Dataset Information

Construction of a nurse shark (Ginglymostoma cirratum) bacterial artificial chromosome (BAC) library and a preliminary genome survey.

ABSTRACT: Sharks are members of the taxonomic class Chondrichthyes, the oldest living jawed vertebrates. Genomic studies of this group, in comparison to representative species in other vertebrate taxa, will allow us to theorize about the fundamental genetic, developmental, and functional characteristics in the common ancestor of all jawed vertebrates.In order to obtain mapping and sequencing data for comparative genomics, we constructed a bacterial artificial chromosome (BAC) library for the nurse shark, Ginglymostoma cirratum.The BAC library consists of 313,344 clones with an average insert size of 144 kb, covering ~4.5 x 1010 bp and thus providing an 11-fold coverage of the haploid genome. BAC end sequence analyses revealed, in addition to LINEs and SINEs commonly found in other animal and plant genomes, two new groups of nurse shark-specific repetitive elements, NSRE1 and NSRE2 that seem to be major components of the nurse shark genome. Screening the library with single-copy or multi-copy gene probes showed 6-28 primary positive clones per probe of which 50-90% were true positives, demonstrating that the BAC library is representative of the different regions of the nurse shark genome. Furthermore, some BAC clones contained multiple genes, making physical mapping feasible.We have constructed a deep-coverage, high-quality, large insert, and publicly available BAC library for a cartilaginous fish. It will be very useful to the scientific community interested in shark genomic structure, comparative genomics, and functional studies. We found two new groups of repetitive elements specific to the nurse shark genome, which may contribute to the architecture and evolution of the nurse shark genome.

SUBMITTER: Luo M

PROVIDER: S-EPMC1513397 | biostudies-literature | 2006 May

REPOSITORIES: biostudies-literature

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Construction of a nurse shark (Ginglymostoma cirratum) bacterial artificial chromosome (BAC) library and a preliminary genome survey.

Luo Meizhong M Kim Hyeran H Kudrna Dave D Sisneros Nicholas B NB Lee So-Jeong SJ Mueller Christopher C Collura Kristi K Zuccolo Andrea A Buckingham E Bryan EB Grim Suzanne M SM Yanagiya Kazuyo K Inoko Hidetoshi H Shiina Takashi T Flajnik Martin F MF Wing Rod A RA Ohta Yuko Y

BMC genomics 20060503

<h4>Background</h4>Sharks are members of the taxonomic class Chondrichthyes, the oldest living jawed vertebrates. Genomic studies of this group, in comparison to representative species in other vertebrate taxa, will allow us to theorize about the fundamental genetic, developmental, and functional characteristics in the common ancestor of all jawed vertebrates.<h4>Aims</h4>In order to obtain mapping and sequencing data for comparative genomics, we constructed a bacterial artificial chromosome (BA ...[more]

PMID: 16672057

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Project description:Many animals of scientific importance lack species-specific reagents (e.g., monoclonal antibodies) for in-depth studies of immune proteins. Mass spectrometry (MS)-based proteomics has emerged as a useful method for monitoring changes in protein abundance and modifications in non-model species. It can be used to quantify hundreds of candidate immune molecules simultaneously without the generation of new reagents. Here, we used MS-based proteomics to identify and quantify candidate immune proteins in the plasma of the nurse shark (Ginglymostoma cirratum), a cartilaginous fish and representative of the most basal extant vertebrate lineage with an immunoglobulin-based immune system. Mass spectrometry-based LC-MS/MS was performed on the blood plasma of nurse sharks immunized with human serum albumin (n=4) or sham immunized (n=1), and sampled at days 0 (baseline control), 1, 2, 3, 5, 7, 14, 21, 28, 25, 42 and 49. An antigen-specific antibody response was experimentally confirmed post-immunization. To provide a high-quality reference to identify proteins, we assembled and annotated a multi-tissue de novo transcriptome integrating long- and short-read sequence data. This comprised 62,682 contigs containing open reading frames (ORFs) with a length >80 amino acids. Using this transcriptome, we reliably identified 626 plasma proteins which were broadly categorized into coagulation, immune, and metabolic functional groups. To assess the feasibility of performing LC-MS/MS proteomics in nurse shark in the absence of species-specific protein annotations, we compared the results to an alternative strategy, mapping peptides to proteins predicted in the genome assembly of a related species, the whale shark (Rhincodon typus). This approach reliably identified 297 proteins, indicating that useful data on the plasma proteome may be obtained in many instances despite the absence of a species-specific reference protein database. Among the plasma proteins defined against the nurse shark transcriptome, fifteen showed consistent changes in abundance across the immunized shark individuals, indicating a role in the immune response. These included alpha-2-macroglobulin (A2M) and a novel protein yet to be characterized in diverse vertebrate lineages. Overall, this study enhances genetic and protein-level resources for nurse shark research and vastly improves our understanding of the elasmobranch plasma proteome, including its remodelling following immune stimulation.

Project description:We present the complete cDNA sequence of shark (Ginglymostoma cirratum) pro-C5 and its molecular characterization with a descriptive analysis of the structural elements necessary for its potential functional role as a potent mediator of inflammation (fragment C5a) and initiator molecule (fragment C5b) for the assembly of the membrane attack complex (MAC) upon activation by C5 convertase. In mammals the three complement activation cascades, the classical, alternative and lectin pathways, converge at the activation of C3, a pivotal complement protein. It is, however, the subsequent activation of the next complement component, C5, which is the focal point at which the initiation of the terminal lytic pathway takes place and involves the stepwise assembly of the MAC. The effector cytolytic function of complement occurs with the insertion of MAC into target membranes causing dough-nut like holes and cell leakage. The lytic activity of shark complement results in structurally similar holes in target membranes suggesting the assembly of a shark MAC that likely involves a functional analogue of C5. The composition of shark MAC remains unresolved and to date conclusive evidence has been lacking for shark C5. The gene has not been cloned nor has the serum protein been characterized for any elasmobranch species. This report is the first to confirm the presence of C5 homologue in the shark. GcC5 is remarkably similar to human C5 in overall structure and domain arrangement. The GcC5 cDNA measured 5160-bp with 5' and 3' UTRs of 35 bp and 79 bp, respectively. Structural analysis of the derived protein sequence predicts a molecule that is a two-chain structure which lacks a thiolester bond and contains a C5 convertase cleavage site indicating that activation will generate two peptides, akin to C5b and C5a. The putative GcC5 molecule also contains the C-terminal C345C/Netrin module that characterizes C3, C4 and C5. Multiple alignment of deduced amino acid sequences shows that GcC5 shares more amino acid identities/similarities with mammals than that with bony fish. We conclude that at the time of emergence of sharks the elaborate mosaic structure of C5 had already evolved.

Dataset Information

Construction of a nurse shark (Ginglymostoma cirratum) bacterial artificial chromosome (BAC) library and a preliminary genome survey.

Publications

Construction of a nurse shark (Ginglymostoma cirratum) bacterial artificial chromosome (BAC) library and a preliminary genome survey.

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