Evolutionary constraints in conserved nongenic sequences of mammals

Mammalian genomes contain many highly conserved nongenic sequences (CNGs) whose functional significance is poorly understood. Sets of CNGs have previously been identified by selecting the most conserved elements from a chromosome or genome, but in these highly selected samples, conservation may be unrelated to purifying selection. Furthermore, conservation of CNGs may be caused by mutation rate variation rather than selective constraints. To account for the effect of selective sampling, we have examined conservation of CNGs in taxa whose evolution is largely independent of the taxa from which the CNGs were initially identified, and we have controlled for mutation rate variation in the genome. We show that selective constraints in CNGs and their flanks are about one-half as strong in hominids as in murids, implying that hominids have accumulated many slightly deleterious mutations in functionally important nongenic regions. This is likely to be a consequence of the low effective population size of hominids leading to a reduced effectiveness of selection. We estimate that there are one and two times as many conserved nucleotides in CNGs as in known protein-coding genes of hominids and murids, respectively. Polymorphism frequencies in CNGs indicate that purifying selection operates in these sequences. During hominid evolution, we estimate that a total of about three deleterious mutations in CNGs and protein-coding genes have been selectively eliminated per diploid genome each generation, implying that deleterious mutations are eliminated from populations non-independently and that sex is necessary for long-term population persistence.     

Genomic characterization of the Bacillus cereus sensu lato species: backdrop to the evolution of Bacillus anthracis

The key genes required for Bacillus anthracis to cause anthrax have been acquired recently by horizontal gene transfer. To understand the genetic background for the evolution of B. anthracis virulence, we obtained high-redundancy genome sequences of 45 strains of the Bacillus cereus sensu lato (s.l.) species that were chosen for their genetic diversity within the species based on the existing multilocus sequence typing scheme. From the resulting data, we called more than 324,000 new genes representing more than 12,333 new gene families for this group. The core genome size for the B. cereus s.l. group was ～1750 genes, with another 2150 genes found in almost every genome constituting the extended core. There was a paucity of genes specific and conserved in any clade. We found no evidence of recent large-scale gene loss in B. anthracis or for unusual accumulation of nonsynonymous DNA substitutions in the chromosome; however, several B. cereus genomes isolated from soil and not previously associated with human disease were degraded to various degrees. Although B. anthracis has undergone an ecological shift within the species, its chromosome does not appear to be exceptional on a macroscopic scale compared with close relatives.     

Small fitness effect of mutations in highly conserved non-coding regions

Comparison of human and mouse genomes has revealed that many non-coding regions have levels of sequence conservation similar to protein-coding genes. These regions have attracted a lot of attention as potentially functional genomic sequences. However, little is known about the effect mutations in these conserved non-coding regions have on fitness and how many of them are present in the human genome as deleterious polymorphisms. To gain insight into the selective constraints imposed on conserved non-coding and protein-coding regions, we compared substitution rates in primate and rodent lineages and analyzed the density and allele frequencies of human polymorphism. Genomic regions conserved between primate and rodent groups show higher relative conservation within rodents than within primates. Thus, our analysis indicates a genome-wide relaxation of selective constraint in the primate lineage, which most likely resulted from a smaller effective population size. We found that this relaxation is much more profound in conserved non-coding regions than in protein-coding regions, and that mutations at a large proportion of sites in conserved non-coding regions are associated with very small fitness effect. Data on human polymorphism are also consistent with very weak selection in conserved non-coding regions. This staggering enrichment in sites at the borderline of neutrality can be explained by assuming an important role for synergistic epistasis in the evolution of non-coding regions. Our results suggest that most individual mutations in conserved non-coding regions are only slightly deleterious but are numerous and may have a significant cumulative impact on fitness.     

Maintenance of chloroplast-derived sequences in the mitochondrial DNA of Gramineae

Evidence for the transfer of DNA from the chloroplast to the mitochondrion has been reported in many higher plants and, in most cases, the transferred chloroplast genes do not have the ability to encode functional products as a consequence of base substitutions and/or multiple rearrangements. We reported previously that the sequence of one end of a chloroplast-derived (ct-derived) fragment of DNA that contained the rps19 and trnH genes has been maintained in most gramineous plants and that its presence seems to be correlated with gene expression in this region. In the present study, we have investigated whether or not the ct-derived sequences in mitochondrial DNA (mtDNA) from some gramineous plants and species of Oryza are conserved, and whether or not such conservation is related to gene expression in these regions. We identified two junctions between ct-derived and mitochondrial sequences that were conserved among some gramineous plants. Around these regions, we found a ct-derived gene for tRNA and the promoter of a mitochondrial gene on the ct-derived sequences, respectively, and these regions were transcribed through the junctions. This result indicates that the junctions and/or regions that are transcribed and functional in mitochondria have been strongly conserved and maintained during their evolution. In Oryza, some junctions between ct-derived and mitochondrial sequences were conserved and other junctions were not. These variations seem to have been caused by deletions and/or rearrangements, and appear to be specific to the type of genome. In the case of Oryza, the timing of deletions and/or rearrangements of ct-derived sequences is likely to have coincided with the divergence of the various genome types. Received: 24 July / 13 August 1997     

Evaluation of conserved and ultra-conserved non-genic sequences in chromosome 15q15-linked periodic catatonia

Conserved and ultra-conserved non-genic sequence elements (CNGs, UCEs) between human and other mammalian genomes seem to constitute a heterogeneous group of functional sequences which likely have important biological function. To determine whether variation in CNGs and UCEs contributes to risk for the schizophrenic subphenotype of periodic catatonia (according to K. Leonhard; OMIM 605419), we evaluated non-coding elements at a critical 7.35?Mb interval on chromosome 15q15 in 8 unrelated cases with periodic catatonia (derived from pedigrees compatible with linkage to chromosome 15q15) and 8 controls, followed by association studies in a cohort of 510 cases and controls. Among 65 CNGs (≥100?bp, 100% identity; human-mouse comparison), 7 CNGs matched criteria for UCE (≥200 bp, 100% identity). A hot spot of 62/65 CNGs (95%) appeared at the MEIS2 locus, which implicates functional importance of associated (ultra-)conserved elements to this early developmental gene, which is present in the human fetal neocortex and associated with metabolic side effects to antipsychotic drugs. Further CNGs were identified at the PLCB2 and DLL4 locus or located intergenic between TYRO3 and MAPKBP1. Automated sequencing revealed genetic variation in 12.3% of CNGs, but frequencies were low (MAF: 0.06-0.4) in cases. Three variants located inside CNGs/UCEs were found in cases only. In a case-control association study we could not confirm a significant association of these three CNG-variants with periodic catatonia. Our results suggest genetic variation in (ultra-)conserved non-genic sequence elements which might alter functional properties. The identified variants are genetically not associated with the phenotype of periodic catatonia.     

Reciprocal chromosome painting illuminates the history of genome evolution of the domestic cat, dog and human

Domestic cats and dogs are important companion animals and model animals in biomedical research. The cat has a highly conserved karyotype, closely resembling the ancestral karyotype of mammals, while the dog has one of the most extensively rearranged mammalian karyotypes investigated so far. We have constructed the first detailed comparative chromosome map of the domestic dog and cat by reciprocal chromosome painting. Dog paints specific for the 38 autosomes and the X chromosomes delineated 68 conserved chromosomal segments in the cat, while reverse painting of cat probes onto red fox and dog chromosomes revealed 65 conserved segments. Most conserved segments on cat chromosomes also show a high degree of conservation in G-banding patterns compared with their canine counterparts. At least 47 chromosomal fissions (breaks), 25 fusions and one inversion are needed to convert the cat karyotype to that of the dog, confirming that extensive chromosome rearrangements differentiate the karyotypes of the cat and dog. Comparative analysis of the distribution patterns of conserved segments defined by dog paints on cat and human chromosomes has refined the human/cat comparative genome map and, most importantly, has revealed 15 cryptic inversions in seven large chromosomal regions of conserved synteny between humans and cats. This revised version was published online in July 2006 with corrections to the Cover Date.     

Centromere satellites from Arabidopsis populations: maintenance of conserved and variable domains

The rapid evolution of centromere sequences between species has led to a debate over whether centromere activity is sequence-dependent. The Arabidopsis thaliana centromere regions contain approximately 20,000 copies of a 178-bp satellite repeat. Here, we analyzed satellites from 41 Arabidopsis ecotypes, providing the first broad population survey of satellite variation within a species. We found highly conserved segments and consistent sequence lengths in the Arabidopsis satellites and in the published collection of human alpha-satellites, supporting models for a functional role. Despite this conservation, polymorphisms are significantly enriched at some sites, yielding variation that could restrict binding proteins to a subset of repeat monomers. Some satellite regions vary considerably; at certain bases, consensus sequences derived from each ecotype diverge significantly from the Arabidopsis consensus, indicating substitutions sweep through a genome in less than 5 million years. Such rapid changes generate more variation within the set of Arabidopsis satellites than in genes from the chromosome arms or from the recombinationally suppressed centromere regions. These studies highlight a balance between the mechanisms that maintain particular satellite domains and the forces that disperse sequence changes throughout the satellite repeats in the genome.     

Enrichment of segmental duplications in regions of breaks of synteny between the human and mouse genomes suggest their involvement in evolutionary rearrangements

The sequence of the mouse genome allows one to compare the conservation of synteny between the human and mouse genome and exploration of regions that might have been involved in major rearrangements during the evolution of these two species (evolutionary genome rearrangements). Recent segmental duplications (or duplicons) are paralogous DNA sequences with high sequence identity that account for about 3.5-5% of the human genome and have emerged during the past approximately 35 million years of evolution. These regions are susceptible to illegitimate recombination leading to rearrangements that result in genomic disorders or genomic mutations. A catalogue of several hundred segmental duplications potentially leading to genomic rearrangements has been reported. The authors and others have observed that some chromosome regions involved in genomic disorders are shuffled in orientation and order in the mouse genome and that regions flanked by segmental duplications are often polymorphic. We have compared the human and mouse genome sequences and demonstrate here that recent segmental duplications correlate with breaks of synteny between these two species. We also observed that nine primary regions involved in human genomic disorders show changes in the order or the orientation of mouse/human synteny segments, were often flanked by segmental duplications in the human sequence. We found that 53% of all evolutionary rearrangement breakpoints associate with segmental duplications, as compared with 18% expected in a random location of breaks along the chromosome (P<0.0001). Our data suggest that segmental duplications have participated in the recent evolution of the human genome, as driving forces for evolutionary rearrangements, chromosome structure polymorphisms and genomic disorders.     

Evolutionarily conserved sequences on human chromosome 21

Comparison of human sequences with the DNA of other mammals is an excellent means of identifying functional elements in the human genome. Here we describe the utility of high-density oligonucleotide arrays as a rapid approach for comparing human sequences with the DNA of multiple species whose sequences are not presently available. High-density arrays representing approximately 22.5 Mb of nonrepetitive human chromosome 21 sequence were synthesized and then hybridized with mouse and dog DNA to identify sequences conserved between humans and mice (human-mouse elements) and between humans and dogs (human-dog elements). Our data show that sequence comparison of multiple species provides a powerful empiric method for identifying actively conserved elements in the human genome. A large fraction of these evolutionarily conserved elements are present in regions on chromosome 21 that do not encode known genes.     

Comparative genomics on mammalian Fgf6-Fgf23 locus

CCND2-C12orf5-FGF23-FGF6 locus at human chromosome 12p13.32 and CCND1-ORAOV1-FGF19-FGF4 locus at human chromosome 11q13.3 are paralogous regions (paralogons) within the human genome. FGF23 is the causative factor for tumor-induced osteomalacia (TIO), a paraneoplastic disorder characterized by hypophosphatemia and skeletal undermineralization, and also for autosomal dominant hypophosphatemic rickets (ADHR). Here, rat Fgf6 and Fgf23 complete coding sequences were determined by using bioinformatics. Rat Fgf6 and Fgf23 genes, consisting of three exons, were located within AC103292.6 rat genome sequence. Rat Fgf6 and Fgf23 genes were clustered in tail-to-head manner with an interval of about 52 kb. Human FGF6 and FGF23 genes were clustered in tail-to-head manner with an interval of about 54 kb. Intergenic conserved region (IGCR) within the FGF6-FGF23 gene cluster was identified based on the evolutionary conservation. Human FGF6-FGF23 IGCR (nucleotide position 111648-112242 of AC008012.8 genome sequence) and rat Fgf6-Fgf23 IGCR (nucleotide position 156318-156894 of AC103292.6 genome sequence) showed 77.6% total nucleotide identity. CP2, E47, CREB and PAX4 binding sites were conserved among human FGF6, rat Fgf6, and mouse Fgf6 promoters. GATA and E47 binding sites were conserved among human FGF23, rat Fgf23, and mouse Fgf23 promoters. Because mouse Fgf23 mRNA was expressed in dendritic cells and activated spleen, tumor infiltrating dendritic cells are candidate sources of FGF23 secretion in TIO patients. This is the first report on comparative genomics analyses on human FGF6-FGF23 gene cluster and rodents Fgf6-Fgf23 gene cluster.     

Ubiquitous selective constraints in the Drosophila genome revealed by a genome-wide interspecies comparison

Non-coding DNA comprises approximately 80% of the euchromatic portion of the Drosophila melanogaster genome. Non-coding sequences are known to contain functionally important elements controlling gene expression, but the proportion of sites that are selectively constrained is still largely unknown. We have compared the complete D. melanogaster and Drosophila simulans genome sequences to estimate mean selective constraint (the fraction of mutations that are eliminated by selection) in coding and non-coding DNA by standardizing to substitution rates in putatively unconstrained sequences. We show that constraint is positively correlated with intronic and intergenic sequence length and is generally remarkably strong in non-coding DNA, implying that more than half of all point mutations in the Drosophila genome are deleterious. This fraction is also likely to be an underestimate if many substitutions in non-coding DNA are adaptively driven to fixation. We also show that substitutions in long introns and intergenic sequences are clustered, such that there is an excess of substitutions <8 bp apart and a deficit farther apart. These results suggest that there are blocks of constrained nucleotides, presumably involved in gene expression control, that are concentrated in long non-coding sequences. Furthermore, we infer that there is more than three times as much functional non-coding DNA as protein-coding DNA in the Drosophila genome. Most deleterious mutations therefore occur in non-coding DNA, and these may make an important contribution to a wide variety of evolutionary processes.     

Estimation of the species-specific mutation rates at the DRB1 locus in humans and chimpanzee

To estimate the species-specific mutation rates at the DRB1 locus in humans and chimpanzee, we analyzed the nucleotide sequence of a 37.6-kb chimpanzee chromosomal segment containing the entire Patr-DRB1*0701 allele and the flanking nongenic region and we compared it with two corresponding human sequences containing the HLA-DRB1*070101 allele using the sequence of HLA-DRB1*04011 as an outgroup. Because the allelic pair of HLA-DRB1*070101 and Patr-DRB1*0701 shows the lowest number of substitutions between the two species, it appears that these sequences diverged close to the time of the humans-chimpanzee divergence (6 million years ago). Alignment of the nucleotide sequences for HLA-DRB1*070101 and Patr-DRB1*0701 alleles showed that they share a high degree of similarity, suggesting that the studied chromosomal segments with these sequences have not been subjected to recombination since the humans-chimpanzee divergence. Comparison of the flanking 10.6 kb of nongenic sequences revealed an average of 41.5 and 83 single nucleotide substitutions in humans and chimpanzee, respectively. Thus, the species-specific nucleotide substitution rates in the flanking nongenic region were estimated to be 6.53 x 10(-10) and 1.31 x 10(-9) per site per year in humans and chimpanzee, respectively. Unexpectedly, the estimated rate in humans was twofold lower than in chimpanzee (P < 10(-3), Tajima's relative rate test) and lower than the average substitution rate in the human genome. Because the nucleotide substitution rate in nongenic regions free from selection is expected to be equal to the mutation rate, the estimated substitution rate should correspond to the species-specific mutation rate at the DRB1 locus. Our results strongly suggest that the mutation rate at DRB1 locus differs among species.     

Relaxation of selective constraint on dog mitochondrial DNA following domestication

The domestication of dogs caused a dramatic change in their way of life compared with that of their ancestor, the gray wolf. We hypothesize that this new life style changed the selective forces that acted upon the species, which in turn had an effect on the dog's genome. We sequenced the complete mitochondrial DNA genome in 14 dogs, six wolves, and three coyotes. Here we show that dogs have accumulated nonsynonymous changes in mitochondrial genes at a faster rate than wolves, leading to elevated levels of variation in their proteins. This suggests that a major consequence of domestication in dogs was a general relaxation of selective constraint on their mitochondrial genome. If this change also affected other parts of the dog genome, it could have facilitated the generation of novel functional genetic diversity. This diversity could thus have contributed raw material upon which artificial selection has shaped modern breeds and may therefore be an important source of the extreme phenotypic variation present in modern-day dogs.     

Conservation of Microstructure between a Sequenced Region of the Genome of Rice and Multiple Segments of the Genome of Arabidopsis thaliana

The nucleotide sequence was determined for a 340-kb segment of rice chromosome 2, revealing 56 putative protein-coding genes. This represents a density of one gene per 6.1 kb, which is higher than was reported for a previously sequenced segment of the rice genome. Sixteen of the putative genes were supported by matches to ESTs. The predicted products of 29 of the putative genes showed similarity to known proteins, and a further 17 genes showed similarity only to predicted or hypothetical proteins identified in genome sequence data. The region contains a few transposable elements: one retrotransposon, and one transposon. The segment of the rice genome studied had previously been identified as representing a part of rice chromosome 2 that may be homologous to a segment of Arabidopsis chromosome 4. We confirmed the conservation of gene content and order between the two genome segments. In addition, we identified a further four segments of the Arabidopsis genome that contain conserved gene content and order. In total, 22 of the 56 genes identified in the rice genome segment were represented in this set of Arabidopsis genome segments, with at least five genes present, in conserved order, in each segment. These data are consistent with the hypothesis that the Arabidopsis genome has undergone multiple duplication events. Our results demonstrate that conservation of the genome microstructure can be identified even between monocot and dicot species. However, the frequent occurrence of duplication, and subsequent microstructure divergence, within plant genomes may necessitate the integration of subsets of genes present in multiple redundant segments to deduce evolutionary relationships and identify orthologous genes.     

A cattle-human comparative map built with cattle BAC-ends and human genome sequence

As a step toward the goal of adding the cattle genome to those available for multispecies comparative genome analysis, 40,224 cattle BAC clones were end-sequenced, yielding 60,547 sequences (BAC end sequences, BESs) after trimming with an average read length of 515 bp. Cattle BACs were anchored to the human and mouse genome sequences by BLASTN search, revealing 29.4% and 10.1% significant hits (E < e-5), respectively. More than 60% of all cattle BES hits in both the human and mouse genomes are located within known genes. In order to confirm in silico predictions of orthologyand their relative position on cattle chromosomes, 84 cattle BESs with similarity to sequences on HSA11 were mapped using a cattle-hamster radiation hybrid (RH) panel. Resulting RH maps of BTA15 and BTA29 cover approximately 85% of HSA11 sequence, revealing a complex patchwork shuffling of segments not explained by a simple translocation followed by internal rearrangements. Overlay of the mouse conserved syntenies onto HSA11 revealed that segmental boundaries appear to be conserved in all three species. The BAC clone-based comparative map provides a foundation for the evolutionary analysis of mammalian karyotypes and for sequencing of the cattle genome.