Shane C Burgess

PMID: 17878436;Abstract:

Since the sequencing of the genome and the development of high-throughput tools for the exploration of functional elements of the genome, the chicken has reached model organism status. Functional genomics focuses on understanding the function and regulation of genes and gene products on a global or genome-wide scale. Systems biology attempts to integrate functional information derived from multiple high-content data sets into a holistic view of all biological processes within a cell or organism. Generation of a large collection (∼600K) of chicken expressed sequence tags, representing most tissues and developmental stages, has enabled the construction of high-density microarrays for transcriptional profiling. Comprehensive analysis of this large expressed sequence tag collection and a set of ∼20K full-length cDNA sequences indicate that the transcriptome of the chicken represents approximately 20,000 genes. Furthermore, comparative analyses of these sequences have facilitated functional annotation of the genome and the creation of several bioinformatic resources for the chicken. Recently, about 20 papers have been published on transcriptional profiling with DNA microarrays in chicken tissues under various conditions. Proteomics is another powerful high-throughput tool currently used for examining the dynamics of protein expression in chicken tissues and fluids. Computational analyses of the chicken genome are providing new insight into the evolution of gene families in birds and other organisms. Abundant functional genomic resources now support large-scale analyses in the chicken and will facilitate identification of transcriptional mechanisms, gene networks, and metabolic or regulatory pathways that will ultimately determine the phenotype of the bird. New technologies such as marker-assisted selection, transgenics, and RNA interference offer the opportunity to modify the phenotype of the chicken to fit defined production goals. This review focuses on functional genomics in the chicken and provides a road map for large-scale exploration of the chicken genome. ©2007 Poultry Science Association Inc.

PMID: 19391180;Abstract:

Experimental identification of expressed proteins by proteomics constitutes the most reliable approach to identify genomic location and structure of protein-coding genes and substantially complements computational genome annotation. Channel catfish herpesvirus (CCV) is a simple comparative model for understanding herpesvirus biology and the evolution of the Herpesviridae. The canonical CCV genome has 76 predicted ORF and only 12 of these have been confirmed experimentally. We describe a modification of a statistical method, which assigns significance measures, q-values, to peptide identifications based on 2-D LC ESI MS/MS, real-decoy database searches and SEQUEST XCorr and DCn scores. We used this approach to identify CCV proteins expressed during its replication in cell culture, to determine protein composition of mature virions and, consequently, to refine the canonical CCVgenome annotation. To complement trypsin, we used partial proteinase K digestion, which yielded greater proteome coverage. At FDR 5%, for peptide identifications, we identified 25/76 previously predicted ORF using trypsin and 31/76 using proteinase K. Furthermore, we identified 17 novel protein-coding regions (7 potential ATG-initiated ORF). Most of these novel ORF encode small proteins (100 amino acids). Directed, strand-specific reverse transcription real-time PCR confirmed RNA expression from 6/7 novel ATG-initiated ORF investigated. © 2009 Wiley-VCH Verlag GmbH & Co. KGaA.

PMID: 17990261;Abstract:

Non-electrophoretic methods based on two-dimensional liquid chromatography followed directly by tandem mass spectrometry (2D-LC/MS²) have become the preferred method for high-throughput expression proteomics and are widely applied to fresh tissues. Pre-fractionation techniques are also used in combination with 2D-LC/MS² to both increase the proteome size and to assign cellular locations. Data from such experiments have become central to systems biology analyses. Here we apply a differential detergent (pre)fractionation (DDF) followed by 2D-LC/MS² to frozen archival tissues. Our results show that by using frozen archival tissues, we do not lose proteome coverage or the ability to assign proteins to cellular compartments. In addition, we were able to assign 'biological process' Gene Ontology (GO) annotations, which will facilitate systems biological modeling of our proteomics data. Copyright © 2007 John Wiley & Sons, Ltd.

PMID: 13679597;Abstract:

Marek's disease virus (MDV) is classified as an oncogenic lymphotropic herpesvirus of chickens. MDV productively and cytolytically infects B, αβT and γδT lymphocytes and latently infects T-helper lymphocytes. The aims of this study were to identify whether MDV infects macrophages in vivo and, if so, whether quantitative differences in macrophage infection are associated with MDV strain virulence. Chickens were infected with either virulent MDV (HPRS-16) or 'hypervirulent' MDV (C12/130). Flow cytometry with monoclonal antibodies recognizing MDV pp38 antigen and leukocyte antigens was used to identify MDV lytically infected cells. Macrophages from HPRS-16- and C12/130-infected chickens were pp38⁺. It is demonstrated that macrophages are pp38⁺ because they are infected and not because they have phagocytosed MDV antigens, as assessed by confocal microscopy using antibodies recognizing MDV antigens of the three herpesvirus kinetic classes: infected cell protein 4 (ICP4, immediate early), pp38 (early) and glycoprotein B (gB, late). Spleen macrophages from MDV-infected chickens were ICP4⁺, pp38⁺ and gB⁺, and ICP4 had nuclear localization denoting infection. Finally, MDV pp38⁺ macrophages had high inherent death rates, confirming cytolytic MDV infection, although production of virus particles has not been detected yet. These results have two fundamental implications for understanding MDV pathogenesis: (i) MDV evolved to perturb innate, in addition to acquired, immunity and (ii) macrophages are excellent candidates for transporting MDV to primary lymphoid organs during the earliest stages of pathogenesis.