The transcriptomic responses of the eastern oyster, Crassostrea virginica, to environmental conditions.

Understanding the mechanisms by which organisms adapt to environmental conditions is a fundamental question for ecology and evolution. In this study, we evaluate changes in gene expression of a marine mollusc, the eastern oyster Crassostrea virginica, associated with the physico-chemical conditions and the levels of metals and other contaminants in their environment. The results indicate that transcript signatures can effectively disentangle the complex interactive gene expression responses to the environment and are also capable of disentangling the complex dynamic effects of environmental factors on gene expression. In this context, the mapping of environment to gene and gene to environment is reciprocal and mutually reinforcing. In general, the response of transcripts to the environment is driven by major factors known to affect oyster physiology such as temperature, pH, salinity, and dissolved oxygen, with pollutant levels playing a relatively small role, at least within the range of concentrations found in the studied oyster habitats. Further, the two environmental factors that dominate these effects (temperature and pH) interact in a dynamic and nonlinear fashion to impact gene expression. Transcriptomic data obtained in our study provide insights into the mechanisms of physiological responses to temperature and pH in oysters that are consistent with the known effects of these factors on physiological functions of ectotherms and indicate important linkages between transcriptomics and physiological outcomes. Should these linkages hold in further studies and in other organisms, they may provide a novel integrated approach for assessing the impacts of climate change, ocean acidification and anthropogenic contaminants on aquatic organisms via relatively inexpensive microarray platforms.

A transcriptomic analysis of the stress induced by capture-release health assessment studies in wild dolphins (Tursiops truncatus).

The health of wild bottlenose dolphins (Tursiops truncatus) is typically evaluated by the study of animals that are captured and released back into the wild after examination. The impact of such studies on gene expression in peripheral blood cells was investigated using microarray and quantitative polymerase chain reaction methods. Significantly increased expression was observed in two major classes of genes: (i) energy metabolism, and (ii) responsiveness to stress and trauma, the latter effect suggesting the initiation of an acute-phase response. The value of data obtained in capture/release studies may need to be weighed against the potential physiological impacts of such studies.

Structure of the catfish IGH locus: analysis of the region including the single functional IGHM gene.

The catfish IGH locus is large ( approximately 1 Mb) and complex, having undergone multiple internal duplications and transpositions. To define the structure of the locus that contains the single expressed IGHM gene, two overlapping bacterial-artificial-chromosome (BAC) clones spanning the most 3' end of the channel catfish immunoglobulin heavy (IGH) chain locus have been completely sequenced. The analyses created a contig of 257,153 bp containing 55 VH, 6 D, 12 JH genes and the IGH constant region genes encoding the functional secreted and membrane forms of IgM and the membrane form of IgD. This analysis revealed three major features. First, no C-region genes were found aside from the previously described IGHM1 and IGHD1, with the latter gene being the most 3' C-region gene of the catfish IGH locus. There was no evidence in the region sequenced for genes that could encode an Ig class similar to the IgZ/IgT described in zebrafish, trout and pufferfish. Second, there are a high number of VH pseudogenes, 28 out of 55 (51%). In contrast, the entire zebrafish IGH locus has 40 functional VH genes and eight pseudogenes (17%). Third, an internal duplication of a 52.4-kb block of VH genes has occurred. These observations suggest that the IGH locus of teleost fish varies significantly from species to species in the diversity of C-region genes as well as the numbers of genes encoding V regions.

The immunoglobulin heavy chain locus of the duck. Genomic organization and expression of D, J, and C region genes.

The region of the duck IgH locus extending from upstream of the proximal diversity (D) segment to downstream of the constant gene cluster has been cloned and mapped. A sequence contig of 48,796 base pairs established that the organization of the genes is D-J(H)-mu-alpha-upsilon. No evidence for a functional homologue (or remnant) of a delta gene was found. The alpha gene is in inverted transcriptional orientation; class switch to IgA expression thus requires inversion of the approximately 27-kilobase pair region that includes both mu and alpha genes. The secreted forms of duck alpha and mu are each encoded by 4 constant region exons, and the hydrophobic C-terminal regions of the membrane receptor forms of alpha and mu are encoded by one and two transmembrane exons, respectively. Putative switch (S) regions were identified for duck mu and upsilon by comparison with chicken Smu and Supsilon sequences and for duck alpha by comparison with mouse Salpha. The duck IgH locus is rich in complex variable number tandem repeats, which occupy approximately 60% of the sequenced region, and occur at a much higher frequency in the IgH locus than in other sequenced regions of the duck genome.

Immune gene discovery by expressed sequence tag analysis of hemocytes and hepatopancreas in the Pacific White Shrimp, Litopenaeus vannamei, and the Atlantic White Shrimp, L. setiferus.

A pilot program was undertaken in immune gene discovery in two sister species of litopenaeid shrimp, the Pacific white shrimp, Litopenaeus vannamei and the Atlantic white shrimp, L. setiferus. RNA from the hemocytes and hepatopancreas of single individuals from each species was recovered, 4 cDNA libraries (one from each tissue/species) were made by a PCR-based method and a total of approximately 2045 randomly selected clones were sequenced. A total of 268 expressed sequence tags (ESTs) were found that corresponded to 44 immune function genes. The most common immune-function ESTs (172) were antimicrobial peptides, which were restricted to the hemocyte libraries. Lectins were the largest group of immune-function ESTs found in the hepatopancreas. Analysis of these libraries indicates that EST approaches are effective for immune gene discovery in shrimp and that the diversity of these PCR-generated libraries would support full-scale EST collection.

Diversity of the immunoglobulin heavy chain in the Atlantic salmon (Salmo salar L.) is contributed by genes from two parallel IgH isoloci.

Immunoglobulin heavy chain (IgH) variable (V) region cDNAs from the Atlantic salmon, Salmo salar L., have been isolated and analysed with respect to diversity and transcription of the two parallel IgH isoloci in this species. A total of nine V(H) families were defined according to the 80% identity criterion, of which seven were highly related (>80% identity) to the V(H) families defined in rainbow trout and arctic charr. The variability of the CDR1 and 2 was low, although mutational hot-spot consensus sequences were accumulated in these regions. The CDR3 showed largest variability, expressing at least eight different groups of D motifs diversified by fusion of the D motifs, possible N and P nucleotide insertions and exonuclease activity. Presumably functional transcripts expressing D motifs in all three reading frames were identified for two of the motifs. The cDNAs were mapped to either of the two parallel loci, and sequence analysis revealed that the repertoire of V(H) segments was contributed by transcription of genes from both of the IgH isoloci. Transcription of genes from both isoloci generated no obvious effects on variability in the CDR3 of the Atlantic salmon IgH chains, although one additional J(H)-segment with altered N-terminal was generated by the process of duplication and divergence. Thus, the issue of biological significance of the two IgH isoloci remains unclear.

An IgH enhancer that drives transcription through basic helix-loop-helix and Oct transcription factor binding motifs. Functional analysis of the E(mu)3' enhancer of the catfish.

The transcriptional enhancer (E(mu)3') of the IgH locus of the channel catfish, Ictalurus punctatus, shows strong B cell-specific activity and differs from the mammalian E(mu) enhancer in both location and structure. It occurs between the mu and delta genes and contains numerous transcription factor binding sites, predominantly octamer and muE5 motifs of consensus and variant sequences. It lacks the classical muA-muE3(CBF)-muB core array of binding motifs seen within mammalian IgH E(mu) enhancers. To determine the functionally important motifs, a series of mutant enhancers was created using sequence-targeted polymerase chain reaction. Whereas the mutation of consensus and variant octamer motifs (individually or in multiples) decreased enhancer function, mutation of a single consensus muE5 motif destroyed the function of this enhancer in mammalian plasmacytomas. Mutation of this consensus muE5 site, combined with mutations of certain octamer sites, destroyed function in catfish B cells. Experiments using artificial enhancers containing multimers of motifs or short regions of the native enhancer suggested that the minimal E(mu)3' enhancer (a) contains a consensus muE5 site and two octamer sites, (b) is B cell-specific, and (c) is active across species. The dependence of an Ig enhancer on sites that bind basic helix-loop-helix and Oct transcription factors has not previously been observed and confirms large differences in structure and function between fish and mammalian IgH enhancers.

Telomerase expression and telomere length in immortal leukocyte lines from channel catfish.

Normal channel catfish leukocytes readily undergo spontaneous in vitro immortalization yielding functionally active diploid cell lines. Since telomerase activation appears to be a critical step in the establishment of immortal mammalian cells, studies were undertaken to determine if and when telomerase expression occurs during the in vitro immortalization process of channel catfish leukocytes. To this end, freshly isolated peripheral blood leukocytes (PBL) from normal fish were shown to exhibit low to undetectable levels of telomerase activity and within four days after culture initiation showed dramatic increases in telomerase activity which typically remained high for at least four weeks. This activity then declined, concomitant with decreases in cellular proliferation and increases in cell death. Cells which escaped this culture "crisis" re-expressed high levels of telomerase activity indefinitely. Although telomerase activity was expressed early in the immortalization process, clonal cell lines derived from these cultures had relatively short telomeres. These results suggest that telomerase expression in catfish leukocytes is activation-induced, and its expression does not necessarily stabilize telomere length until a critically, albeit ill-defined, short length is reached.

Catfish Oct2 binding affinity and functional preference for octamer motifs, and interaction with OBF-1.

The DNA-binding (POU) domain of the catfish Oct2 transcription factor was shown, by electromobility shift assays and surface plasmon resonance techniques, to have an affinity for the consensus octamer motif (ATGCAAAT) that was slightly higher than its affinity for a variant motif (ATGtAAAT). This observation is consistent with the transcriptional activation potentials of catfish Oct2 alpha and Oct2 beta, which were shown to activate transcription in catfish B and T cell lines to an equivalent extent from both the consensus and variant octamer motifs. When tested in a mouse plasmacytoma cell line, catfish Oct2 alpha and Oct2 beta, as well as mouse Oct2, showed higher transcriptional activation with the variant, as compared to the consensus, octamer motif. Catfish Oct2 was shown to function synergistically with the mammalian co-activator, OBF-1, activating octamer-dependent transcription in catfish T cells. The strong transcriptional activity of OBF-1 in catfish cells was dependent on the presence of octamer motif(s) at the proximal (promoter) rather than the distal (enhancer) position.

Light chain variable region diversity in Atlantic cod (Gadus morhua L.).

This study was undertaken to determine if a lack of V(L) domain variability could explain, in part, the failure of Atlantic cod to respond to immunization with the production of specific antibodies. The variability of cod V(L) regions was studied in 33 cDNA and two genomic clones. The variability of the CDRs was estimated by the Shannon entropy method and compared with that in other species. It was found to be lowest in the little skate (Raja erinacea), higher in cod, and highest in Xenopus and mouse. While the variability of the CDRs is slightly lower in cod than in Xenopus and mouse, it is spread over broader areas of the amino acid sequence. The length of CDR1 and CDR3 in cod is equal to or exceeds that found in skate, Xenopus, chicken and mammals. Isoelectric points and hydrophobicity vary substantially among the studied Ig light chain domains. Thus, neither the length, nor the variability, nor the physicochemical properties (pI and hydrophobicity) of the L chain CDRs can explain the absence of antibody response to immunization in cod.

Coding sequences of the MHC II beta chain of homozygous rainbow trout (Oncorhynchus mykiss).

Six lines of homozygous rainbow trout (Oncorhynchus mikiss) from different genetic and geographical backgrounds have been produced as aquatic models for biomedical research by the chromosome set manipulation techniques of androgenesis and gynogenesis. Messenger RNA from spleens was extracted. and the MHC II B cDNA sequences, amplified by RT PCR, were cloned into plasmids. Sequences of the MHC II beta2 domains were highly conserved between the different plasmids from the same and different lines of trout. Most of the variability among sequences was found in the amino terminal half of the beta1 domain, which corresponds with the peptide binding region of the MHC II molecule. This diversity suggests that the different lines of trout may exhibit differences in immune response. Rainbow trout MHC II B sequences were similar to the MHC II B sequences of the Pacific salmon (O. gorbuscha, O. tshawytscha, O. nerka, O. miasou, O. kisutch). Southern blot analysis performed on the restricted DNA of the OSU and Hot Creek trout, and the doubled haploid progeny produced by androgenesis from OSU x Hot Creek hybrids indicates that two distinct genes encode the MHC II B sequences and that these genes are unlinked.

Duck lymphocytes. VIII. T-lymphoblastoid cell lines from reticuloendotheliosis virus-induced tumours.

The T strain of reticuloendotheliosis virus (REV-T) obtained, along with the helper chicken syncytia virus (CSV), from the CSO4 cell line was highly oncogenic and rapidly fatal in ducks. Tumours were mainly seen in spleen, but neoplastic cells were observed microscopically in many organs. In vitro REV transformation of duck lymphocytes failed to yield stable cell lines, so cells from organs (blood, bone marrow, spleen, lymph node, bursa of Fabricius) of infected birds were used to establish cell lines. Some of these cell lines have been cloned. The success rates of establishment and cloning were increased if cells were cultured in a range of media containing different supplements; however, medium containing 5% foetal calf serum (FCS) and 5% duck serum was generally most efficacious for initial establishment, while spent medium from the parental line supplemented with a further 20% FCS gave best results for cloning. Cloned cell lines had the morphology of lymphoblastoid cells, with irregular nuclei and diffuse chromatin. Analysis of mRNA extracted from these cell lines showed that the uncloned lines were strongly expressing the ß chain of the T cell antigen receptor (TCR) and weakly expressing immunoglobulin (Ig) polypeptides [λ light chain and µ, υ, υ (ΔFc) and α heavy chains in various proportions], suggesting the presence of T and B cells. The cloned cell lines that could be classified were TCR ß+ ve T cells. This is the first report of the establishment, cloning and partial characterization of duck lymphoblastoid cell lines.

T-cell receptors in channel catfish: structure and expression of TCR alpha and beta genes.

Herein are reported full length cDNA sequences for TCR alpha- and beta-chains of the channel catfish. Included are sequences belonging to four Valpha and six Vbeta families which share hallmarks in common with the Valpha and Vbeta genes of other species. Similar to the situation in other vertebrates, the catfish Calpha and Cbeta sequences exhibit distinct immunoglobulin, connecting peptide, transmembrane and cytoplasmic domains. However, the catfish TCR Calpha and Cbeta regions are shorter than those of mammals and the catfish Cbeta chain lacks a cysteine in its connecting peptide region. Two different catfish Cbeta cDNA sequences were identified, suggesting the existence of either two Cbeta loci or allotypes. Based on Southern blot analyses, each of the catfish TCR gene loci appear to be arranged in a translocon (as opposed to multicluster) organization with multiple V elements and a single or few copies of C region DNA. At the deduced amino acid level, the catfish Cbeta sequence exhibits 42% identity with the Cbeta of Atlantic salmon, 41% identity with the Cbeta of rainbow trout and 26% identity with Cbeta of the horned shark. The catfish Calpha amino acid sequence exhibits 44 and 29% identity with Calpha of the rainbow trout and southern pufferfish, respectively. TCRalpha and beta messages are selectively expressed and rearranged in a catfish clonal cell line that appears to be of the T lineage. This TCR alpha/beta expressing clonal lymphocyte line, designated 28S.1, has T-cell like function in that it constitutively produces a supernatant factor(s) with growth promoting activity. These findings should facilitate functional studies of fish TCRs and T cells in ways not previously possible with other 'lower' vertebrate models.

Mitogen and growth factor-induced activation of a STAT-like molecule in channel catfish lymphoid cells.

This article describes the identification of a putative STAT molecule in the channel catfish (Ictalurus punctatus), the first report of such a molecule in a 'lower' vertebrate. A monoclonal antibody against human STAT6 recognizes an approximately 100 kDa molecule that becomes activated and translocates to the nucleus upon both growth factor and mitogen stimulation of catfish leukocytes. This presumed catfish STAT binds the mammalian interferon-gamma activation site, a known motif of mammalian STAT binding, as shown by electromobility shift assays. Purification of the proteins present in these DNA complexes confirms that the catfish reactive molecule binds to the interferon-gamma activation site sequence. These results suggest that STAT molecules have been highly conserved in vertebrate evolution.

Characterization of Oct2 from the channel catfish: functional preference for a variant octamer motif.

The Ig heavy chain enhancer of the channel catfish (Ictalurus punctatus) has an unusual position and structure, being found in the 3' region of the mu gene and containing eight functional octamer motifs of consensus (ATGCAAAT) and variant sequences. The presence of multiple octamer motifs suggests that an Oct2 homologue may play an important role in driving expression of the Ig heavy chain locus in a teleost fish. To test this hypothesis, two catfish Oct2 cDNAs (alpha and beta) were cloned by screening a catfish B cell cDNA library. Catfish Oct2 alpha and beta isoforms are derived by alternative RNA splicing; as determined by Southern analysis, Oct2 is a single copy gene. In comparisons with mammalian Oct2, the catfish Oct2 isoforms show high sequence conservation in their N-terminal regions and POU domains, but extensive divergence in their C-terminal regions. Catfish Oct2 a and beta are tissue restricted, bind both consensus and variant octamer motifs, and activate transcription in both catfish and murine cells. In contrast, mouse Oct2 activated transcription in mouse but not catfish cells. Catfish Oct2 beta is a more potent transcriptional activator than Oct2 alpha. In transient expression assays, catfish Oct2 beta showed a marked preference for the octamer variant, ATGtAAAT, which occurs twice in the catfish enhancer. Mouse Oct2 also showed increased activity with the variant octamer when tested in mouse B cells. Gel-shift analysis competition assays indicated that catfish Oct2 binds the consensus octamer motif with an apparently higher affinity than it does the variant motif.