Enhancer and promoter activity in the JH to IGHM intron of the Pekin duck, Anas platyrhynchos.

A transcriptional enhancer, Emu, was defined in the IGH locus of the Pekin duck, Anas platyrhynchos. Regions of DNA from the JH to IGHM intron were cloned into reporter constructs containing the SV40 promoter and transiently transfected into chicken B and T lymphocytes. A strong transcriptional activity, of several hundred-fold greater than that of a reporter construct with the promoter alone, was localized to a 281bp region that contains 2 E-box motifs, CAGCTG. This fragment showed enhancer activity in both orientations and was active in chicken B cells but not in T cells. When the activity of the enhancer was tested in constructs without a promoter, it showed high transcriptional activity in the forward orientation, but much less activity (by two orders of magnitude) when tested in the reverse orientation. This suggests that the fragment contains not only enhancer activity but may contain promoter activity analogous to that of the Imu promoter described in mammals. Thus it appears that the location, but not the fine structure, of the Emu enhancer was established before the evolutionary divergence of the avian and mammalian lineages some 300Myr ago.

A dolphin peripheral blood leukocyte cDNA microarray for studies of immune function and stress reactions.

A microarray focused on stress response and immune function genes of the bottlenosed dolphin has been developed. Random expressed sequence tags (ESTs) were isolated and sequenced from two dolphin peripheral blood leukocyte (PBL) cDNA libraries biased towards T- and B-cell gene expression by stimulation with IL-2 and LPS, respectively. A total of 2784 clones were sequenced and contig analysis yielded 1343 unigenes (archived and annotated at ). In addition, 52 dolphin genes known to be important in innate and adaptive immune function and stress responses of terrestrial mammals were specifically targeted, cloned and added to the unigene collection. The set of dolphin sequences printed on a cDNA microarray comprised the 1343 unigenes, the 52 targeted genes and 2305 randomly selected (but unsequenced) EST clones. This set was printed in duplicate spots, side by side, and in two replicates per slide, such that the total number of features per microarray slide was 19,200, including controls. The dolphin arrays were validated and transcriptomic profiles were generated using PBL from a wild dolphin, a captive dolphin and dolphin skin cells. The results demonstrate that the array is a reproducible and informative tool for assessing differential gene expression in dolphin PBL and in other tissues.

Characterization of the immunoglobulin A heavy chain gene of the Atlantic bottlenose dolphin (Tursiops truncatus).

Immunoglobulin constant region heavy chain genes of the dolphin (Tursiops truncatus) have been described for IgM and IgG but not for IgA. Here, the heavy chain sequence of dolphin IgA has been cloned and sequenced as cDNA. RT-PCR amplification from blood peripheral lymphocytes was carried out using degenerate primers and a single sequence was detected. The inferred heavy chain structure shows conserved features typical of mammalian IgA heavy chains, including three constant (C) regions, a hinge region between constant region domain 1 (C1) and constant region domain 2 (C2), and conserved residues for interaction with the Fc alpha R1 and N-glycosylation sites. Comparisons of the deduced amino acid sequences of the IgA heavy chain for the dolphin and the evolutionarily related artiodactyl species showed high similarity. In cattle and sheep, as in dolphins, a single IgA subclass has been identified. Southern blot analysis as well as genomic PCR confirmed the presence of multiple IGHA sequences suggesting that IGHA pseudogenes may be present in the dolphin genome.

Evolution of antibody class switching: identification and transcriptional control of an Inu exon in the duck (Anas platyrhynchos).

Immunoglobulin class switching is characteristic to the tetrapod lineage, but the nature of this process has been elucidated only in mammals, where I-exon transcription initiates and directs the recombination in the IgH locus. Here, it is shown that an I-exon occurs 5' of the nu (IgY constant region) gene of the duck (Anas platyrhynchos): it is longer than mammalian I-exons and comprised primarily of tandem repeats. The Inu promoter was identified and shown to be responsive to stimulation with IL-4 but not LPS. It contains Oct, LYF-1, ATF, and C/EBP motifs. Site directed mutagenesis indicates that 2 C/EBP motifs are uniquely necessary for the response of the promoter to IL-4, as tested in the mouse pre-B cell line, 70Z/3. These results support the conclusion that the signal transduction pathways controlling I-exon promoter responses to cytokines have been highly conserved in vertebrate evolution.

Immunoglobulins of the non-galliform birds: antibody expression and repertoire in the duck.

Galliform and non-galliform birds express three immunoglobulin isotypes, IgM, IgA and IgY. Beyond this we should not generalize because differences in gene organization may have functional consequences reflected in the immune response. At present, studies on non-galliform birds are largely restricted to ducks. Ducks express an alternatively spliced form of their IgY heavy chain (upsilon) gene, the IgY(DeltaFc), that lacks the Fc region and Fc-associated secondary effector functions. It is not known how common the expression of the IgY(DeltaFc) is among birds, nor the functional consequences. It is also not known whether the unusual organization of the duck IgH locus, also shared with the chicken, having the gene order of mu, alpha and upsilon, with alpha inverted in the locus, is unique to the galloanseriform lineage. Ducks, like chickens, have a single immunoglobulin light chain of the lambda (lambda) type. Evidence suggests that ducks, like chickens, generate their immunoglobulin repertoire through a single functional rearrangement of the variable (V) region, and generate diversity through gene conversion from a pool of pseudogenes. In Southern blots of germline and rearranged bursal DNA, both the heavy and light chain loci of ducks appear to each undergo one major rearrangement event. For both heavy and light chains, the functional V region element and the pseudogenes appear to consist of a single gene family. Further analysis of 26 heavy chain joining (JH) and 27 light chain JL segments shows there is use of a single J segment in ducks, which is diversified presumably through somatic mutations and gene conversion events. Despite this limitation on the rearrangement of immunoglobulin genes, analysis of 26 DH and 122 VL sequences suggests that extensive sequence diversity is generated.

New resources for marine genomics: bacterial artificial chromosome libraries for the Eastern and Pacific oysters (Crassostrea virginica and C. gigas).

Large-insert genomic bacterial artificial chromosome (BAC) libraries of two culturally and economically important oyster species, Crassostrea virginica and C. gigas, have been developed as part of an international effort to develop tools and reagents that will advance our ability to conduct genetic and genomic research. A total of 73,728 C. gigas clones with an average insert size of 152 kb were picked and arrayed representing an 11.8-fold genome coverage. A total of 55,296 clones with an average insert size of 150 kb were picked and arrayed for C. virginica, also representing an 11.8-fold genome coverage. The C. gigas and C. virginica libraries were screened with probes derived from selected oyster genes using high-density BAC colony filter arrays. The probes identified 4 to 25 clones per gene for C. virginica and 5 to 50 clones per gene for C. gigas. We conducted a preliminary analysis of genetic polymorphism represented in the C. gigas library. The results suggest that the degree of divergence among similar sequences is highly variable and concentrated in intronic regions. Evidence supporting allelic polymorphism is reported for two genes and allelic and/or locus specific polymorphism for several others. Classical inheritance studies are needed to confirm the nature of these polymorphisms. The oyster BAC libraries are publicly available to the research community on a cost-recovery basis at (www.genome.clemson.edu).

The Immunoglobulin G Heavy Chain (IGHG) genes of the Atlantic bottlenose dolphin, Tursiops truncatus.

Dolphin Immunoglobulin G Heavy Chain (IGHG) sequences were obtained by PCR amplification of cDNA from peripheral blood leukocytes using degenerate primers. Analysis of full-length sequences indicated the presence of two expressed isotypes, IGHG1 and IGHG2 that differ mainly in the hinge region of the molecule. Genomic Southern blot analysis indicated that the IGHG1 and IGHG2 genes are most likely present in single copies. The inferred amino acid sequences show greatest similarity between the dolphin and other closely related artiodactyl species. The genetic structure of the IGHG genes were deduced through genomic PCR and revealed that the hinge regions of both IGHG1 and IGHG2 are encoded by a single exon. The transmembrane region of the dolphin IGHG chain shows similarity to the transmembrane region of other mammalian IGHG chains with a canonical CART motif. This is in contrast to the unusual Ser to Gly substitution previously found in the dolphin IGHM transmembrane region, and the functional significance of this variation for B cell antigen-receptor dimer activation remains unknown.

Cloning of the IgM heavy chain of the bottlenose dolphin (Tursiops truncatus), and initial analysis of VH gene usage.

Clones encoding the dolphin IgM heavy (micro) chain gene were isolated from a cDNA library of peripheral blood leukocytes. Genomic Southern blot analyses showed that the dolphin IGHM gene is most likely present in a single copy, and its sequence shows greatest similarity to those of the IGHM gene of the sheep, pig and cow, evolutionarily related artiodactyls. The transmembrane (TM) form of the IGHM chain was isolated by 3' RACE. While showing similarities to the TM regions of other mammalian IGHM chains, the highly conserved Ser residue of the CART motif is substituted with a Gly in the dolphin. In contrast to the pig and cow, which utilize only a single VH family, the dolphin expresses at least two distinct VH families, belonging to the mammalian VH clans I and III. At least two JH genes were identified in the dolphin. Some CDR3 regions of the dolphin VH are long (up to 21 amino acids), and contain multiple Cys residues, hypothesized to stabilize the CDR3 structure through disulfide bond formation.

Sampling the skin transcriptome of the North Atlantic right whale.

As an initial step in defining the transcriptome of the North Atlantic right whale (Eubalaena glacialis) and developing functional genomic tools to study right whale health at the molecular physiological level, a cDNA library has been constructed from a skin biopsy. 2496 randomly selected clones (expressed sequence tags, ESTs) have been sequenced, and genes identified as important in the response to stress and immune challenges have been cloned by targeted RT-PCR from skin cDNA. The analysis of the EST collection (archived at www.marinegenomics.org and GenBank) showed a 34.79% redundancy, yielding 1578 unigenes and 27 potential microsatellite markers. 96 genes were cloned by targeted PCR; moreover, 52 of these genes are stress and immune function related. A Gene Ontology analysis of the unigene collection indicates that the skin is a rich source of expressed genes with diverse functions, suggesting an important role in multiple physiological processes including those related to immunity and stress response.