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The genetic basis for the acquisition and retention of innovative morphological structures is of wide interest to evolutionary biologists. Mammals possess specific anatomical features that are not found in other vertebrates, such as external ears, placenta, and hair. It has been inferred that genetic changes in developmental regulatory pathways may contribute, in part, to the evolution of such innovations. In particular, duplicated developmental genes (paralogs) play an important role in generating variations in evolutionarily important morphological structures via deployment of novel cis-regulatory elements. The use of comparative genomics together with mouse transgenic methods provides an effective strategy for interrogating the regulatory landscape of duplicated loci of major developmental genes, as reported here for one of the Dlx gene clusters. The Distal-less (Dlx) genes comprise a homeobox-containing multigene family. The mammalian Dlx gene family consists of six members that are orthologous to the Drosophila Distal-less gene and which are organized into three convergently transcribed (tail-to-tail) bigene clusters; it is presumed that the ancestral bigene cluster was generated initially by tandem gene duplication followed by whole genome duplications (Stock et al., ‘96). Bigene cluster pairs are Dlx1 and Dlx2, Dlx6 and Dlx5, and Dlx41 and Dlx3 (Simeone et al., ‘94; McGuinness et al., ‘96; Nakamura et al., ‘96; Ellies et al., ‘97b; Zerucha and Ekker, 2000), each of which is originally linked to a HOX cluster (HOXD, HOXA, and HOXB, respectively). Dlx expression patterns and functional studies by targeted Dlx deletions provide strong evidence that these transcription factors play major roles in patterning of multiple developmental structures (Merlo et al., 2000; Quint et al., 2000; Zerucha and Ekker, 2000; Zerucha et al., 2000; Depew et al., 2002; Panganiban and Rubenstein, 2002). Though vertebrate Distal-less genes were originally described as being primarily “head” homeobox genes (Duboule, ‘94), they have been shown also to be involved in development of many other structures, that is, limbs, peripheral neurons, whiskers/hair follicles, otic vesicles, teeth, median fins in zebrafish, cement glands in Xenopus, keratinocytes in Xenopus and mice, placenta, genital tubercle and hematopoietic cells in humans (Dirksen et al., ‘93; Akimenko et al., ‘94; Morasso et al., ‘94, ‘99; Porteus et al., ‘94; Shimamoto et al., ‘97; Zerucha et al., ‘97; Park and Morasso, ‘99; Hwang et al., 2008). cis-Regulatory elements confer cell and tissue specificity with regard to the transcriptional machinery and have been implicated as being important for morphological evolution, particularly in the context of embryonic transcription factor genes (Shashikant et al., ‘98; Carroll, 2008; Cretekos et al., 2008; Levine, 2010). Since Dlx genes have been co-opted recurrently in vertebrate evolution by acquisition of novel expression sites, their cis-regulatory element functionality and evolutionary history are of great interest in terms of bauplan divergences in mammals. Delineation of the genomic organization and overlapping expression patterns of the Dlx genes provides a window into understanding the genomic control of development of the many structures described above. Expression patterns of linked Dlx genes have suggested that cis-regulatory elements controlling spatio-temporal expression exist within their respective intergenic regions (Ellies et al., ‘97a). Functional analyses of the intergenic region between paired Dlx genes showed that this region indeed harbors important cis-regulatory elements (Zerucha et al., 2000; Sumiyama et al., 2002; Ghanem et al., 2003). In the Dlx3–4 cluster there are several evolutionarily conserved elements within about 20 kb of the intergenic non-coding region of human and mouse. Moreover, one of these was shown to be a vital enhancer element for branchial arch expression in a Dlx3 specific manner (Sumiyama and Ruddle, 2003). Global alignments using genomic sequences from multiple species is an effective means to identify potentially functional elements in the non-coding regions. The ENCODE project report has shown that a total of 5% of the bases in the genome can be confidently identified as being under evolutionary constraint in mammals, and that approximately 60% of these bases are biologically functional on the basis of experimental assays (ENCODE, 2007). The purpose of the current study was to investigate the rate of change of molecular evolution of cis-elements possibly related to mammalian specific characters of the Dlx3–4 bigene cluster using an extended dataset of 12 diverse mammalian species, including a marsupial and a monotreme. Large-scale sequence comparisons and subsequent constrained motif extraction were conducted with the aim of identifying molecular evolutionary trends in the respective elements. The analyses show that conserved nucleotide elements in intergenic region evolve at markedly different rates and highlight varying patterns of evolutionary divergence among the species. In transgenic mice experiments, we demonstrated that I37-2 cis-regulatory element for branchial arches showed theria-specific conservation and enhancer activity, whereas that of the platypus showed no enhancer activity in branchial arches. The findings serve as guideposts for our ultimate goal to examine the nature in which the cis-regulatory modules in the Dlx bigene clusters act to bring about developmental and gross morphological changes in vertebrate evolution.

Here, we demonstrate how comparative sequence analysis facilitates genome-wide base-pair-level interpretation of individual genetic variation and address two questions of importance for human personal genomics: first, whether an individual's functional variation comes mostly from noncoding or coding polymorphisms; and, second, whether population-specific or globally-present polymorphisms contribute more to functional variation in any given individual. Neither has been definitively answered by analyses of existing variation data because of a focus on coding polymorphisms, ascertainment biases in favor of common variation, and a lack of base-pair-level resolution for identifying functional variants. We resequenced 575 amplicons within 432 individuals at genomic sites enriched for evolutionary constraint and also analyzed variation within three published human genomes. We find that single-site measures of evolutionary constraint derived from mammalian multiple sequence alignments are strongly predictive of reductions in modern-day genetic diversity across a range of annotation categories and across the allele frequency spectrum from rare (10% minor allele frequency). Furthermore, we show that putatively functional variation in an individual genome is dominated by polymorphisms that do not change protein sequence and that originate from our shared ancestral population and commonly segregate in human populations. These observations show that common, noncoding alleles contribute substantially to human phenotypes and that constraint-based analyses will be of value to identify phenotypically relevant variants in individual genomes.

The ENCyclopedia Of DNA Elements (ENCODE) Project aims to identify all functional elements in the human genome sequence. The pilot phase of the Project is focused on a specified 30 megabases (∼1%) of the human genome sequence and is organized as an international consortium of computational and laboratory-based scientists working to develop and apply high-throughput approaches for detecting all sequence elements that confer biological function. The results of this pilot phase will guide future efforts to analyze the entire human genome.

Chromosome 5 is one of the largest human chromosomes and contains numerous intrachromosomal duplications, yet it has one of the lowest gene densities. This is partially explained by numerous gene-poor regions that display a remarkable degree of noncoding conservation with non-mammalian vertebrates, suggesting that they are functionally constrained. In total, we compiled 177.7 million base pairs of highly accurate finished sequence containing 923 manually curated protein-coding genes including the protocadherin and interleukin gene families. We also completely sequenced versions of the large chromosome-5-specific internal duplications. These duplications are very recent evolutionary events and probably have a mechanistic role in human physiological variation, as deletions in these regions are the cause of debilitating disorders including spinal muscular atrophy.

The dramatic radiation of sticklebacks in different post-glacial environments provides a unique opportunity to study the molecular mechanisms that underlie rapid evolutionary change in vertebrates. We have developed a number of genomic and genetic tools to facilitate further study of a wide range of morphological, physiological and behavioral traits in sticklebacks. A large collection of microsatellite markers has previously been developed for use in genome-wide linkage mapping of interesting traits in crosses between different stickleback forms. cDNA libraries have been generated and EST sequencing projects have begun to isolate stickleback homologs of developmental control genes. Large insert BAC libraries have been built to compare chromosome regions of interest from both anadromous and freshwater stickleback populations. Large scale fingerprinting of one of these libraries has been used to assemble overlapping contigs of BAC clones for chromosome walking and positional cloning. Together with recent development of methods to make transgenic sticklebacks, these tools should make it possible to identify the molecular basis of many different evolutionary traits in stickleback, and to begin to answer longstanding questions about the numbers and types of mutations that control the appearance of new morphological, physiological, and behavioral traits during vertebrate evolution.

Attempts to identify regulatory sequences in the human genome have involved experimental and computational methods such as cross-species sequence comparisons and the detection of transcription factor binding-site motifs in coexpressed genes. Although these strategies provide information on which genomic regions are likely to be involved in gene regulation, they do not give information on their functions. We have developed a functional selection for promoter regions in the human genome that uses a retroviral plasmid library-based system. This approach enriches for and detects promoter function of isolated DNA fragments in an in vitro cell culture assay. By using this method, we have discovered likely promoters of known and predicted genes, as well as many other putative promoter regions based on the presence of features such as CpG islands. Comparison of sequences of 858 plasmid clones selected by this assay with the human genome draft sequence indicates that a significantly higher percentage of sequences align to the 500-bp segment upstream of the transcription start sites of known genes than would be expected from random genomic sequences. We also observed enrichment for putative promoter regions of genes predicted in at least two annotation databases and for clones overlapping with CpG islands. Functional validation of randomly selected clones enriched by this method showed that a large fraction of these putative promoters can drive the expression of a reporter gene in transient transfection experiments. This method promises to be a useful genome-wide function-based approach that can complement existing methods to look for promoters.

Large-scale genetic screens in zebrafish have identified thousands of mutations in hundreds of essential genes. The genetic mapping of these mutations is necessary to link DNA sequences to the gene functions defined by mutant phenotypes. Here, we report two advances that will accelerate the mapping of zebrafish mutations: (1) The construction of a first generation single nucleotide polymorphism (SNP) map of the zebrafish genome comprising 2035 SNPs and 178 small insertions/deletions, and (2) the development of a method for mapping mutations in which hundreds of SNPs can be scored in parallel with an oligonucleotide microarray. We have demonstrated the utility of the microarray technique in crosses with haploid and diploid embryos by mapping two known mutations to their previously identified locations. We have also used this approach to localize four previously unmapped mutations. We expect that mapping with SNPs and oligonucleotide microarrays will accelerate the molecular analysis of zebrafish mutations.[Supplemental material is available online atwww.genome.org. The sequence data described in this paper have been submitted to dbSNP under accession nos. 5103507–5105537. The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: J. Postlethwait, C.-B. Chien, C. Kimmel, L. Maves, and M. Westerfield.]

Marine stickleback fish have colonized and adapted to thousands of streams and lakes formed since the last ice age, providing an exceptional opportunity to characterize genomic mechanisms underlying repeated ecological adaptation in nature. Here we develop a high-quality reference genome assembly for threespine sticklebacks. By sequencing the genomes of twenty additional individuals from a global set of marine and freshwater populations, we identify a genome-wide set of loci that are consistently associated with marine–freshwater divergence. Our results indicate that reuse of globally shared standing genetic variation, including chromosomal inversions, has an important role in repeated evolution of distinct marine and freshwater sticklebacks, and in the maintenance of divergent ecotypes during early stages of reproductive isolation. Both coding and regulatory changes occur in the set of loci underlying marine–freshwater evolution, but regulatory changes appear to predominate in this well known example of repeated adaptive evolution in nature., National Human Genome Research Institute (U.S.), National Human Genome Research Institute (U.S.) (NHGRI CEGS Grant P50-HG002568)

The living coelacanth is a lobe-finned fish that represents an early evolutionary departure from the lineage that led to land vertebrates, and is of extreme interest scientifically. It has changed very little in appearance from fossilized coelacanths of the Cretaceous (150 to 65 million years ago), and is often referred to as a “living fossil.” An important general question is whether long-term stasis in morphological evolution is associated with stasis in genome evolution. To this end we have used targeted genome sequencing for acquiring 1,612,752 bp of high quality finished sequence encompassing the four HOX clusters of the Indonesian coelacanth Latimeria menadoensis . Detailed analyses were carried out on genomic structure, gene and repeat contents, conserved noncoding regions, and relative rates of sequence evolution in both coding and noncoding tracts. Our results demonstrate conclusively that the coelacanth HOX clusters are evolving comparatively slowly and that this taxon should serve as a viable outgroup for interpretation of the genomes of tetrapod species.

Since its start, the Mammalian Gene Collection (MGC) has sought to provide at least one full-protein-coding sequence cDNA clone for every human and mouse gene with a RefSeq transcript, and at least 6200 rat genes. The MGC cloning effort initially relied on random expressed sequence tag screening of cDNA libraries. Here, we summarize our recent progress using directed RT-PCR cloning and DNA synthesis. The MGC now contains clones with the entire protein-coding sequence for 92% of human and 89% of mouse genes with curated RefSeq (NM-accession) transcripts, and for 97% of human and 96% of mouse genes with curated RefSeq transcripts that have one or more PubMed publications, in addition to clones for more than 6300 rat genes. These high-quality MGC clones and their sequences are accessible without restriction to researchers worldwide.

We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view about chromatin structure has emerged, including its interrelationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded novel mechanistic and evolutionary insights about the functional landscape of the human genome. Together, these studies are defining a path forward to pursue a more-comprehensive characterisation of human genome function.

Major phenotypic changes evolve in parallel in nature by molecular mechanisms that are largely unknown. Here, we use positional cloning methods to identify the major chromosome locus controlling armor plate patterning in wild threespine sticklebacks. Mapping, sequencing, and transgenic studies show that the Ectodysplasin (EDA) signaling pathway plays a key role in evolutionary change in natural populations and that parallel evolution of stickleback low-plated phenotypes at most freshwater locations around the world has occurred by repeated selection of Eda alleles derived from an ancestral low-plated haplotype that first appeared more than two million years ago. Members of this clade of low-plated alleles are present at low frequencies in marine fish, which suggests that standing genetic variation can provide a molecular basis for rapid, parallel evolution of dramatic phenotypic change in nature.

The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5′-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

Human chromosome 16 features one of the highest levels of segmentally duplicated sequence among the human autosomes. We report here the 78,884,754 base pairs of finished chromosome 16 sequence, representing over 99.9% of its euchromatin. Manual annotation revealed 880 protein-coding genes confirmed by 1,670 aligned transcripts, 19 transfer RNA genes, 341 pseudogenes and three RNA pseudogenes. These genes include metallothionein, cadherin and iroquois gene families, as well as the disease genes for polycystic kidney disease and acute myelomonocytic leukaemia. Several large-scale structural polymorphisms spanning hundreds of kilobase pairs were identified and result in gene content differences among humans. Whereas the segmental duplications of chromosome 16 are enriched in the relatively gene-poor pericentromere of the p arm, some are involved in recent gene duplication and conversion events that are likely to have had an impact on the evolution of primates and human disease susceptibility.

Background: Many different environmental and genetic sex-determination mechanisms are found in nature. Closely related species can use different master sex-determination switches, suggesting that these developmental pathways can evolve very rapidly. Previous cytological studies suggest that recently diverged species of stickleback fish have different sex chromosome complements. Here, we investigate the genetic and chromosomal mechanisms that underlie sex determination in the threespine stickleback ( Gasterosteus aculeatus ). Results: Genome-wide linkage mapping identifies a single chromosome region at the distal end of linkage group (LG) 19, which controls male or female sexual development in threespine sticklebacks. Although sex chromosomes are not cytogenetically visible in this species, several lines of evidence suggest that LG 19 is an evolving sex chromosome system, similar to the XX female/XY male system in many other species: (1) males are consistently heterozygous for unique alleles in this region; (2) recombination between loci linked to the sex-determination region is reduced in male meiosis relative to female meiosis; (3) sequence analysis of X- and Y-specific bacterial artificial chromosome (BAC) clones from the sex-determination region reveals many sequence differences between the X- and Y-specific clones; and (4) the Y chromosome has accumulated transposable elements and local duplications. Conclusions: Taken together, our data suggest that threespine sticklebacks have a simple chromosomal mechanism for sex determination based on a nascent Y chromosome that is less than 10 million years old. Further analysis of the stickleback system will provide an exciting window into the evolution of sex-determination pathways and sex chromosomes in vertebrates.

The synaptic cell adhesion molecules encoded by the protocadherin gene cluster are hypothesized to provide a molecular code involved in the generation of synaptic complexity in the developing brain. Variation in copy number and sequence content of protocadherin cluster genes among vertebrate species could reflect adaptive differences in protocadherin function. We have completed an analysis of zebrafish protocadherin cluster genes. Zebrafish have two unlinked protocadherin clusters, DrPcdh1 and DrPcdh2. Like mammalian protocadherin clusters, DrPcdh1 has both α and γ variable and constant region exons. A consensus protocadherin promoter motif sequence identified in mammals is also conserved in zebrafish. Few orthologous relationships, however, are apparent between zebrafish and mammalian protocadherin proteins. Here we show that protocadherin cluster genes in human, mouse, rat, and zebrafish are subject to striking gene conversion events. These events are restricted to regions of the coding sequence, particularly the coding sequences of ectodomain 6 and the cytoplasmic domain. Diversity among paralogs is restricted to particular ectodomains that are excluded from conversion events. Conversion events are also strongly correlated with an increase in third-position GC content. We propose that the combination of lineage-specific duplication, restricted gene conversion, and adaptive variation in diversified ectodomains drives vertebrate protocadherin cluster evolution.

Chromosome 19 has the highest gene density of all human chromosomes, more than double the genome-wide average. The large clustered gene families, corresponding high G + C content, CpG islands and density of repetitive DNA indicate a chromosome rich in biological and evolutionary significance. Here we describe 55.8 million base pairs of highly accurate finished sequence representing 99.9% of the euchromatin portion of the chromosome. Manual curation of gene loci reveals 1,461 protein-coding genes and 321 pseudogenes. Among these are genes directly implicated in mendelian disorders, including familial hypercholesterolaemia and insulin-resistant diabetes. Nearly one-quarter of these genes belong to tandemly arranged families, encompassing more than 25% of the chromosome. Comparative analyses show a fascinating picture of conservation and divergence, revealing large blocks of gene orthology with rodents, scattered regions with more recent gene family expansions and deletions, and segments of coding and non-coding conservation with the distant fish species Takifugu.

Six strains of human cytomegalovirus have been sequenced, including two laboratory strains (AD169 and Towne) that have been extensively passaged in fibroblasts and four clinical isolates that have been passaged to a limited extent in the laboratory (Toledo, FIX, PH, and TR). All of the sequenced viral genomes have been cloned as infectious bacterial artificial chromosomes. A total of 252 ORFs with the potential to encode proteins have been identified that are conserved in all four clinical isolates of the virus. Multiple sequence alignments revealed substantial variation in the amino acid sequences encoded by many of the conserved ORFs.

As the final sequencing of the human genome has now been completed, we present the results of the largest examination of the quality of the finished DNA sequence. The completed study covers the major contributing sequencing centres and is based on a rigorous combination of laboratory experiments and computational analysis.

The genomic organization of the human protocadherin α, β, and γ gene clusters (designated Pcdhα [gene symbol PCDHA], Pcdhβ [PCDHB], and Pcdhγ [PCDHG]) is remarkably similar to that of immunoglobulin and T-cell receptor genes. The extracellular and transmembrane domains of each protocadherin protein are encoded by an unusually large “variable” region exon, while the intracellular domains are encoded by three small “constant” region exons located downstream from a tandem array of variable region exons. Here we report the results of a comparative DNA sequence analysis of the orthologous human (750 kb) and mouse (900 kb) protocadherin gene clusters. The organization of Pcdhα andPcdhγ gene clusters in the two species is virtually identical, whereas the mouse Pcdhβ gene cluster is larger and contains more genes than the human Pcdhβ gene cluster. We identified conserved DNA sequences upstream of the variable region exons, and found that these sequences are more conserved between orthologs than between paralogs. Within this region, there is a highly conserved DNA sequence motif located at about the same position upstream of the translation start codon of each variable region exon. In addition, the variable region of each gene cluster contains a rich array of CpG islands, whose location corresponds to the position of each variable region exon. These observations are consistent with the proposal that the expression of each variable region exon is regulated by a distinct promoter, which is highly conserved between orthologous variable region exons in mouse and human.[The sequence data described in this paper have been submitted to the GenBank/EMBL/DDBJ data library under accession nos. AY013756–AY013813,AY013873–AY013878, AF332005, and AF332006.]

BACKGROUND: Corus CAD is a clinically validated test based on age, sex, and expression levels of 23 genes in whole blood that provides a score (1-40 points) proportional to the likelihood of obstructive coronary disease. Clinical laboratory process variability was examined using whole blood controls across a 24 month period: Intra-batch variability was assessed using sample replicates; inter-batch variability examined as a function of laboratory personnel, equipment, and reagent lots. METHODS/RESULTS: To assess intra-batch variability, five batches of 132 whole blood controls were processed; inter-batch variability was estimated using 895 whole blood control samples. ANOVA was used to examine inter-batch variability at 4 process steps: RNA extraction, cDNA synthesis, cDNA addition to assay plates, and qRT-PCR. Operator, machine, and reagent lots were assessed as variables for all stages if possible, for a total of 11 variables. Intra- and inter-batch variations were estimated to be 0.092 and 0.059 Cp units respectively (SD); total laboratory variation was estimated to be 0.11 Cp units (SD). In a regression model including all 11 laboratory variables, assay plate lot and cDNA kit lot contributed the most to variability (p = 0.045; 0.009 respectively). Overall, reagent lots for RNA extraction, cDNA synthesis, and qRT-PCR contributed the most to inter-batch variance (52.3%), followed by operators and machines (18.9% and 9.2% respectively), leaving 19.6% of the variance unexplained. CONCLUSION: Intra-batch variability inherent to the PCR process contributed the most to the overall variability in the study while reagent lot showed the largest contribution to inter-batch variability.

Regions of extensive linkage disequilibrium (LD) appear to be a common feature of the human genome. However, the mechanisms that maintain these regions are unknown. In an effort to understand whether gene density contributes to LD, we determined the degree of promoter sequence variation in a large tandem-arrayed gene family, the human protocadherin alpha cluster, on chromosome 5. These genes are expressed at synaptic junctions in the developing brain and the adult brain and may be involved in the determination of synaptic complexity. We sequenced the promoters of all 13 alpha protocadherin genes in 96 European Americans and identified polymorphisms in the promoters alpha 1, alpha 3, alpha 4, alpha 5, alpha 7, alpha 9, alpha 11, and alpha 13. In these promoters, 11 common SNPs are in extensive LD, forming two 48-kb haplotypes of equal frequency, in this population, that extend from the alpha1 through alpha 7 genes. We sequenced these promoters in East Asians and African Americans, and we estimated haplotype frequencies and calculated LD statistics for all three populations. Our results indicate that, although extensive LD is an ancient feature of the alpha cluster, it has eroded over time. SNPs 3' of alpha 7 are involved in ancestral recombination events in all populations, and overall alpha-cluster LD is reduced in African Americans. We obtained significant positive values for Tajima's D test for all alpha promoter SNPs in Europeans (D=3.03) and East Asians (D=2.64), indicating an excess of intermediate-frequency variants, which is a signature of balancing selection. We also discovered a 16.7-kb deletion that truncates the alpha 8 gene and completely removes the alpha 9 and alpha 10 genes. This deletion appears in unaffected individuals from multiple populations, suggesting that a reduction in protocadherin gene number is not obviously deleterious.