| Vol. 101(Suppl. I) October 2006 |  |
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A bacterial artificial chromosome library for Biomphalaria glabrata, intermediate snail host of Schistosoma mansoni Vol. 101(Suppl. I): 167-177, 2006 Coen M Adema/+, Mei-Zhong Luo*, Ben Hanelt, Lynn A Hertel†, Jennifer J Marshall, Si-Ming Zhang, Randall J DeJong/++, Hye-Ran Kim*, David Kudrna*, Rod A Wing*, Cari Soderlund**, Matty Knight***, Fred A Lewis***, Roberta Lima Caldeira****, Liana K Jannotti-Passos****, Omar dos Santos Carvalho****, Eric S Loker Department of Biology, University of New Mexico,
Albuquerque, NM, US *Arizona Genomics Institute, Department of Plant
Sciences **Arizona Genomics Computational Laboratory, BIO5 Institute,
University of Arizona, Tucson, US ***Biomedical Research Institute,
Rockville, MD, US ****Laboratório de Helmintoses Intestinais,
Centro de Pesquisas To provide a novel resource for analysis of the genome of Biomphalaria glabrata, members of the international Biomphalaria glabrata Genome Initiative (biology.unm.edu/biomphalaria-genome.html), working with the Arizona Genomics Institute (AGI) and supported by the National Human Genome Research Institute (NHGRI), produced a high quality bacterial artificial chromosome (BAC) library. The BB02 strain B. glabrata, a field isolate (Belo Horizonte, Minas Gerais, Brasil) that is susceptible to several strains of Schistosoma mansoni, was selfed for two generations to reduce haplotype diversity in the offspring. High molecular weight DNA was isolated from ovotestes of 40 snails, partially digested with HindIII, and ligated into pAGIBAC1 vector. The resulting B. glabrata BAC library (BG_BBa) consists of 61824 clones (136.3 kb average insert size) and provides 9.05 ´ coverage of the 931 Mb genome. Probing with single/low copy number genes from B. glabrata and fingerprinting of selected BAC clones indicated that the BAC library sufficiently represents the gene complement. BAC end sequence data (514 reads, 299860 nt) indicated that the genome of B. glabrata contains ~ 63% AT, and disclosed several novel genes, transposable elements, and groups of high frequency sequence elements. This BG_BBa BAC library, available from AGI at cost to the research community, gains in relevance because BB02 strain B. glabrata is targeted whole genome sequencing by NHGRI. Key words: genomics - gene discovery - fingerprinting - schistosomiasis - medical malacology
The application of molecular approaches continues to contribute novel insights into the biology, including genomics of molluscs (Zhang et al. 2004, Mitta et al. 2005). To date, several mitochondrial genomes of molluscs have been sequenced (DeJong et al. 2004, Mizi et al. 2005), but the nuclear genome of a representative of the Phylum Mollusca remains to be fully characterized. In fact, lophotrochozoan protostomes of which mollusca represent the largest group (Rouse 1999), are underrepresented among the animals from which the current assembly of fully sequenced genomes has been obtained. Thus, genomic data from a mollusc will help fill a gap in the information on the evolutionary history of animal life (Collins et al. 2003). Molluscs are a highly diverse group that includes some of the largest, longest living, and most intelligent invertebrates. Genome information will instruct on several remarkable properties of molluscs such as shell formation (biomineralization; Milet et al. 2004), the evolution of body asymmetry (Schilthuizen & Davison 2005), and hermaphrodism (Paraense & Corrêa 1988). Molluscs are also being used to study pharmo-toxicology (Terlau & Olivera 2004); neuroendocrinology (Altelaar et al. 2005); parthenogenesis (Jokela et al. 2003); and the molecular basis of behavior and learning (Williamson & Chrachri 2004, Zhurov et al. 2005). Molluscs serve as bioindicators for monitoring of the environment (Zhao et al. 2005), and (snails especially) are useful for understanding how natural selection operates (Vermeij 2002). Furthermore, molluscs are economically important as a major source of food, can destroy crops, colonize and impact new habitats as invasive species (Pointier et al. 2005), and transmit medically important pathogens. The latter applies to the freshwater gastropod Biom-phalaria glabrata (Planorbidae, Basommatophora). This snail serves as one of the most important intermediate hosts for a widespread pathogen of humans, the digenetic trematode Schistosoma mansoni (Paraense & Corrêa 1963, Morgan et al. 2001). This parasite causes intestinal schistosomiasis, a debilitating disease that afflicts over 50 million humans (Chitsulo et al. 2004). To a large extent, the geographic distribution of B. glabrata defines the distribution of S. mansoni in the Neotropics (Paraense 1986, DeJong et al. 2003). Genetic determinants affect the susceptibility of B. glabrata for S. mansoni (Lewis et al. 2001), and heterogeneity in genetic composition of B. glabrata on smaller scales may further influence the transmission patterns of schistosomiasis (Theron & Coustau 2005). More comprehensive genome sequence data for B. glabrata would enable novel investigative approaches to study determinants of transmission, especially in light of an advancing genome project for S. mansoni (Loverde et al. 2004). B. glabrata also hosts a variety of other digenetic trematodes and has been adopted as the most commonly used model host to study the basic biology of digenean-snail interactions (Lie 1982, Adema & Loker 1997, Vergote et al. 2005). As one example, B. glabrata has been found to produce after exposure to digeneans a unique family of hemolymph molecules termed FREPs (fibrinogen-related proteins). FREPs consist of a juxtaposition of fibrinogen and immunoglobulin superfamily domains, and have proven to be remarkably diverse in their composition. B. glabrata thus serves as a new model system to examine the nature and diversity of non-self recognition molecules produced by invertebrates (Zhang et al. 2004). Information on the genome of B. glabrata will also have relevance for several other Biomphalaria species and for yet other species of molluscs which serve as hosts for schistosomes and for a number of other trematode, and some nematode, infectious agents. Besides schistosomiasis, diseases such as fascioliasis, clonorchiasis, and paragonimiasis represent only a few of the snail transmitted diseases with worldwide medical and economic impact (Lockyer et al. 2004a). In 2001, an international consortium, "the Biom-phalaria glabrata genome initiative" was founded to develop genome-type projects for this particular pulmonate gastropod species (http://biology.unm.edu/biomphalaria-genome/index.html). Members of this consortium have contributed several gene discovery projects (Jones et al. 2001, Miller et al. 2001, Schneider & Zelck 2001, Raghavan et al. 2003, Lockyer et al. 2004b, Nowak et al. 2004, Jung et al. 2005, Mitta et al. 2005), the full-length sequence of the mitochondrial genome of B. glabrata (DeJong et al. 2004), and an estimate of 931 Mb for the size of the nuclear genome of B. glabrata (Gregory 2003). A novel resource for genomic studies became available when the National Human Genome Research Institute (NHGRI) awarded a white paper application (http://www.genome.gov/Pages/Research/Sequencing/ BACLibrary/ BgBACprops.pdf) for funding of the production a high quality bacterial artificial chromosome (BAC) library for B. glabrata (http://www.genome.gov/page.cfm ?pageID=10001852). Such a library provides access to large regions of the genome of B. glabrata, in an experimentally manageable fashion. Significantly, the NHGRI support guaranteed high quality standards for the finished library, and also made the BAC library publicly available at cost to the research community. The actual development of the BAC library was a collaboration between the Arizona Genomics Institute (AGI; part of the National Institutes of Health BAC Resource Network) and members of B. glabrata genome initiative. The genomic DNA from a recent B. glabrata field isolate from a schistosomiasis endemic area in Brazil, shown to be susceptible to S. mansoni, was used to ensure that the BAC library provides data that are relevant in the context of parasite-snail compatibility. This report describes the B. glabrata strain used, and both the production and characterization of the BAC library. Lastly, analysis of sequence data obtained provides first glimpses into the genomic make-up of B. glabrata. Acknowledgments Ulrike Zelck (Tûbingen University, Germany) provided helpful discussion for selection of low copy genes. At the University of Arizona, Chris Mueller, Kristi Collura, Nick Sisneros, Marina Wissotski, Dan Smart, David Campos and Kiran Rao provided excellent technical assistance. At the University of New Mexico, George Rosenberg assisted with sequencing of BACs, technical support was provided by the Molecular Biology Facility, which is supported by NIH grant number 1P20RR18754 from the Institute Development Development Award (IDeA) Program of the National Center for Research Resources. Fig. 1 | Fig. 2 | Fig. 3 | Fig. 4 | Fig. 5 | Fig. 6 | Table I | Table II | Table III | Table IV
Financial support: the production and distribution of the BG_BBa BAC library at AGI was supported by the funding from the National Human Genome Research Institute under the BAC Library Production program (grant 5U01HG002525; RAW). Parts of this study were supported by NIH grants AI024340 (ESL), AI052363 (CMA), and Fiocruz +Corresponding author: coenadem@unm.edu. †Deceased 2 April 2005 ++Present address: Laboratory of Malaria and Vector Research, NIAID/NIH, Twinbrook III, Room 2E-20 MSC 8132 Bethesda, MD 20892, US Received 25 May 2006 Accepted 26 June 2006
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