G.W. Warr

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.

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.

Immunoglobulin Isotypes: Structure, Function, and Genetics

Immunoglobulin (Ig) classes (in mammals, IgM, IgA, IgD, IgG, IgE) are defined by the isotypes of heavy (H) chains (µ, α, δ, γ, and e). Each isotype is in turn distinguished by unique structures in its constant region domains. These different structures confer distinctive functions on the Ig classes. When two or more Ig classes are very similar, as occurs with the four different types of IgG found in man and mouse, they are usually termed subclasses. Each isotype is encoded by a distinct gene and multiple heavy chain isoforms can be produced by alternative pathways of RNA processing, such as the secreted (slg) and membrane (mlg) forms of all H chains, or the full-length and truncated H chain isoforms of certain avian antibodies. Allelic variation in the constant (C) regions gives rise to allotypes. The different types of light (L) chains (in mammals, к and λ) are also typically referred to as isotypes. This system of classification of Igs was developed from studies of man and his immunological understudy, the mouse, and has proven useful not only in these two species, but also in other mammalian species. Although the classification of mammalian Ig classes and isotypes is quite clear, the situation with Igs from nonmammalian vertebrates is not. For example, is the shark molecule referred to as IgM really IgM? Should we call the predominant low molecular weight Ig in chickens IgG or IgY? This chapter discusses the ways in which these and similar questions have been approached.

Coding sequences of the MHC ii β chain of homozygous rainbow trout (Oncorhynchus mykiss)1

Abstract Six lines of homozygous rainbow trout ( Oncorhynchus mykiss ) 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 β 2 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 β 1 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 masou 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×Hot Creek hybrids indicates that two distinct genes encode the MHC II B sequences and that these genes are unlinked

Purification of duck immunoglobulins: an evaluation of protein A and protein G affinity chromatography.

Duck serum proteins binding to protein A Sepharose CL-4B and protein G Sepharose 4 Fast Flow and eluted at pH 2.8 or 11.5 were characterized by sodium dodecyl sulphate polyacrylamide gel electrophoresis, radial/immunodiffusion against defined anti-immunoglobulin (Ig) reagents, and by the reactivity in immunoelectrophoresis of antisera raised in rabbits inoculated with the eluates. The results indicated that IgY (previous nomenclature 7.8S IgG) and IgY (delta Fc) (previously 5.7S IgG) bound to protein A efficiently and to protein G weakly, while IgM bound to protein A and protein G weakly. Some binding of non-Ig proteins also occurred. Attempts to separate the non-Ig proteins from the Igs by elution at different pHs (5.0, 4.0, 3.0 and 2.5) were unsuccessful, but it was found that precipitation of Igs in day-old duck serum with Na2SO4, followed by chromatography on protein A Sepharose, yielded relatively pure IgY. The efficient binding of the duck IgYs to protein A resembles high affinity binding of mammalian Igs but cannot be attributed to the Fc, as it is in mammals, since the IgY (delta Fc) does not have an Fc region. Instead, binding probably occurs through unique histidine residues occurring predominantly in the CH1 domain.

Putative immunoglobulin VH genes of the goldfish,Carassius auratus, detected by heterologous cross-hybridization with a murine VH probe

By using a defined cDNA probe for the VH region of a murine phosphocholine-binding myeloma protein (S107) we have defined a family of distinct cross-hybridizing DNA sequences in genomic DNA of the goldfish. The estimated number of the goldfish putative VH family detectable by the S107 probe is about 36. By using two putative goldfish VH probes to analyze, by hybridization, the relationships among seven of the goldfish genomic clones, we have determined that the putative goldfish VH genes recognized by the S107 probe comprise at least several distinct families that are not closely related.

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 β 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 β+ ve T cells. This is the first report of the establishment, cloning and partial characterization of duck lymphoblastoid cell lines.

Brief review of McDowell and Simon

All multicellular organisms are in an arms race against the vast armies of rapidly mutating microbial pathogens that are seeking access to their rich stores of nutrients. The challenges for the organism are two-fold. First, to develop appropriate immune defense molecules, and second, to generate the diversity needed to combat a rapidly changing pathogen population. Immunologists have had much success in understanding the varied means by which animals accomplish this, but our integrated vision of immunity generally does not stray across the line that divides animals from plants. However, it is obvious that plants can resist many infectious disease agents very effectively, and in this article McDowell and Simon review the multiple lines of defense that plants use against pathogenic microbes. In some cases higher plants use immune function proteins that are structurally similar to major components in the innate immune systems of animals (e.g. the NOD proteins), and they can also generate diversity in their clustered immune genes by mechanisms that include crossovers, largeand small-scale deletions and duplications of multiple genes or sections of genes, sequence exchanges and gene conversion.