Biomedical subjects

Joan Burnside

Publications and source records attributed to Joan Burnside.

15 recordsLinked to original sources

PubMed2006

Marek's disease virus up-regulates major histocompatibility complex class II cell surface expression in infected cells.

Many herpesviruses modulate major histocompatibility complex (MHC) expression on the cell surface as an immune evasion mechanism. We report here that Marek's disease virus (MDV), a lymphotrophic avian alphaherpesvirus, up-regulates MHC class II cell surface expression in infected cells, contrary to all other herpesviruses examined to date. This MDV-induced class II up-regulation was detected both in vitro and in vivo. This effect was not solely an indirect effect of interferon, which is a highly potent natural inducer of MHC class II expression, since MHC class II up-regulation in cultured primary fibroblast cells was confined to the infected cells only. MHC class II up-regulation was also observed in infected cells of the bursa of Fabricius during the lytic phase of MDV infection in birds and upon reactivation of MDV from latency in an MDV-transformed cell line. As MDV is a strictly cell-associated virus and requires activated T cells for its life cycle, this up-regulation of MHC class II in infected cells may contribute to virus spread within the infected host by increasing the chance of contact between productively infected cells and susceptible activated T cells.

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PubMed2006

Chicken genomics resource: sequencing and annotation of 35,407 ESTs from single and multiple tissue cDNA libraries and CAP3 assembly of a chicken gene index.

Its accessibility, unique evolutionary position, and recently assembled genome sequence have advanced the chicken to the forefront of comparative genomics and developmental biology research as a model organism. Several chicken expressed sequence tag (EST) projects have placed the chicken in 10th place for accrued ESTs among all organisms in GenBank. We have completed the single-pass 5'-end sequencing of 37,557 chicken cDNA clones from several single and multiple tissue cDNA libraries and have entered 35,407 EST sequences into GenBank. Our chicken EST sequences and those found in public databases (on July 1, 2004) provided a total of 517,727 public chicken ESTs and mRNAs. These sequences were used in the CAP3 assembly of a chicken gene index composed of 40,850 contigs and 79,192 unassembled singlets. The CAP3 contigs show a 96.7% match to the chicken genome sequence. The University of Delaware (UD) EST collection (43,928 clones) was assembled into 19,237 nonredundant sequences (13,495 contigs and 5,742 unassembled singlets). The UD collection contains 6,223 unique sequences that are not found in other public EST collections but show a 76% match to the chicken genome sequence. Our chicken contig and singlet sequences were annotated according to the highest BlastX and/or BlastN hits. The UD CAP3 contig assemblies and singlets are searchable by nucleotide sequence or key word (http://cogburn.dbi.udel.edu), and the cDNA clones are readily available for distribution from the chick EST website and clone repository (http://www.chickest.udel.edu). The present paper describes the construction and normalization of single and multiple tissue chicken cDNA libraries, high-throughput EST sequencing from these libraries, the CAP3 assembly of a chicken gene index from all public ESTs, and the identification of several nonredundant chicken gene sets for production of custom DNA microarrays.

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PubMed2006

Marek's disease virus encodes MicroRNAs that map to meq and the latency-associated transcript.

MicroRNAs (miRNAs) are a class of small (approximately 22-nucleotide) regulatory molecules that block translation or induce degradation of target mRNAs. These have been identified in a wide range of organisms, including viruses. In particular, the oncogenic gammaherpesviruses Kaposi's sarcoma herpesvirus and Epstein-Barr virus encode miRNAs that could potentially regulate either viral or host genes. To determine if Marek's disease virus (MDV), an oncogenic alphaherpesvirus of chickens, encodes miRNAs, we isolated small RNAs from MDV-infected chicken embryo fibroblasts (CEF) and used the 454 Life Sciences sequencing technology to obtain the sequences of 13,679 candidate host and viral small RNAs. Eight miRNAs were found, five of which flank the meq oncogene and three that map to the latency-associated transcript (LAT) region of the genome. The meq gene is unique to pathogenic serotypes of MDV and is transcriptionally active during latency and transformation, and the LAT region of the MDV genome is antisense to the immediate-early gene ICP4. Secondary structure analysis predicted that the regions flanking the miRNAs could form hairpin precursors. Northern blot analysis confirmed expression of all miRNAs in MDV-infected CEF, MDV-induced tumors, and MDV lymphoblastoid cell lines. We propose that the MDV miRNAs function to enable MDV pathogenesis and contribute to MDV-induced transformation of chicken T cells.

Amino Acid Sequence↗

PubMed2005

Development of a cDNA array for chicken gene expression analysis.

BACKGROUND: The application of microarray technology to functional genomic analysis in the chicken has been limited by the lack of arrays containing large numbers of genes. RESULTS: We have produced cDNA arrays using chicken EST collections generated by BBSRC, University of Delaware and the Fred Hutchinson Cancer Research Center. From a total of 363,838 chicken ESTs representing 24 different adult or embryonic tissues, a set of 11,447 non-redundant ESTs were selected and added to an existing collection of clones (4,162) from immune tissues and a chicken bursal cell line (DT40). Quality control analysis indicates there are 13,007 useable features on the array, including 160 control spots. The array provides broad coverage of mRNAs expressed in many tissues; in addition, clones with expression unique to various tissues can be detected. CONCLUSIONS: A chicken multi-tissue cDNA microarray with 13,007 features is now available to academic researchers from http://genomics@fhcrc.org. Sequence information for all features on the array is in GenBank, and clones can be readily obtained. Targeted users include researchers in comparative and developmental biology, immunology, vaccine and agricultural technology. These arrays will be an important resource for the entire research community using the chicken as a model.

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PubMed2005

Conservation of biological properties of the CD40 ligand, CD154 in a non-mammalian vertebrate.

Signals delivered by the CD40 ligand, CD154, have crucial roles in immune responses in mammals, being required for development of germinal centres, maturation of T-dependent antibody responses, and generation of B-cell memory. To determine whether these functions were conserved in a non-mammalian species, a putative chicken CD 154 cDNA was used to make an oligomeric fusion protein, and to raise monoclonal antibodies. The antibodies detected surface expression on activated T-cells. The fusion protein detected expression of a receptor on B-cells, thrombocytes and macrophages. Biological effects of the fusion protein included induction of NO synthesis in a macrophage cell line, enhancement of splenic B-cell survival, and induction of apoptosis in a bursal lymphoma cell line. These observations demonstrated substantial functional equivalence with mammalian CD 154 and thus provided evidence for the early evolutionary emergence of the set of functions associated with this molecule, and its central role in the vertebrate immune system.

Amino Acid Sequence↗

PubMed2004

Transcriptome analysis for the chicken based on 19,626 finished cDNA sequences and 485,337 expressed sequence tags.

We present an analysis of the chicken (Gallus gallus) transcriptome based on the full insert sequences for 19,626 cDNAs, combined with 485,337 EST sequences. The cDNA data set has been functionally annotated and describes a minimum of 11,929 chicken coding genes, including the sequence for 2260 full-length cDNAs together with a collection of noncoding (nc) cDNAs that have been stringently filtered to remove untranslated regions of coding mRNAs. The combined collection of cDNAs and ESTs describe 62,546 clustered transcripts and provide transcriptional evidence for a total of 18,989 chicken genes, including 88% of the annotated Ensembl gene set. Analysis of the ncRNAs reveals a set that is highly conserved in chickens and mammals, including sequences for 14 pri-miRNAs encoding 23 different miRNAs. The data sets described here provide a transcriptome toolkit linked to physical clones for bioinformaticians and experimental biologists who wish to use chicken systems as a low-cost, accessible alternative to mammals for the analysis of vertebrate development, immunology, and cell biology.

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PubMed2004

Microarray analysis of Lyn-deficient B cells reveals germinal center-associated nuclear protein and other genes associated with the lymphoid germinal center.

Lyn is the only member of the Src family expressed in DT40 B cells, which provide a unique model to study the singular contribution of this protein tyrosine kinase (PTK) family to cell signaling. In these cells, gene ablation of Lyn leads to defective B cell receptor signaling. Complementary DNA array analysis of Lyn-deficient DT40 cells shows that the absence of Lyn leads to down-regulation of numerous genes encoding proteins involved in B cell receptor signaling, proliferation, control of transcription, immunity/inflammation response, and cytoskeletal organization. Most of these expression changes have not been previously associated with Lyn PTK signaling. They include alterations in mRNA levels of germinal center-associated nuclear protein (germinal center-associated DNA primase) (GANP), CD74, CD22, NF-kappaB, elongation factor 1alpha, CD79b, octamer binding factor 1, Ig H chain, stathmin, and gamma-actin. Changes in GANP expression were also confirmed in Lyn-deficient mice, suggesting that Lyn PTK has a unique function not compensated for by other Src kinases. Because Lyn-deficient mice have impaired development of germinal centers in spleen, the decreased expression of GANP in the Lyn-deficient DT40 cell line and Lyn-deficient mice suggests that Lyn controls the formation and proliferation of germinal centers via GANP. GANP promoter activity was higher in wild-type vs Lyn-deficient cells. Mutation of the PU.1 binding site reduced activity in wild-type cells and had no effect in Lyn-deficient cells. The presence of Lyn enhanced PU.1 expression in a Northern blot. Thus, the following new signaling pathway has been described: Lyn-->PU.1-->GANP.

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PubMed2004

Identification and molecular cloning of functional chicken IL-12.

By a combination of large-scale sequencing, bioinformatics, and traditional molecular biology, we identified the long-searched-for cDNA sequences encoding the homologues of the chicken IL-12p35 and IL-12p40 chains. These molecules are the first discovered nonmammalian IL-12 subunits. The homologies of the chicken IL-12p35 and IL-12p40 proteins to the corresponding known subunits of various species, i.e., humans, sheep, horse, cat, bovine, mouse, and woodchuck, ranged between 21 and 42%, respectively. The expression of IL-12 subunits was observed in lymphoid cells and proved to be dependent on the cell type and stimulus, while expression was not detected in stimulated primary chicken embryo fibroblast cells. Following transient expression of both molecules in COS-7 cells, we confirmed the necessity of heterodimerization into IL-12p70 to yield bioactivity as was also shown for its mammalian counterparts. The chicken IL-12p70 molecule, generated either by transient coexpression of monomeric IL-12p35 and monomeric IL-12p40 or as a fusion protein (as in a fusion linker construct), induced IFN-gamma synthesis and proliferative activity of freshly exposed chicken splenocytes. The high degree of functional similarity between chicken IL-12 and IL-12 of higher mammalian vertebrates, despite their poor sequence homology, illustrates the conservation and vital importance of the IL-12 molecule since the evolutionary dichotomy of birds and mammals >300 million years ago. In this article, we describe the first nonmammalian IL-12 molecule and show that this chicken IL-12 molecule is bioactive.

Amino Acid Sequence↗

PubMed2004

Herpesvirus of turkeys: microarray analysis of host gene responses to infection.

Herpesvirus of turkeys (HVT) provides an economically important live vaccine for prevention of Marek's disease (MD) of chickens. MD, characterized by both immunosuppression and T-cell lymphoma, is caused by another herpesvirus termed Marek's disease virus (MDV). Microarrays were used to investigate the response of chicken embryonic fibroblasts (CEF) to infection with HVT. Genes responding to HVT infection include several induced by interferon along with others modulating signal transduction, transcription, scaffolding proteins, and the cytoskeleton. Results are compared with earlier studies examining the responses of CEF cells to infection with MDV.

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PubMed2004

Patterns of gene expression in the developing chick thymus.

Gene expression in thymic T cells during late embryogenesis and early growth in chicks was examined using cDNA microarrays. Gene expression patterns were profiled into nine clusters by using self-organizing maps (SOM) clustering analysis. The expression patterns for a set of genes confirmed current information on the development of immune response. Expression of cell surface markers (MHC class I alpha chain, MHC class II associated invariant chain, CD8 beta chain, CD18, and beta2-microglobulin), and genes involved in the innate immune response (NK lysin-like) increased with age, and these patterns were consistent with an increase in the immune responsiveness of the young chick. The expression of cytokine receptor common gamma chain (gammac), death receptor-3 (DR3), and TCR alpha chain increased up to 1 day of age and then decreased. DR3 could play a role in the apoptosis during T-cell maturation, while the differential expression of TCR genes could reflect regulation of the rearrangement of TCR genes and TCR-mediated signal transduction during T cell development. Three genes coding for previously uncharacterized proteins are included in the clusters. These gene expression profiling studies provide background information on the developing chick immune system and provide preliminary functional information on unknown proteins.

Amino Acid Sequence↗

PubMed2003

Five hundred sixty-five triples of chicken, human, and mouse candidate orthologs.

The Human Genome Project has provided abundant gene sequence information on human and important model organisms. The chicken is well positioned from an evolutionary standpoint to serve as a link between higher and lower organisms, particularly mammals, and amphibia and fish. In this study we used stringent criteria to select 565 triples of chicken, human, and mouse candidate orthologs. We analyze the sequences with respect to nucleotide and amino acid similarities. This analysis also allows measurement of evolutionary distances of different proteins. We found that chicken-human and chicken-mouse sequence identities are highly correlated; similarly for chicken-human and chicken-mouse evolutionary distances. With chicken as the out-group, we found that mouse has a higher substitution rate than human, supporting the generation-time effect hypothesis. We also described the transversion bias, which is the preference for some transversions than others in nucleotide substitutions. We demonstrated that there are statistically significant properties in the differences of orthologous sequences. The differential patterns, in combination with sequence similarity analysis, may lead to the identification of genes that are very divergent from the mammalian orthologs.

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PubMed2002

Sulfhydryl oxidases: emerging catalysts of protein disulfide bond formation in eukaryotes.

Members of the Quiescin-sulfhydryl oxidase (QSOX) family utilize a thioredoxin domain and a small FAD-binding domain homologous to the yeast ERV1p protein to oxidize sulfhydryl groups to disulfides with the reduction of oxygen to hydrogen peroxide. QSOX enzymes are found in all multicellular organisms for which complete genomes exist and in Trypanosoma brucei, but are not found in yeast. The avian QSOX is the best understood enzymatically: its preferred substrates are peptides and proteins, not monothiols such as glutathione. Mixtures of avian QSOX and protein disulfide isomerase catalyze the rapid insertion of the correct disulfide pairings in reduced RNase. Immunohistochemical studies of human tissues show a marked and highly localized concentration of QSOX in cell types associated with heavy secretory loads. Consistent with this role in the formation of disulfide bonds, QSOX is typically found in the cell in the endoplasmic reticulum and Golgi and outside the cell. In sum, this review suggests that QSOX enzymes play a significant role in oxidative folding of a large variety of proteins in a wide range of multicellular organisms.

Amino Acid Sequence↗

PubMed2002

Jak3-regulated genes: DNA array analysis of concanavalin a-interleukin-2-activated chicken T cells treated with a specific jak3 inhibitor.

Janus kinase 3 (Jak3) is important in the activation and proliferation of lymphoid cells and binds to the common gamma subunit of several cytokine receptors, including the interleukin-2 (IL-2) receptor (IL-2R). DNA arrays were used to measure mRNA levels of a large number of genes regulated by signaling through the Jak3 tyrosine kinase pathway by blocking concanavalin A (ConA)-IL-2-activated chicken splenic T cells with a specific Jak3 inhibitor (WHI-P154). Of the 635 genes detected by arrays containing about 1200 cDNAs, 12 were upregulated in control cells compared with inhibitor-treated cells, and 6 were expressed at higher levels in the inhibitor-treated group. By identifying genes that are directly or indirectly regulated by Jak3, we can gain insight into the roles of this key intermediate in avian T cell activation and further our understanding of intracellular signaling networks in the immune response.

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PubMed2002

A novel interferon regulatory factor (IRF), IRF-10, has a unique role in immune defense and is induced by the v-Rel oncoprotein.

The cloning and functional characterization of a novel interferon regulatory factor (IRF), IRF-10, are described. IRF-10 is most closely related to IRF-4 but differs in both its constitutive and inducible expression. The expression of IRF-10 is inducible by interferons (IFNs) and by concanavalin A. In contrast to that of other IRFs, the inducible expression of IRF-10 is characterized by delayed kinetics and requires protein synthesis, suggesting a unique role in the later stages of an antiviral defense. Accordingly, IRF-10 is involved in the upregulation of two primary IFN-gamma target genes (major histocompatibility complex [MHC] class I and guanylate-binding protein) and interferes with the induction of the type I IFN target gene for 2',5'-oligo(A) synthetase. IRF-10 binds the interferon-stimulated response element site of the MHC class I promoter. In contrast to that of IRF-1, which has some of the same functional characteristics, the expression of IRF-10 is not cytotoxic for fibroblasts or B cells. The expression of IRF-10 is induced by the oncogene v-rel, the proto-oncogene c-rel, and IRF-4 in a tissue-specific manner. Moreover, v-Rel and IRF-4 synergistically cooperate in the induction of IRF-10 in fibroblasts. The level of IRF-10 induction in lymphoid cell lines by Rel proteins correlates with Rel transformation potential. These results suggest that IRF-10 plays a role in the late stages of an immune defense by regulating the expression some of the IFN-gamma target genes in the absence of a cytotoxic effect. Furthermore, IRF-10 expression is regulated, at least in part, by members of the Rel/NF-kappa B and IRF families.

Amino Acid Sequence↗

PubMed2000

Differential cytokine expression in avian cells in response to invasion by Salmonella typhimurium, Salmonella enteritidis and Salmonella gallinarum.

Salmonella enterica is a facultative intracellular pathogen that is capable of causing disease in a range of hosts. Although human salmonellosis is frequently associated with consumption of contaminated poultry and eggs, and the serotypes Salmonella gallinarum and Salmonella pullorum are important world-wide pathogens of poultry, little is understood of the mechanisms of pathogenesis of Salmonella in the chicken. Type III secretion systems play a key role in host cell invasiveness and trigger the production of pro-inflammatory cytokines during invasion of mammalian hosts. This results in a polymorphonuclear cell influx that contributes to the resulting enteritis. In this study, a chicken primary cell culture model was used to investigate the cytokine responses to entry by the broad host range serotypes S. enteritidis and S. typhimurium, and the host specific serotype S. gallinarum, which rarely causes disease outside its main host, the chicken. The cytokines interleukin (IL)-1ss, IL-2, IL-6 and interferon (IFN)-gamma were measured by quantitative RT-PCR, and production of IL-6 and IFN-gamma was also determined through bioassays. All serotypes were invasive and had little effect on the production of IFN-gamma compared with non-infected cells; S. enteritidis invasion caused a slight down-regulation of IL-2 production. For IL-1ss production, infection with S. typhimurium had little effect, whilst infection with S. gallinarum or S. enteritidis caused a reduction in IL-1ss mRNA levels. Invasion of S. typhimurium and S. enteritidis caused an eight- to tenfold increase in production of the pro-inflammatory cytokine IL-6, whilst invasion by S. gallinarum caused no increase. These findings correlate with the pathogenesis of Salmonella in poultry. S. typhimurium and S. enteritidis invasion produces a strong inflammatory response, that may limit the spread of Salmonella largely to the gut, whilst S. gallinarum does not induce an inflammatory response and may not be limited by the immune system, leading to the severe systemic disease fowl typhoid.

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