A 44K microarray dataset of the changing transcriptome in developing Atlantic salmon (Salmo salar L.)

<p>Abstract</p> <p>Background</p> <p>Atlantic salmon (<it>Salmo salar </it>L.) is an environmentally and economically important organism and its gene content is reasonably well characterized. From a transcriptional standpoint, it is important to characterize the changes in gene expression over the course of unperturbed early development, from fertilization through to the parr stage.</p> <p>Findings</p> <p><it>S. salar </it>samples were taken at 17 time points from 2 to 89 days post fertilization. Total RNA was extracted and cRNA was synthesized and hybridized to a newly developed 44K oligo salmonid microarray platform. Quantified results were subjected to preliminary data analysis and submitted to NCBI's Gene Expression Omnibus (GEO). Data can be found under the GEO accession number GSE25938. <url>http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE25938</url></p> <p>Conclusions</p> <p>Throughout the entire period of development, several thousand genes were found to be differentially regulated. This work represents the trancriptional characterization of a very large geneset that will be extremely valuable in further examination of the transcriptional changes in Atlantic salmon during the first few months of development. The expression profiles can help to annotate salmon genes in addition to being used as references against any number of experimental variables to which developing salmonids might be subjected.</p

Expression and genomic organization of zonadhesin-like genes in three species of fish give insight into the evolutionary history of a mosaic protein

BACKGROUND: The mosaic sperm protein zonadhesin (ZAN) has been characterized in mammals and is implicated in species-specific egg-sperm binding interactions. The genomic structure and testes-specific expression of zonadhesin is known for many mammalian species. All zonadhesin genes characterized to date consist of meprin A5 antigen receptor tyrosine phosphatase mu (MAM) domains, mucin tandem repeats, and von Willebrand (VWD) adhesion domains. Here we investigate the genomic structure and expression of zonadhesin-like genes in three species of fish. RESULTS: The cDNA and corresponding genomic locus of a zonadhesin-like gene (zlg) in Atlantic salmon (Salmo salar) were sequenced. Zlg is similar in adhesion domain content to mammalian zonadhesin; however, the domain order is altered. Analysis of puffer fish (Takifugu rubripes) and zebrafish (Danio rerio) sequence data identified zonadhesin (zan) genes that share the same domain order, content, and a conserved syntenic relationship with mammalian zonadhesin. A zonadhesin-like gene in D. rerio was also identified. Unlike mammalian zonadhesin, D. rerio zan and S. salar zlg were expressed in the gut and not in the testes. CONCLUSION: We characterized likely orthologs of zonadhesin in both T. rubripes and D. rerio and uncovered zonadhesin-like genes in S. salar and D. rerio. Each of these genes contains MAM, mucin, and VWD domains. While these domains are associated with several proteins that show prominent gut expression, their combination is unique to zonadhesin and zonadhesin-like genes in vertebrates. The expression patterns of fish zonadhesin and zonadhesin-like genes suggest that the reproductive role of zonadhesin evolved later in the mammalian lineage

Regulatory Processes That Control Haploid Expression of Salmon Sperm mRNAs

Objective&nbsp; Various stages of mRNA processing are necessary for functionally important genes required during late-stage sperm differentiation. Protein–RNA complexes form that edit, stabilize, store, deliver, localize and regulate translation of sperm mRNAs. These regulatory processes are often directed by recognition sequence elements and the particular composition of the proteins associated with the mRNAs. Previous work has shown that the cAMP response element modulator (CREM), estrogen receptor-alpha (ERα) and forkhead box L2A (FOXL2A) proteins are present in late-stage salmon sperm. Here we investigate whether these and other regulatory proteins might control processing of mRNAs not expressed until the haploid stage of development. We also examine regulatory processes that prepare and present mRNAs that generate unique products essential for differentiating sperm (i.e. for flagellar assembly and function). Results&nbsp; We provide evidence for potential sperm-specific recognition elements in 5′-untranslated regions (utrs) that may bind CREM, ERα, FOXL2A, Y-box and other proteins. We show that changes within the 5′-utrs and open reading frames of some sperm genes lead to distinct protein termini that may provide specific interfaces necessary for localization and function within the paternal gamete

Regulation and Expression of Sexual Differentiation Factors in Embryonic and Extragonadal Tissues of Atlantic salmon

Background: The products of cyp19, dax, foxl2, mis, sf1 and sox9 have each been associated with sex-determiningprocesses among vertebrates. We provide evidence for expression of these regulators very early in salmoniddevelopment and in tissues outside of the hypothalamic-pituitary-adrenal/gonadal (HPAG) axis. Although thefunction of these factors in sexual differentiation have been defined, their roles in early development before sexualfate decisions and in tissues beyond the brain or gonad are essentially unknown.Results: Bacterial artificial chromosomes containing salmon dax1 and dax2, foxl2b and mis were isolated and theregulatory regions that control their expression were characterized. Transposon integrations are implicated in theshaping of the dax and foxl2 loci. Splice variants for cyp19b1 and mis in both embryonic and adult tissues weredetected and characterized. We found that cyp19b1 transcripts are generated that contain 5’-untranslated regionsof different lengths due to cryptic splicing of the 3’-end of intron 1. We also demonstrate that salmon mistranscripts can encode prodomain products that present different C-termini and terminate before translation of theMIS hormone. Regulatory differences in the expression of two distinct aromatases cyp19a and cyp19b1 are exerted,despite transcription of their transactivators (ie; dax1, foxl2, sf1) occurring much earlier during embryonicdevelopment.Conclusions: We report the embryonic and extragonadal expression of dax, foxl2, mis and other differentiationfactors that indicate that they have functions that are more general and not restricted to steroidogenesis andgonadogenesis. Spliced cyp19b1 and mis transcripts are generated that may provide regulatory controls for tissueordevelopment-specific activities. Selection of cyp19b1 transcripts may be regulated by DAX-1, FOXL2 and SF-1complexes that bind motifs in intron 1, or by signals within exon 2 that recruit splicing factors, or both. Thepotential translation of proteins bearing only the N-terminal MIS prodomain may modulate the functions of otherTGF b family members in different tissues. The expression patterns of dax1 early in salmon embryogenesisimplicate its role as a lineage determination factor. Other roles for these factors during embryogenesis and outsidethe HPAG axis are discussed

Evolution of duplicated IgH loci in Atlantic salmon, Salmo salar

<p>Abstract</p> <p>Background</p> <p>The Atlantic salmon (<it>Salmo salar</it>) immunoglobulin heavy chain (<it>IgH</it>) locus possesses two parallel <it>IgH </it>isoloci (<it>IGH-A </it>and <it>IGH-B</it>), that are related to the genomic duplication event in the family Salmonidae. These duplicated <it>IgH </it>loci in Atlantic salmon provide a unique opportunity to examine the mechanisms of genome diversity and genome evolution of the <it>IgH </it>loci in vertebrates. In this study, we defined the structure of these loci in Atlantic salmon, and sequenced 24 bacterial artificial chromosome (BAC) clones that were assembled into the <it>IGH-A </it>(1.1 Mb) and <it>IGH-B </it>(0.9 Mb) loci. In addition, over 7,000 cDNA clones from the <it>IgH </it>variable (V<smcaps>H</smcaps>) region have been sequenced and analyzed.</p> <p>Results</p> <p>The present study shows that the genomic organization of the duplicated <it>IgH </it>loci in Atlantic salmon differs from that in other teleosts and other vertebrates. The loci possess multiple Cτ genes upstream of the Cμ region, with three of the Cτ genes being functional. Moreover, the duplicated loci possess over 300 V<smcaps>H</smcaps> segments which could be classified into 18 families. This is the largest number of V<smcaps>H</smcaps> families currently defined in any vertebrate. There were significant structural differences between the two loci, indicating that both <it>IGH-A </it>and <it>-B </it>loci have evolved independently in the short time after the recent genome duplication approximately 60 mya.</p> <p>Conclusions</p> <p>Our results indicate that the duplication of the <it>IgH </it>loci in Atlantic salmon significantly contributes to the increased diversity of the antibody repertoire, as compared with the single <it>IgH </it>locus in other vertebrates.</p

Sequencing the genome of the Atlantic salmon (Salmo salar)

The International Collaboration to Sequence the Atlantic Salmon Genome (ICSASG) will produce a genome sequence that identifies and physically maps all genes in the Atlantic salmon genome and acts as a reference sequence for other salmonids

Fish and chips: Various methodologies demonstrate utility of a 16,006-gene salmonid microarray

BACKGROUND: We have developed and fabricated a salmonid microarray containing cDNAs representing 16,006 genes. The genes spotted on the array have been stringently selected from Atlantic salmon and rainbow trout expressed sequence tag (EST) databases. The EST databases presently contain over 300,000 sequences from over 175 salmonid cDNA libraries derived from a wide variety of tissues and different developmental stages. In order to evaluate the utility of the microarray, a number of hybridization techniques and screening methods have been developed and tested. RESULTS: We have analyzed and evaluated the utility of a microarray containing 16,006 (16K) salmonid cDNAs in a variety of potential experimental settings. We quantified the amount of transcriptome binding that occurred in cross-species, organ complexity and intraspecific variation hybridization studies. We also developed a methodology to rapidly identify and confirm the contents of a bacterial artificial chromosome (BAC) library containing Atlantic salmon genomic DNA. CONCLUSION: We validate and demonstrate the usefulness of the 16K microarray over a wide range of teleosts, even for transcriptome targets from species distantly related to salmonids. We show the potential of the use of the microarray in a variety of experimental settings through hybridization studies that examine the binding of targets derived from different organs and tissues. Intraspecific variation in transcriptome expression is evaluated and discussed. Finally, BAC hybridizations are demonstrated as a rapid and accurate means to identify gene content

Isolation, characterization and comparison of Atlantic and Chinook salmon growth hormone 1 and 2

<p>Abstract</p> <p>Background</p> <p>Growth hormone (GH) is an important regulator of skeletal growth, as well as other adapted processes in salmonids. The GH gene (<it>gh</it>) in salmonids is represented by duplicated, non-allelic isoforms designated as <it>gh1 </it>and <it>gh2</it>. We have isolated and characterized <it>gh</it>-containing bacterial artificial chromosomes (BACs) of both Atlantic and Chinook salmon (<it>Salmo salar </it>and <it>Oncorhynchus tshawytscha</it>) in order to further elucidate our understanding of the conservation and regulation of these loci.</p> <p>Results</p> <p>BACs containing <it>gh1 </it>and <it>gh2 </it>from both Atlantic and Chinook salmon were assembled, annotated, and compared to each other in their coding, intronic, regulatory, and flanking regions. These BACs also contain the genes for skeletal muscle sodium channel oriented in the same direction. The sequences of the genes for interferon alpha-1, myosin alkali light chain and microtubule associated protein Tau were also identified, and found in opposite orientations relative to <it>gh1 </it>and <it>gh2</it>. Viability of each of these genes was examined by PCR. We show that transposon insertions have occurred differently in the promoters of <it>gh</it>, within and between each species. Other differences within the promoters and intronic and 3'-flanking regions of the four <it>gh </it>genes provide evidence that they have distinct regulatory modes and possibly act to function differently and/or during different times of salmonid development.</p> <p>Conclusion</p> <p>A core proximal promoter for transcription of both <it>gh1 </it>and <it>gh2 </it>is conserved between the two species of salmon. Nevertheless, transposon integration and regulatory element differences do exist between the promoters of <it>gh1 </it>and <it>gh2</it>. Additionally, organization of transposon families into the BACs containing <it>gh1 </it>and for the BACs containing <it>gh2</it>, are very similar within orthologous regions, but much less clear conservation is apparent in comparisons between the <it>gh1</it>- and <it>gh2</it>-containing paralogous BACs for the two fish species. This is consistent with the hypothesis that a burst of transposition activity occurred during the speciation events which led to Atlantic and Pacific salmon. The Chinook and other <it>Oncorhynchus </it>GH1s are strikingly different in comparison to the other GHs and this change is not apparent in the surrounding non-coding sequences.</p

Salmo salar and Esox lucius full-length cDNA sequences reveal changes in evolutionary pressures on a post-tetraploidization genome

<p>Abstract</p> <p>Background</p> <p>Salmonids are one of the most intensely studied fish, in part due to their economic and environmental importance, and in part due to a recent whole genome duplication in the common ancestor of salmonids. This duplication greatly impacts species diversification, functional specialization, and adaptation. Extensive new genomic resources have recently become available for Atlantic salmon (<it>Salmo salar</it>), but documentation of allelic versus duplicate reference genes remains a major uncertainty in the complete characterization of its genome and its evolution.</p> <p>Results</p> <p>From existing expressed sequence tag (EST) resources and three new full-length cDNA libraries, 9,057 reference quality full-length gene insert clones were identified for Atlantic salmon. A further 1,365 reference full-length clones were annotated from 29,221 northern pike (<it>Esox lucius</it>) ESTs. Pairwise d_N/d_Scomparisons within each of 408 sets of duplicated salmon genes using northern pike as a diploid out-group show asymmetric relaxation of selection on salmon duplicates.</p> <p>Conclusions</p> <p>9,057 full-length reference genes were characterized in <it>S. salar </it>and can be used to identify alleles and gene family members. Comparisons of duplicated genes show that while purifying selection is the predominant force acting on both duplicates, consistent with retention of functionality in both copies, some relaxation of pressure on gene duplicates can be identified. In addition, there is evidence that evolution has acted asymmetrically on paralogs, allowing one of the pair to diverge at a faster rate.</p

A Salmonid EST Genomic Study: Genes, Duplications, Phylogeny and Microarrays

Background: Salmonids are of interest because of their relatively recent genome duplication, and their extensive usein wild fisheries and aquaculture. A comprehensive gene list and a comparison of genes in some of the different speciesprovide valuable genomic information for one of the most widely studied groups of fish.Results: 298,304 expressed sequence tags (ESTs) from Atlantic salmon (69% of the total), 11,664 chinook, 10,813sockeye, 10,051 brook trout, 10,975 grayling, 8,630 lake whitefish, and 3,624 northern pike ESTs were obtained in thisstudy and have been deposited into the public databases. Contigs were built and putative full-length Atlantic salmonclones have been identified. A database containing ESTs, assemblies, consensus sequences, open reading frames, genepredictions and putative annotation is available. The overall similarity between Atlantic salmon ESTs and those of rainbowtrout, chinook, sockeye, brook trout, grayling, lake whitefish, northern pike and rainbow smelt is 93.4, 94.2, 94.6, 94.4,92.5, 91.7, 89.6, and 86.2% respectively. An analysis of 78 transcript sets show Salmo as a sister group to Oncorhynchusand Salvelinus within Salmoninae, and Thymallinae as a sister group to Salmoninae and Coregoninae within Salmonidae.Extensive gene duplication is consistent with a genome duplication in the common ancestor of salmonids. Using all of theavailable EST data, a new expanded salmonid cDNA microarray of 32,000 features was created. Cross-specieshybridizations to this cDNA microarray indicate that this resource will be useful for studies of all 68 salmonid species.Conclusion: An extensive collection and analysis of salmonid RNA putative transcripts indicate that Pacific salmon,Atlantic salmon and charr are 94–96% similar while the more distant whitefish, grayling, pike and smelt are 93, 92, 89 and86% similar to salmon. The salmonid transcriptome reveals a complex history of gene duplication that is consistent withan ancestral salmonid genome duplication hypothesis. Genome resources, including a new 32 K microarray, providevaluable new tools to study salmonids