Shark Genes Research Articles

Substitutions in the Glycogenin-1 Gene Are Associated with the Evolution of Endothermy in Sharks and Tunas.

Despite 400–450 million years of independent evolution, a strong phenotypic convergence has occurred between two groups of fish: tunas and lamnid sharks. This convergence is characterized by centralization of red muscle, a distinctive swimming style (stiffened body powered through tail movements) and elevated body temperature (endothermy). Furthermore, both groups demonstrate elevated white muscle metabolic capacities. All these traits are unusual in fish and more likely evolved to support their fast-swimming, pelagic, predatory behavior. Here, we tested the hypothesis that their convergent evolution was driven by selection on a set of metabolic genes. We sequenced white muscle transcriptomes of six tuna, one mackerel, and three shark species, and supplemented this data set with previously published RNA-seq data. Using 26 species in total (including 7,032 tuna genes plus 1,719 shark genes), we constructed phylogenetic trees and carried out maximum-likelihood analyses of gene selection. We inferred several genes relating to metabolism to be under selection. We also found that the same one gene, glycogenin-1, evolved under positive selection independently in tunas and lamnid sharks, providing evidence of convergent selective pressures at gene level possibly underlying shared physiology.

Sequencing and Analysis of Full-Length cDNAs, 5′-ESTs and 3′-ESTs from a Cartilaginous Fish, the Elephant Shark (Callorhinchus milii)

Cartilaginous fishes are the most ancient group of living jawed vertebrates (gnathostomes) and are, therefore, an important reference group for understanding the evolution of vertebrates. The elephant shark (Callorhinchus milii), a holocephalan cartilaginous fish, has been identified as a model cartilaginous fish genome because of its compact genome (∼910 Mb) and a genome project has been initiated to obtain its whole genome sequence. In this study, we have generated and sequenced full-length enriched cDNA libraries of the elephant shark using the ‘oligo-capping’ method and Sanger sequencing. A total of 6,778 full-length protein-coding cDNA and 10,701 full-length noncoding cDNA were sequenced from six tissues (gills, intestine, kidney, liver, spleen, and testis) of the elephant shark. Analysis of their polyadenylation signals showed that polyadenylation usage in elephant shark is similar to that in mammals. Furthermore, both coding and noncoding transcripts of the elephant shark use the same proportion of canonical polyadenylation sites. Besides BLASTX searches, protein-coding transcripts were annotated by Gene Ontology, InterPro domain, and KEGG pathway analyses. By comparing elephant shark genes to bony vertebrate genes, we identified several ancient genes present in elephant shark but differentially lost in tetrapods or teleosts. Only ∼6% of elephant shark noncoding cDNA showed similarity to known noncoding RNAs (ncRNAs). The rest are either highly divergent ncRNAs or novel ncRNAs. In addition to full-length transcripts, 30,375 5′-ESTs and 41,317 3′-ESTs were sequenced and annotated. The clones and transcripts generated in this study are valuable resources for annotating transcription start sites, exon-intron boundaries, and UTRs of genes in the elephant shark genome, and for the functional characterization of protein sequences. These resources will also be useful for annotating genes in other cartilaginous fishes whose genomes have been targeted for whole genome sequencing.

Unresolved orthology and peculiar coding sequence properties of lamprey genes: the KCNA gene family as test case

BackgroundIn understanding the evolutionary process of vertebrates, cyclostomes (hagfishes and lamprey) occupy crucial positions. Resolving molecular phylogenetic relationships of cyclostome genes with gnathostomes (jawed vertebrates) genes is indispensable in deciphering both the species tree and gene trees. However, molecular phylogenetic analyses, especially those including lamprey genes, have produced highly discordant results between gene families. To efficiently scrutinize this problem using partial genome assemblies of early vertebrates, we focused on the potassium voltage-gated channel, shaker-related (KCNA) family, whose members are mostly single-exon.ResultsSeven sea lamprey KCNA genes as well as six elephant shark genes were identified, and their orthologies to bony vertebrate subgroups were assessed. In contrast to robustly supported orthology of the elephant shark genes to gnathostome subgroups, clear orthology of any sea lamprey gene could not be established. Notably, sea lamprey KCNA sequences displayed unique codon usage pattern and amino acid composition, probably associated with exceptionally high GC-content in their coding regions. This lamprey-specific property of coding sequences was also observed generally for genes outside this gene family.ConclusionsOur results suggest that secondary modifications of sequence properties unique to the lamprey lineage may be one of the factors preventing robust orthology assessments of lamprey genes, which deserves further genome-wide validation. The lamprey lineage-specific alteration of protein-coding sequence properties needs to be taken into consideration in tackling the key questions about early vertebrate evolution.

Parathyroid hormone gene family in a cartilaginous fish, the elephant shark (Callorhinchus milii)

The development of bone was a major step in the evolution of vertebrates. A bony skeleton provided structural support and a calcium reservoir essential for the movement from an aquatic to a terrestrial environment. Cartilaginous fishes are the oldest living group of jawed vertebrates. In this study we have identified three members of the parathyroid hormone (Pth) gene family in a cartilaginous fish, the elephant shark (Callorhinchus milii). The three genes include two Pth genes, designated as Pth1 and Pth2, and a Pthrp gene. Phylogenetic analysis suggested that elephant shark Pth2 is an ancient gene whose orthologue is lost in bony vertebrates. The Pth1 and Pth2 genes have the same structure as the Pth gene in bony vertebrates, whereas the structure of the Pthrp gene is more complex in tetrapods compared with elephant shark. The three elephant shark genes showed distinct patterns of expression, with Pth2 being expressed only in the brain and spleen. This contrasts with localization of the corresponding proteins, which showed considerable overlap in their distribution. There were conserved sites of localization for Pthrp between elephant shark and mammals, including tissues such as kidney, skin, skeletal and cardiac muscle, pancreas, and cartilage. The elephant shark Pth1(1-34) and Pthrp(1-34) peptides were able to stimulate cAMP accumulation in mammalian UMR106.01 cells. However, Pth2(1-34) peptide did not show such PTH-like biologic activity. The presence of Pth and Pthrp genes in the elephant shark indicates that these genes played fundamental roles before their recruitment to bone development in bony jawed vertebrates.

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.

Cloning, distribution and up-regulation of the teleost fish MHC class II alpha suggests a role for granulocytes as antigen-presenting cells

The major histocompatibility complex (MHC) class II alpha chain gene of the teleost fish gilthead seabream ( Sparus aurata), Spau-DAA, has been characterized. We cloned, sequenced and studied its polymorphism, before evaluating its expression in resting seabream leucocytes, tissues and tumor cells as well as in primed leucocytes. A complete allele was obtained by overlapping sequence fragments obtained by RT-PCR. The full-length Spau-DAA*101 comprises 1840 bp with a 5′-UTR region of 84 bp, an ORF of 729 bp and a 3′-UTR of 1027 bp. The putative protein of 242 residues shows homology with known MHC class II alpha genes, varying from 71 to 28% in other fish and humans, respectively. The protein sequence showed all the important features: leader peptide, α1, α2 and CP/TM/CYT regions, conserved cysteines and N-glycosylation site. The phylogenetic tree shows that it is included in the cluster containing the Percomorpha subclass and far from the human and shark genes. It is polymorphic, as seen when we sequenced the complete ORF of 11 alleles showing most of the amino acidic changes in the α1 domain, where the peptide-binding region (PBR) is found. Spau-DAA mRNA expression was mainly found in peritoneal exudate leucocytes, head-kidney, spleen, thymus and gill. Minor expression was detected in gut, brain, liver and PBLs. RT-PCR expression studies in isolated leucocyte subpopulations revealed, for the first time in the literature, that acidophilic granulocytes show high MHC class II gene expression. Apart from these granulocytes lymphocytes also express the Spau-DAA gene, although other cell types may also do the same. Finally, incubation of head-kidney leucocytes with yeast cells or pathogenic bacteria up-regulates Spau-DAA gene expression whilst incubation with ConA, ConA + LPS or PHA does not. The possible involvement of the seabream MHC class II alpha gene in the fish defence and antigen presentation are discussed.

Antibody repertoire development in cartilaginous fish

There are 3 H chain and 3 L chain isotypes in the cartilaginous fish, all encoded by genes in the so-called cluster (VDDJ, VJ) organization. The H chain isotypes IgM and IgNAR, are readily detected at the protein level in most species. The third is readily identified at the protein level in skates (IgR) but only via immunoprecipitation or at the transcript level in sharks (IgW). High levels of diversity in CDR3 and up to 200 germline genes have been detected for IgM depending upon the species examined. IgNAR displays very high levels of CDR3 diversity but almost none in the germline. At least IgNAR and L chain genes have been shown to hypermutate to very high levels, apparently in response to antigen. The mutation footprints are similar to those in mammals except that the shark genes uniquely mutate nucleotide residues in tandem. A conspicuous feature of cartilaginous fish Ig genes is the presence of germline-joined genes, which are a result of RAG activity in germ cells. Such genes are expressed early in ontogeny and then extinguished or expressed at lower levels. 19S IgM and IgW expression precede that of 7S IgM and IgNAR during ontogeny. The ‘switch’ from 19S to 7S IgM, the regulation of expression of the Ig clusters, and the microenvironments for mutation/selection of cartilaginous fish B cells are all areas of ongoing research.

Appearance and disappearance of Syk family protein-tyrosine kinase genes during metazoan evolution.

Syk family protein-tyrosine kinases are essential components of immunoreceptor signaling in mammalian lymphocytes. The absence of Syk genes from the Caenorhabditis elegans genome suggests that this kinase family is of recent evolutionary origin. Surprisingly, we have found that Hydra vulgaris, a member of the early diverging animal phylum Cnidaria, contains a gene encoding a Syk kinase. Phylogenetic analysis indicates that a single Syk family gene was present in animals prior to the gene duplication that gave rise to Syk and ZAP-70, the two mammalian Syk family genes. C. elegans also lacks a Shark protein-tyrosine kinase gene, which we show is a member of a sister group to the Syk family. We conclude that both Syk and Shark genes were lost from the genome of an ancestor of C. elegans. This natural gene knockout result indicates that neither Syk nor Shark kinases are essential for processes held in common between the nematode and other metazoans. The Hydra Syk gene is expressed in epithelial cells, a site consistent with a role for Hydra Syk in recognition of foreign cells.