Genome-wide characterization of centromeric satellites from multiple mammalian genomes

Despite its importance in cell biology and evolution, the centromere has remained the final frontier in genome assembly and annotation due to its complex repeat structure. However, isolation and characterization of the centromeric repeats from newly sequenced species are necessary for a complete understanding of genome evolution and function. In recent years, various genomes have been sequenced, but the characterization of the corresponding centromeric DNA has lagged behind. Here, we present a computational method (RepeatNet) to systematically identify higher-order repeat structures from unassembled whole-genome shotgun sequence and test whether these sequence elements correspond to functional centromeric sequences. We analyzed genome datasets from six species of mammals representing the diversity of the mammalian lineage, namely, horse, dog, elephant, armadillo, opossum, and platypus. We define candidate monomer satellite repeats and demonstrate centromeric localization for five of the six genomes. Our analysis revealed the greatest diversity of centromeric sequences in horse and dog in contrast to elephant and armadillo, which showed high-centromeric sequence homogeneity. We could not isolate centromeric sequences within the platypus genome, suggesting that centromeres in platypus are not enriched in satellite DNA. Our method can be applied to the characterization of thousands of other vertebrate genomes anticipated for sequencing in the near future, providing an important tool for annotation of centromeres.     

Quantitative estimates of sequence divergence for comparative analyses of mammalian genomes

Comparative sequence analyses on a collection of carefully chosen mammalian genomes could facilitate identification of functional elements within the human genome and allow quantification of evolutionary constraint at the single nucleotide level. High-resolution quantification would be informative for determining the distribution of important positions within functional elements and for evaluating the relative importance of nucleotide sites that carry single nucleotide polymorphisms (SNPs). Because the level of resolution in comparative sequence analyses is a direct function of sequence diversity, we propose that the information content of a candidate mammalian genome be defined as the sequence divergence it would add relative to already-sequenced genomes. We show that reliable estimates of genomic sequence divergence can be obtained from small genomic regions. On the basis of a multiple sequence alignment of approximately 1.4 megabases each from eight mammals, we generate such estimates for five unsequenced mammals. Estimates of the neutral divergence in these data suggest that a small number of diverse mammalian genomes in addition to human, mouse, and rat would allow single nucleotide resolution in comparative sequence analyses.     

Characterization of the opossum immune genome provides insights into the evolution of the mammalian immune system

The availability of the first marsupial genome sequence has allowed us to characterize the immunome of the gray short-tailed opossum (Monodelphis domestica). Here we report the identification of key immune genes, including the highly divergent chemokines, defensins, cathelicidins, and Natural Killer cell receptors. It appears that the increase in complexity of the mammalian immune system occurred prior to the divergence of the marsupial and eutherian lineages approximately 180 million years ago. Genomes of ancestral mammals most likely contained all of the key mammalian immune gene families, with evolution on different continents, in the presence of different pathogens leading to lineage specific expansions and contractions, resulting in some minor differences in gene number and composition between different mammalian lineages. Gene expansion and extensive heterogeneity in opossum antimicrobial peptide genes may have evolved as a consequence of the newborn young needing to survive without an adaptive immune system in a pathogen laden environment. Given the similarities in the genomic architecture of the marsupial and eutherian immune systems, we propose that marsupials are ideal model organisms for the study of developmental immunology.     

Using genomic data to unravel the root of the placental mammal phylogeny

The phylogeny of placental mammals is a critical framework for choosing future genome sequencing targets and for resolving the ancestral mammalian genome at the nucleotide level. Despite considerable recent progress defining superordinal relationships, several branches remain poorly resolved, including the root of the placental tree. Here we analyzed the genome sequence assemblies of human, armadillo, elephant, and opossum to identify informative coding indels that would serve as rare genomic changes to infer early events in placental mammal phylogeny. We also expanded our species sampling by including sequence data from >30 ongoing genome projects, followed by PCR and sequencing validation of each indel in additional taxa. Our data provide support for a sister-group relationship between Afrotheria and Xenarthra (the Atlantogenata hypothesis), which is in turn the sister-taxon to Boreoeutheria. We failed to recover any indels in support of a basal position for Xenarthra (Epitheria), which is suggested by morphology and a recent retroposon analysis, or a hypothesis with Afrotheria basal (Exafricoplacentalia), which is favored by phylogenetic analysis of large nuclear gene data sets. In addition, we identified two retroposon insertions that also support Atlantogenata and none for the alternative hypotheses. A revised molecular timescale based on these phylogenetic inferences suggests Afrotheria and Xenarthra diverged from other placental mammals approximately 103 (95-114) million years ago. We discuss the impacts of this topology on earlier phylogenetic reconstructions and repeat-based inferences of phylogeny.     

Evolutionary dynamics of transposable elements in the short-tailed opossum Monodelphis domestica

The genome of the gray short-tailed opossum Monodelphis domestica is notable for its large size ( approximately 3.6 Gb). We characterized nearly 500 families of interspersed repeats from the Monodelphis. They cover approximately 52% of the genome, higher than in any other amniotic lineage studied to date, and may account for the unusually large genome size. In comparison to other mammals, Monodelphis is significantly rich in non-LTR retrotransposons from the LINE-1, CR1, and RTE families, with >29% of the genome sequence comprised of copies of these elements. Monodelphis has at least four families of RTE, and we report support for horizontal transfer of this non-LTR retrotransposon. In addition to short interspersed elements (SINEs) mobilized by L1, we found several families of SINEs that appear to use RTE elements for mobilization. In contrast to L1-mobilized SINEs, the RTE-mobilized SINEs in Monodelphis appear to shift from G+C-rich to G+C-low regions with time. Endogenous retroviruses have colonized approximately 10% of the opossum genome. We found that their density is enhanced in centromeric and/or telomeric regions of most Monodelphis chromosomes. We identified 83 new families of ancient repeats that are highly conserved across amniotic lineages, including 14 LINE-derived repeats; and a novel SINE element, MER131, that may have been exapted as a highly conserved functional noncoding RNA, and whose emergence dates back to approximately 300 million years ago. Many of these conserved repeats are also present in human, and are highly over-represented in predicted cis-regulatory modules. Seventy-six of the 83 families are present in chicken in addition to mammals.     

Analyses of deep mammalian sequence alignments and constraint predictions for 1% of the human genome

A key component of the ongoing ENCODE project involves rigorous comparative sequence analyses for the initially targeted 1% of the human genome. Here, we present orthologous sequence generation, alignment, and evolutionary constraint analyses of 23 mammalian species for all ENCODE targets. Alignments were generated using four different methods; comparisons of these methods reveal large-scale consistency but substantial differences in terms of small genomic rearrangements, sensitivity (sequence coverage), and specificity (alignment accuracy). We describe the quantitative and qualitative trade-offs concomitant with alignment method choice and the levels of technical error that need to be accounted for in applications that require multisequence alignments. Using the generated alignments, we identified constrained regions using three different methods. While the different constraint-detecting methods are in general agreement, there are important discrepancies relating to both the underlying alignments and the specific algorithms. However, by integrating the results across the alignments and constraint-detecting methods, we produced constraint annotations that were found to be robust based on multiple independent measures. Analyses of these annotations illustrate that most classes of experimentally annotated functional elements are enriched for constrained sequences; however, large portions of each class (with the exception of protein-coding sequences) do not overlap constrained regions. The latter elements might not be under primary sequence constraint, might not be constrained across all mammals, or might have expendable molecular functions. Conversely, 40% of the constrained sequences do not overlap any of the functional elements that have been experimentally identified. Together, these findings demonstrate and quantify how many genomic functional elements await basic molecular characterization.     

Taxonomy of lice and their endosymbiotic bacteria in the post-genomic era

Recent studies of molecular and genomic data from the parasitic lice of birds and mammals, as well as their mutualistic endosymbiotic bacteria, are changing the phylogenetic relationships and taxonomy of these organisms. Phylogenetic studies of lice suggest that vertebrate parasitism arose multiple times from free-living book and bark lice. Molecular clocks show that the major families of lice arose in the late Mesozoic and radiated in the early Cenozoic, following the radiation of mammals and birds. The recent release of the human louse genome has provided new opportunities for research. The genome is being used to find new genetic markers for phylogenetics and population genetics, to understand the complex evolutionary relationships of mitochondrial genes, and to study genome evolution. Genomes are informing us not only about lice, but also about their obligate endosymbiotic bacteria. In contrast to lice and their hosts, lice and their endosymbionts do not share common evolutionary histories, suggesting that endosymbionts are either replaced over time or that there are multiple independent origins of symbiosis in lice. Molecular phylogenetics and whole genome sequencing have recently provided the first insights into the phylogenetic placement and metabolic characteristics of these distantly related bacteria. Comparative genomics between distantly related louse symbionts can provide insights into conserved metabolic functions and can help to explain how distantly related species are fulfilling their role as mutualistic symbionts. In lice and their endosymbionts, molecular data and genome sequencing are driving our understanding of evolutionary relationships and classification, and will for the foreseeable future.     

Locating protein-coding sequences under selection for additional, overlapping functions in 29 mammalian genomes

The degeneracy of the genetic code allows protein-coding DNA and RNA sequences to simultaneously encode additional, overlapping functional elements. A sequence in which both protein-coding and additional overlapping functions have evolved under purifying selection should show increased evolutionary conservation compared to typical protein-coding genes--especially at synonymous sites. In this study, we use genome alignments of 29 placental mammals to systematically locate short regions within human ORFs that show conspicuously low estimated rates of synonymous substitution across these species. The 29-species alignment provides statistical power to locate more than 10,000 such regions with resolution down to nine-codon windows, which are found within more than a quarter of all human protein-coding genes and contain ～2% of their synonymous sites. We collect numerous lines of evidence that the observed synonymous constraint in these regions reflects selection on overlapping functional elements including splicing regulatory elements, dual-coding genes, RNA secondary structures, microRNA target sites, and developmental enhancers. Our results show that overlapping functional elements are common in mammalian genes, despite the vast genomic landscape.     

Integrated cytogenetic BAC map of the genome of the gray,short-tailed opossum, <Emphasis Type="Italic">Monodelphis domestica</Emphasis>

The generation of high-quality genome assemblies for numerous species is advancing at a rapid pace. As the number of genome assemblies increases, so does our ability to investigate genome relationships and their contributions to unraveling complex biological, evolutionary, and biomedical processes. A key process in the generation of a genome assembly is to determine and verify the precise physical location and order of the large sequence blocks (scaffolds) that result from the assembly. For organisms of relatively recent common ancestry this process may be achieved largely through comparative sequence alignment. However, as the evolutionary distance between species lengthens, the use of comparative sequence alignment becomes increasingly less reliable. Simultaneous cytogenetic mapping, using multicolor fluorescence in-situ hybridization (FISH) analysis, offers an alternative means to define the cytogenetic location and relative order of DNA sequences, thereby anchoring the genome sequence to the karyotype. In this article we report the molecular cytogenetic locations of 415 bacterial artificial chromosome (BAC) clones that served to anchor sequence scaffolds of the gray, short-tailed opossum (Monodelphis domestica) to its karyotype, which enabled accurate integration of these regions into the genome assembly.     

Emergence of mammals by emergency: exaptation

With the development of molecular embryology and the coming of the post‐genomic era, the molecular mechanisms of morphological evolution have recently begun to be elucidated. Whole genome sequences of many vertebrate species have been determined, and comparative genomics has suggested that one source of biodiversity is conserved non‐coding elements (CNEs), which may be involved in generating new networks of gene expression. Nishihara et al. (Genome Res. 2006; 16, 864) discovered retroposon (AmnSINE1s)‐derived CNEs in the human genome, and suggested that the AmnSINE1s obtained their function (i.e., exapted) in a common mammalian ancestor and are involved in generating mammalian‐specific morphology. Therefore, investigation of the function of AmnSINE1‐derived CNEs in morphogenesis helps us understand the molecular events of how mammals obtained their specific morphological characters by exaptation that occurred when the first mammalian ancestor emerged about 250 Ma (million years ago). Because there are more than 100 AmnSINE1‐derived CNE loci in the mammalian genome, a burst of exaptation of AmnSINE1s must have occurred, possibly triggered by the Permian‐Triassic mass extinction 250 Ma. In this review, we discuss morphological evolution of the mammalian‐specific characters including brain that were exapted after retrotransposition of AmnSINE1s by referring to two CNE loci described by Sasaki et al. (Proc. Natl. Acad. Sci. USA 2008; 105, 4220).     

Diversity and evolution of transposable elements in Arabidopsis

Transposable elements are mobile genetic elements that have successfully populated eukaryotic genomes and show diversity in their structure and transposition mechanisms. Although first viewed solely as selfish, transposable elements are now known as important vectors to drive the adaptation and evolution of their host genome. Transposable elements can affect host gene structures, gene copy number, gene expression, and even as a source for novel genes. For example, a number of transposable element sequences have been co-opted to contribute to evolutionary innovation, such as the mammalian placenta and the vertebrate immune system. In plants, the need to adapt rapidly to changing environmental conditions is essential and is reflected, as will be discussed, by genome plasticity and an abundance of diverse, active transposon families. This review focuses on transposable elements in plants, particularly those that have beneficial effects on the host. We also emphasize the importance of having proper tools to annotate and classify transposons to better understand their biology.     

A microsatellite-based, physically anchored linkage map for the gray, short-tailed opossum (Monodelphis domestica).

The genome of the gray, short-tailed opossum, Monodelphis domestica, will be the first of any marsupial to be fully sequenced. The utility of this sequence will be greatly enhanced by construction and integration of detailed genetic and physical maps. Therefore, it is important to verify the unusual recombinational characteristics that were suggested by the 'first-generation' M. domestica linkage map; specifically, very low levels of recombination and severely reduced female recombination, both of which are contrary to patterns in other vertebrates. We constructed a new linkage map based on a different genetic cross, using a new and much larger set of map markers, and physically anchored and oriented the linkage groups onto chromosomes via fluorescence in-situ hybridization mapping. This map includes 150 loci in eight autosomal linkage groups corresponding to the eight autosome pairs, and spans 86-89% of the autosomal genome. The sex-averaged autosomal map covers 715 cM, with a full-length estimate of 866 cM; the shortest full-length linkage map reported for any vertebrate. The sex-specific maps confirmed severely reduced female recombination in all linkage groups, and an overall F/M map ratio = 0.54. These results greatly extend earlier findings, and provide an improved microsatellite-based linkage map for this species.     

Genomic catastrophism and the origin of vertebrate immunity

Genomic catastrophism is the belief that unique genetic events, unlike those observed in recent evolutionary history, played a key role in the origin of vertebrate adaptations. Catastrophist hypotheses have been particularly popular is accounting for the origin of vertebrate specific immunity. Two major such hypotheses involve genome duplication by polyploidization and horizontal gene transfer. Recent analyses lead to decisive rejection of the widely cited hypothesis that the vertebrate genome underwent two rounds of genome duplication, and theoretical considerations suggest that genome duplication is unlikely to lead to new adaptive advances. Likewise, the evidence that key elements of the vertebrate immune system arose by horizontal transfer from a bacterium or by incorporation of a transposable element into the vertebrate genome remains relatively weak. Thus, at present, a uniformitarian view of the origin of the vertebrate immune system seems more reasonable, especially given the longer time-frame for vertebrate evolution indicated by molecular data.     

自然选择及其分子信号

摘要：本综述拟介绍有关自然选择的一些基本概念。在人类进化过程中,自然选择会在基因组中留下一些分子信号,我们重点分析如何利用这些信号来识别自然选择尤其是正向选择,因为发生正向选择作用的基因组区域一定具有重要的功能。     

Distribution and intensity of constraint in mammalian genomic sequence

Comparisons of orthologous genomic DNA sequences can be used to characterize regions that have been subject to purifying selection and are enriched for functional elements. We here present the results of such an analysis on an alignment of sequences from 29 mammalian species. The alignment captures approximately 3.9 neutral substitutions per site and spans approximately 1.9 Mbp of the human genome. We identify constrained elements from 3 bp to over 1 kbp in length, covering approximately 5.5% of the human locus. Our estimate for the total amount of nonexonic constraint experienced by this locus is roughly twice that for exonic constraint. Constrained elements tend to cluster, and we identify large constrained regions that correspond well with known functional elements. While constraint density inversely correlates with mobile element density, we also show the presence of unambiguously constrained elements overlapping mammalian ancestral repeats. In addition, we describe a number of elements in this region that have undergone intense purifying selection throughout mammalian evolution, and we show that these important elements are more numerous than previously thought. These results were obtained with Genomic Evolutionary Rate Profiling (GERP), a statistically rigorous and biologically transparent framework for constrained element identification. GERP identifies regions at high resolution that exhibit nucleotide substitution deficits, and measures these deficits as "rejected substitutions". Rejected substitutions reflect the intensity of past purifying selection and are used to rank and characterize constrained elements. We anticipate that GERP and the types of analyses it facilitates will provide further insights and improved annotation for the human genome as mammalian genome sequence data become richer.     

Reconstructing large regions of an ancestral mammalian genome in silico

It is believed that most modern mammalian lineages arose from a series of rapid speciation events near the Cretaceous-Tertiary boundary. It is shown that such a phylogeny makes the common ancestral genome sequence an ideal target for reconstruction. Simulations suggest that with methods currently available, we can expect to get 98% of the bases correct in reconstructing megabase-scale euchromatic regions of an eutherian ancestral genome from the genomes of approximately 20 optimally chosen modern mammals. Using actual genomic sequences from 19 extant mammals, we reconstruct 1.1 Mb of ancient genome sequence around the CFTR locus. Detailed examination suggests the reconstruction is accurate and that it allows us to identify features in modern species, such as remnants of ancient transposon insertions, that were not identified by direct analysis. Tracing the predicted evolutionary history of the bases in the reconstructed region, estimates are made of the amount of DNA turnover due to insertion, deletion, and substitution in the different placental mammalian lineages since the common eutherian ancestor, showing considerable variation between lineages. In coming years, such reconstructions may help in identifying and understanding the genetic features common to eutherian mammals and may shed light on the evolution of human or primate-specific traits.     

A genetic linkage map for zebrafish: comparative analysis and localization of genes and expressed sequences

Genetic screens in zebrafish (Danio rerio) have isolated mutations in hundreds of genes with essential functions. To facilitate the identification of candidate genes for these mutations, we have genetically mapped 104 genes and expressed sequence tags by scoring single-strand conformational polymorphisms in a panel of haploid siblings. To integrate this map with existing genetic maps, we also scored 275 previously mapped genes, microsatellites, and sequence-tagged sites in the same haploid panel. Systematic phylogenetic analysis defined likely mammalian orthologs of mapped zebrafish genes, and comparison of map positions in zebrafish and mammals identified significant conservation of synteny. This comparative analysis also identified pairs of zebrafish genes that appear to be orthologous to single mammalian genes, suggesting that these genes arose in a genome duplication that occurred in the teleost lineage after the divergence of fish and mammal ancestors. This comparative map analysis will be useful in predicting the locations of zebrafish genes from mammalian gene maps and in understanding the evolution of the vertebrate genome.