Survey Sequencing and Comparative Analysis of the Elephant Shark (Callorhinchus milii) Genome

Owing to their phylogenetic position, cartilaginous fishes (sharks, rays, skates, and chimaeras) provide a critical reference for our understanding of vertebrate genome evolution. The relatively small genome of the elephant shark, Callorhinchus milii, a chimaera, makes it an attractive model cartilaginous fish genome for whole-genome sequencing and comparative analysis. Here, the authors describe survey sequencing (1.4× coverage) and comparative analysis of the elephant shark genome, one of the first cartilaginous fish genomes to be sequenced to this depth. Repetitive sequences, represented mainly by a novel family of short interspersed element–like and long interspersed element–like sequences, account for about 28% of the elephant shark genome. Fragments of approximately 15,000 elephant shark genes reveal specific examples of genes that have been lost differentially during the evolution of tetrapod and teleost fish lineages. Interestingly, the degree of conserved synteny and conserved sequences between the human and elephant shark genomes are higher than that between human and teleost fish genomes. Elephant shark contains putative four Hox clusters indicating that, unlike teleost fish genomes, the elephant shark genome has not experienced an additional whole-genome duplication. These findings underscore the importance of the elephant shark as a critical reference vertebrate genome for comparative analysis of the human and other vertebrate genomes. This study also demonstrates that a survey-sequencing approach can be applied productively for comparative analysis of distantly related vertebrate genomes.     

Survey sequencing and comparative analysis of the elephant shark (Callorhinchus milii) genome

Owing to their phylogenetic position, cartilaginous fishes (sharks, rays, skates, and chimaeras) provide a critical reference for our understanding of vertebrate genome evolution. The relatively small genome of the elephant shark, Callorhinchus milii, a chimaera, makes it an attractive model cartilaginous fish genome for whole-genome sequencing and comparative analysis. Here, the authors describe survey sequencing (1.4× coverage) and comparative analysis of the elephant shark genome, one of the first cartilaginous fish genomes to be sequenced to this depth. Repetitive sequences, represented mainly by a novel family of short interspersed element–like and long interspersed element–like sequences, account for about 28% of the elephant shark genome. Fragments of approximately 15,000 elephant shark genes reveal specific examples of genes that have been lost differentially during the evolution of tetrapod and teleost fish lineages. Interestingly, the degree of conserved synteny and conserved sequences between the human and elephant shark genomes are higher than that between human and teleost fish genomes. Elephant shark contains putative four Hox clusters indicating that, unlike teleost fish genomes, the elephant shark genome has not experienced an additional whole-genome duplication. These findings underscore the importance of the elephant shark as a critical reference vertebrate genome for comparative analysis of the human and other vertebrate genomes. This study also demonstrates that a survey-sequencing approach can be applied productively for comparative analysis of distantly related vertebrate genomes.     

Gene and genome duplications in vertebrates: the one-to-four (-to-eight in fish) rule and the evolution of novel gene functions

One important mechanism for functional innovation during evolution is the duplication of genes and entire genomes. Evidence is accumulating that during the evolution of vertebrates from early deuterostome ancestors entire genomes were duplicated through two rounds of duplications (the 'one-to-two-to-four' rule). The first genome duplication in chordate evolution might predate the Cambrian explosion. The second genome duplication possibly dates back to the early Devonian. Recent data suggest that later in the Devonian, the fish genome was duplicated for a third time to produce up to eight copies of the original deuterostome genome. This last duplication took place after the two major radiations of jawed vertebrate life, the ray-finned fish (Actinopterygia) and the sarcopterygian lineage, diverged. Therefore the sarcopterygian fish, which includes the coelacanth, lungfish and all land vertebrates such as amphibians, reptiles, birds and mammals, tend to have only half the number of genes compared with actinopterygian fish. Although many duplicated genes turned into pseudogenes, or even 'junk' DNA, many others evolved new functions particularly during development. The increased genetic complexity of fish might reflect their evolutionary success and diversity.     

Evolution of the vertebrate paralemmin gene family: ancient origin of gene duplicates suggests distinct functions

Paralemmin-1 is a protein implicated in plasma membrane dynamics, the development of filopodia, neurites and dendritic spines, as well as the invasiveness and metastatic potential of cancer cells. However, little is known about its mode of action, or about the biological functions of the other paralemmin isoforms: paralemmin-2, paralemmin-3 and palmdelphin. We describe here evolutionary analyses of the paralemmin gene family in a broad range of vertebrate species. Our results suggest that the four paralemmin isoform genes (PALM1, PALM2, PALM3 and PALMD) arose by quadruplication of an ancestral gene in the two early vertebrate genome duplications. Paralemmin-1 and palmdelphin were further duplicated in the teleost fish specific genome duplication. We identified a unique sequence motif common to all paralemmins, consisting of 11 highly conserved residues of which four are invariant. A single full-length paralemmin homolog with this motif was identified in the genome of the sea lamprey Petromyzon marinus and an isolated putative paralemmin motif could be detected in the genome of the lancelet Branchiostoma floridae. This allows us to conclude that the paralemmin gene family arose early and has been maintained throughout vertebrate evolution, suggesting functional diversification and specific biological roles of the paralemmin isoforms. The paralemmin genes have also maintained specific features of gene organisation and sequence. This includes the occurrence of closely linked downstream genes, initially identified as a readthrough fusion protein with mammalian paralemmin-2 (Palm2-AKAP2). We have found evidence for such an arrangement for paralemmin-1 and -2 in several vertebrate genomes, as well as for palmdelphin and paralemmin-3 in teleost fish genomes, and suggest the name paralemmin downstream genes (PDG) for this new gene family. Thus, our findings point to ancient roles for paralemmins and distinct biological functions of the gene duplicates.     

Turning the clock back on ancient genome duplication

Complete genome sequence data led rapidly to the conclusion that ancient genome duplications had shaped the genomes of the model organisms Saccharomyces cerevisiae and Arabidopsis thaliana. Recent contributions have gone on to refine date estimates for these duplications and, in the case of Arabidopsis, to infer additional, more ancient, rounds of duplication by reconstructing gene order before the most recent duplication event. It is becoming widely accepted that an ancient duplication occurred before the radiation of the ray-finned fish. However, despite methodological advances and the availability of complete genome sequence data the debate over whether very ancient genome duplications have occurred early in the vertebrate lineage has not yet been fully resolved.     

Genome evolution and biodiversity in teleost fish

Teleost fish, which roughly make up half of the extant vertebrate species, exhibit an amazing level of biodiversity affecting their morphology, ecology and behaviour as well as many other aspects of their biology. This huge variability makes fish extremely attractive for the study of many biological questions, particularly of those related to evolution. New insights gained from different teleost species and sequencing projects have recently revealed several peculiar features of fish genomes that might have played a role in fish evolution and speciation. There is now substantial evidence that a round of tetraploidization/rediploidization has taken place during the early evolution of the ray-finned fish lineage, and that hundreds of duplicate pairs generated by this event have been maintained over hundreds of millions of years of evolution. Differential loss or subfunction partitioning of such gene duplicates might have been involved in the generation of fish variability. In contrast to mammalian genomes, teleost genomes also contain multiple families of active transposable elements, which might have played a role in speciation by affecting hybrid sterility and viability. Finally, the amazing diversity of sex determination systems and the plasticity of sex chromosomes observed in teleost might have been involved in both pre- and postmating reproductive isolation. Comparison of data generated by current and future genome projects as well as complementary studies in other species will allow one to approach the molecular and evolutionary mechanisms underlying genome diversity in fish, and will certainly significantly contribute to our understanding of gene evolution and function in humans and other vertebrates.     

Isochore patterns and gene distributions in fish genomes

The compositional approach developed in our laboratory many years ago revealed a large-scale compositional heterogeneity in vertebrate genomes, in which GC-rich and GC-poor regions, the isochores, were found to be characterized by high and low gene densities, respectively. Here we mapped isochores on fish chromosomes and assessed gene densities in isochore families. Because of the availability of sequence data, we have concentrated our investigations on four species, zebrafish (Brachydanio rerio), medaka (Oryzias latipes), stickleback (Gasterosteus aculeatus), and pufferfish (Tetraodon nigroviridis), which belong to four distant orders and cover almost the entire GC range of fish genomes. These investigations produced isochore maps that were drastically different not only from those of mammals (in that only two major isochore families were essentially present in each genome vs five in the human genome) but also from each other (in that different isochore families were represented in different genomes). Gene density distributions for these fish genomes were also obtained and shown to follow the expected increase with increasing isochore GC. Finally, we discovered a remarkable conservation of the average size of the isochores (which match replicon clusters in the case of human chromosomes) and of the average GC levels of isochore families in both fish and human genomes. Moreover, in each genome the GC-poorest isochore families comprised a group of "long isochores" (2-20 Mb in size), which were the lowest in GC and varied in size distribution and relative amount from one genome to the other.     

A family of Tc1-like transposons from the genomes of fishes and frogs: evidence for horizontal transmission.

Tc1-like transposons are very widely distributed within the genomes of animal species. They consist of an inverted repeat sequence flanking a transposase gene with homology to the mobile DNA element, Tc1 of the nematode Caenorhabditis elegans. These elements seem particularly to infest the genomes of fish and amphibian species where they can account for 1% of the total genome. However, all vertebrate Tc1-like elements isolated so far are non-functional in that they contain multiple frameshifts within their transposase coding regions. Here I describe a Tc1-like transposon (PPTN) from the genome of a marine flatfish species (Pleuronectes platessa) which bears conserved inverted repeats flanking an apparently intact transposase gene. Closely related, although degenerate, Tc1-like transposons were also isolated from the genomes of Atlantic salmon (SSTN, Salmo salar) and frog (RTTN, Rana temporaria). Consensual nucleic acid sequences were derived by comparing several individual isolates from each species and conceptual amino acid sequences were thence derived for their transposases. Phylogenetic analysis of these sequences with previously isolated Tc1-like transposases shows that the elements from plaice, salmon and frog comprise a new subfamily of Tc1-like transposons. Each member is distinct in that it is not found in the genomes of the other species tested. Plaice genomes contain about 300 copies of PPTN, salmon 1200 copies of SSTN and frog genomes about 500 copies of RTTN. The presence of these closely related elements in the genomes of fish and frog species, representing evolutionary lines, which diverged more than 400 million years ago, is not consistent with a vertical transmission model for their distributions.     

Codons AGA and AGG are read as glycine in ascidian mitochondria

Summary A 1.2-kb DNA fragment of the cytochrome oxidase subunit I (CO I) gene of mitochondria isolated from an ascidian,Halocynthia roretzi, was amplified by polymerase chain reaction (PCR) and sequenced. Codons AGA and AGG appeared in its reading frame, indicating that these are sense codons in this organelle. Sequence comparisons with the corresponding regions of other animal mitochondrial CO I genes suggest that codons AGA and AGG correspond to glycine in the ascidian mitochondrial genome, but not to serine as in most invertebrate genomes, nor to stops as in vertebrate genomes. The other codons are identical to those of vertebrate mitochondria.     

Cloning, physical mapping and genome organization of mitochondrial DNA from Cyprinus carpio oocytes

Summary The mitochondrial genome from Cyprinus carpio oocytes is a 10.5 megadalton, circular DNA molecule. The carp mitochondrial DNA was cloned in pBR325. Three recombinant plasmids accounted for the entire genome. Mapping of this DNA using 11 different restriction endonucleases is reported here. Both the large and small rRNA genes were then localized using Southern blot analysis. The subunit I of the cytochrome oxidase, the cytochrome b, the tRNA^Glu and the URF 4 genes were localized by nucleotide sequence analysis and homology studies with human mtDNA.Our results suggest that a similar gene order has been maintained in the mitochondrial genomes of Chordata and support the hypothesis of a common ancestor for all vertebrate organelle genomes.This study constitutes the first report on the genome organization of a fish mtDNA and provides information for further investigation in connection with sequence determination, replication, and gene expression in carp mitochondria.This work was supported by proyect RS-82-21 from the Universidad Austral de Chile and Grant N^o 1116 from Fondo Nacional de Desarrollo Cientifico y Tecnologico     

The gain and loss of genes during 600 million years of vertebrate evolution

Background  

Gene duplication is assumed to have played a crucial role in the evolution of vertebrate organisms. Apart from a continuous mode of duplication, two or three whole genome duplication events have been proposed during the evolution of vertebrates, one or two at the dawn of vertebrate evolution, and an additional one in the fish lineage, not shared with land vertebrates. Here, we have studied gene gain and loss in seven different vertebrate genomes, spanning an evolutionary period of about 600 million years.     

Hox gene duplication in fish

The study of Hox gene clusters continues to serve as a paradigm for those interested in vertebrate genome evolution. Recent exciting discoveries about Hox gene composition in fishes challenges conventional views about vertebrate Hox gene evolution, and has initiated lively debates concerning the evolutionary events making the divergence of the major vertebrate lineages. Comparative analyses indicate that Hox cluster duplications occurred in early vertebrate evolution, and again within the order Cypriniformes of teleost fish. Loss of Hox genes was more widespread than duplication during fish evolution.     

Paleogenomics in vertebrates, or the recovery of lost genomes from the mist of time

Knowledge of the structure of ancestral genomes provides the basis of a new framework to better represent and interpret results from genomic and evolutionary studies. Because these ancestors lived tens of hundreds of million years ago, this knowledge will inevitably take the form of abstract representations, reconstructed on the basis both of experimental evidence collected on extant genomes and of our understanding of evolutionary processes. This is the field of Paleogenomics, a young discipline that is providing an increasingly precise picture of our ancestral vertebrate genomes based on cytogenetic data, genome sequences and new algorithmic developments. Many recent studies have focused on the ancestral placental mammal and teleost fish genomes, although the outlines of even more distant pre-vertebrate ancestors are being reported.     

Interspecies Insertion Polymorphism Analysis Reveals Recent Activity of Transposable Elements in Extant Coelacanths

Coelacanths are lobe-finned fish represented by two extant species, Latimeria chalumnae in South Africa and Comoros and L. menadoensis in Indonesia. Due to their intermediate phylogenetic position between ray-finned fish and tetrapods in the vertebrate lineage, they are of great interest from an evolutionary point of view. In addition, extant specimens look similar to 300 million-year-old fossils; because of their apparent slowly evolving morphology, coelacanths have been often described as « living fossils ». As an underlying cause of such a morphological stasis, several authors have proposed a slow evolution of the coelacanth genome. Accordingly, sequencing of the L. chalumnae genome has revealed a globally low substitution rate for protein-coding regions compared to other vertebrates. However, genome and gene evolution can also be influenced by transposable elements, which form a major and dynamic part of vertebrate genomes through their ability to move, duplicate and recombine. In this work, we have searched for evidence of transposition activity in coelacanth genomes through the comparative analysis of orthologous genomic regions from both Latimeria species. Comparison of 5.7 Mb (0.2%) of the L. chalumnae genome with orthologous Bacterial Artificial Chromosome clones from L. menadoensis allowed the identification of 27 species-specific transposable element insertions, with a strong relative contribution of CR1 non-LTR retrotransposons. Species-specific homologous recombination between the long terminal repeats of a new coelacanth endogenous retrovirus was also detected. Our analysis suggests that transposon activity is responsible for at least 0.6% of genome divergence between both Latimeria species. Taken together, this study demonstrates that coelacanth genomes are not evolutionary inert: they contain recently active transposable elements, which have significantly contributed to post-speciation genome divergence in Latimeria.     

Discovery of a large number of previously unrecognized mitochondrial pseudogenes in fish genomes

Nuclear inserted copies of mitochondrial origin (numts) vary widely among eukaryotes, with human and plant genomes harboring the largest repertoires. Numts were previously thought to be absent from fish species, but the recent release of three fish nuclear genome sequences provides the resource to obtain a more comprehensive insight into the extent of mtDNA transfer in fishes. From the sequence analyses of the genomes of Fugu rubripes, Tetraodon nigroviridis, and Danio rerio, we have identified 2, 5, and 10 recent numt integrations, respectively, which integrated into those genomes less than 0.6 million years (Myr) ago. Such results contradict the hypothesis of absence or rarity of numts in fishes, as (i) the ratio of numts to the total size of the nuclear genome in T. nigroviridis was superior to the ratio observed in several higher vertebrate species (e.g., chicken, mouse, and rat), and only surpassed by humans, and (ii) the mtDNA coverage transferred to the nuclear genome of D. rerio is exceeded only by human and mouse, within the whole range of eukaryotic genomes surveyed for numts. Additionally, 335, 336, and 471 old numts (>12.5 Myr) were detected in F. rubripes, T. nigroviridis, and D. rerio, respectively. Surprisingly, old numts are inserted preferentially into known or predicted genes, as inferred for recent numts in human. However, because in fish genomes such integrations are old, they are likely to represent evolutionary successes and they may be considered a potential important evolutionary mechanism for the enhancement of genomic coding regions.     

The DNA sequence of medaka chromosome LG22

We report the genomic DNA sequence of a single chromosome (linkage group 22; LG22) of the small teleost fish medaka (Oryzias latipes) as a first whole chromosome sequence from a non-mammalian vertebrate. The order and orientation of 633 protein-coding genes were deduced from 18,803,338 bp of DNA sequence, providing the opportunity to analyze chromosome evolution of vertebrate genomes by direct comparison with the human genome. The average number of genes in the "conserved gene cluster" (CGC), a strict definition of "synteny" at the sequence basis, between medaka and human was 1.6. These and other data suggest that approximately 38.8% of pair-wise gene relationships would have been broken from their common ancestor in the human and medaka lineages and further imply that approx 20,000 (15,520-23,280) breaks would have occurred from the entire genome of the common ancestor. These breaks were generated mainly by intra-chromosomal shufflings at a specific era in the vertebrate lineage. These precise comparative genomics allowed us to identify the pieces of ancient chromosomes of the common vertebrate ancestor and estimate chromosomal evolution in the vertebrate lineage.     

Yesterday's polyploids and the mystery of diploidization

Thirty years after Susumu Ohno proposed that vertebrate genomes are degenerate polyploids, the extent to which genome duplication contributed to the evolution of the vertebrate genome, if at all, is still uncertain. Sequence-level studies on model organisms whose genomes show clearer evidence of ancient polyploidy are invaluable because they indicate what the evolutionary products of genome duplication can look like. The greatest mystery is the molecular basis of diploidization, the evolutionary process by which a polyploid genome turns into a diploid one.