in the context of analyzing large genomic datasets, and noisy position weight matrices which characterize binding sites. Here, we try to address this problem by using a framework to maximally utilize the information content of the genomic DNA in the region of query, taking cues from values of various biologically meaningful genetic and epigenetic factors in the query region such as clade-specific evolutionary parameters, presence/absence of nearby coding regions, etc. We present a new method for TFBS prediction in metazoan genomes that utilizes both the CRM architecture of sequences and a variety of features of individual motifs. Our proposed approach is based on a discriminative probabilistic model known as conditional random fields that explicitly optimizes the predictive probability of motif presence in large sequences, based on the joint effect of all such features.Results: This model overcomes weaknesses in earlier methods based on less effective statistical formalisms that are sensitive to spurious signals in the data. We evaluate our method on both simulated CRMs and real Drosophila sequences in comparison with a wide spectrum of existing models, and outperform the state of the art by 22% in F1 score.Availability and Implementation: The code is publicly available at http://www.sailing.cs.cmu.edu/discover.html.Contact: epxing@cs.cmu.eduSupplementary information: Supplementary data are available at Bioinformatics online.

Genomic analysis of a sexually-selected character: EST sequencing and microarray analysis of eye-antennal imaginal discs in the stalk-eyed fly Teleopsis dalmanni (Diopsidae).

BackgroundMany species of stalk-eyed flies (Diopsidae) possess highly-exaggerated, sexually dimorphic eye-stalks that play an important role in the mating system of these flies. Eye-stalks are increasingly being used as a model system for studying sexual selection, but little is known about the genetic mechanisms producing variation in these ornamental traits. Therefore, we constructed an EST database of genes expressed in the developing eye-antennal imaginal disc of the highly dimorphic species Teleopsis dalmanni. We used this set of genes to construct microarray slides and compare patterns of gene expression between lines of flies with divergent eyespan.ResultsWe generated 33,229 high-quality ESTs from three non-normalized libraries made from the developing eye-stalk tissue at different developmental stages. EST assembly and annotation produced a total of 7,066 clusters comprising 3,424 unique genes with significant sequence similarity to a protein in either Drosophila melanogaster or Anopheles gambiae. Comparisons of the transcript profiles at different stages reveal a developmental shift in relative expression from genes involved in anatomical structure formation, transcription, and cell proliferation at the larval stage to genes involved in neurological processes and cuticle production during the pupal stages. Based on alignments of the EST fragments to homologous sequences in Drosophila and Anopheles, we identified 20 putative gene duplication events in T. dalmanni and numerous genes undergoing significantly faster rates of evolution in T. dalmanni relative to the other Dipteran species. Microarray experiments identified over 350 genes with significant differential expression between flies from lines selected for high and low relative eyespan but did not reveal any primary biological process or pathway that is driving the expression differences.ConclusionThe catalogue of genes identified in the EST database provides a valuable framework for a comprehensive examination of the genetic basis of eye-stalk variation. Several candidate genes, such as crooked legs, cdc2, CG31917 and CG11577, emerge from the analysis of gene duplication, protein evolution and microarray gene expression. Additional comparisons of expression profiles between, for example, males and females, and species that differ in eye-stalk sexual dimorphism, are now enabled by these resources.

Pol II promoter prediction using characteristic 4-mer motifs: a machine learning approach

BackgroundEukaryotic promoter prediction using computational analysis techniques is one of the most difficult jobs in computational genomics that is essential for constructing and understanding genetic regulatory networks. The increased availability of sequence data for various eukaryotic organisms in recent years has necessitated for better tools and techniques for the prediction and analysis of promoters in eukaryotic sequences. Many promoter prediction methods and tools have been developed to date but they have yet to provide acceptable predictive performance. One obvious criteria to improve on current methods is to devise a better system for selecting appropriate features of promoters that distinguish them from non-promoters. Secondly improved performance can be achieved by enhancing the predictive ability of the machine learning algorithms used.ResultsIn this paper, a novel approach is presented in which 128 4-mer motifs in conjunction with a non-linear machine-learning algorithm utilising a Support Vector Machine (SVM) are used to distinguish between promoter and non-promoter DNA sequences. By applying this approach to plant, Drosophila, human, mouse and rat sequences, the classification model has showed 7-fold cross-validation percentage accuracies of 83.81%, 94.82%, 91.25%, 90.77% and 82.35% respectively. The high sensitivity and specificity value of 0.86 and 0.90 for plant; 0.96 and 0.92 for Drosophila; 0.88 and 0.92 for human; 0.78 and 0.84 for mouse and 0.82 and 0.80 for rat demonstrate that this technique is less prone to false positive results and exhibits better performance than many other tools. Moreover, this model successfully identifies location of promoter using TATA weight matrix.ConclusionThe high sensitivity and specificity indicate that 4-mer frequencies in conjunction with supervised machine-learning methods can be beneficial in the identification of RNA pol II promoters comparative to other methods. This approach can be extended to identify promoters in sequences for other eukaryotic genomes.

The Adhesion GPCR GPR125 is specifically expressed in the choroid plexus and is upregulated following brain injury

BackgroundGPR125 belongs to the family of Adhesion G protein-coupled receptors (GPCRs). A single copy of GPR125 was found in many vertebrate genomes. We also identified a Drosophila sequence, DmCG15744, which shares a common ancestor with the entire Group III of Adhesion GPCRs, and also contains Ig, LRR and HBD domains which were observed in mammalian GPR125.ResultsWe found specific expression of GPR125 in cells of the choroid plexus using in situ hybridization and protein-specific antibodies and combined in situ/immunohistochemistry co-localization using cytokeratin, a marker specific for epithelial cells. Induction of inflammation by LPS did not change GPR125 expression. However, GPR125 expression was transiently increased (almost 2-fold) at 4 h after traumatic brain injury (TBI) followed by a decrease (approximately 4-fold) from 2 days onwards in the choroid plexus as well as increased expression (2-fold) in the hippocampus that was delayed until 1 day after injury.ConclusionThese findings suggest that GPR125 plays a functional role in choroidal and hippocampal response to injury.

Annotation and expression profiling of apoptosis-related genes in the yellow fever mosquito, Aedes aegypti

Apoptosis has been extensively studied in Drosophila by both biochemical and genetic approaches, but there is a lack of knowledge about the mechanisms of apoptosis regulation in other insects. In mosquitoes, apoptosis occurs during Plasmodium and arbovirus infection in the midgut, suggesting that apoptosis plays a role in mosquito innate immunity. We searched the Aedes aegypti genome for apoptosis-related genes using Drosophila and Anopheles gambiae protein sequences as queries. In this study we have identified eleven caspases, three inhibitor of apoptosis (IAP) proteins, a previously unreported IAP antagonist, and orthologs of Drosophila Ark, Dnr1, and BG4 (also called dFadd). While most of these genes have been previously annotated, we have improved the annotation of several of them, and we also report the discovery of four previously unannotated apoptosis-related genes. We examined the developmental expression profile of these genes in Ae. aegypti larvae, pupae and adults, and we also studied the function of a novel IAP antagonist, IMP. Expression of IMP in mosquito cells caused apoptosis, indicating that it is a functional pro-death protein. Further characterization of these genes will help elucidate the molecular mechanisms of apoptosis regulation in Ae. aegypti.

Purifying Selection Maintains Highly Conserved Noncoding Sequences in Drosophila

The majority of metazoan genomes consist of nonprotein-coding regions, although the functional significance of most noncoding DNA sequences remains unknown. Highly conserved noncoding sequences (CNSs) have proven to be reliable indicators of functionally constrained sequences such as cis-regulatory elements and noncoding RNA genes. However, CNSs may arise from nonselective evolutionary processes such as genomic regions with extremely low mutation rates known as mutation "cold spots." Here we combine comparative genomic data from recently completed insect genome projects with population genetic data in Drosophila melanogaster to test predictions of the mutational cold spot model of CNS evolution in the genus Drosophila. We find that point mutations in intronic and intergenic CNSs exhibit a significant reduction in levels of divergence relative to levels of polymorphism, as well as a significant excess of rare derived alleles, compared with either the nonconserved spacer regions between CNSs or with 4-fold silent sites in coding regions. Controlling for the effects of purifying selection, we find no evidence of positive selection acting on Drosophila CNSs, although we do find evidence for the action of recurrent positive selection in the spacer regions between CNSs. We estimate that approximately 85% of sites in Drosophila CNSs are under constraint with selection coefficients (N(e)s) on the order of 10-100, and thus, the estimated strength and number of sites under purifying selection is greater for Drosophila CNSs relative to those in the human genome. These patterns of nonneutral molecular evolution are incompatible with the mutational cold spot hypothesis to explain the existence of CNSs in Drosophila and, coupled with similar findings in mammals, argue against the general likelihood that CNSs are generated by mutational cold spots in any metazoan genome.