Biomedical subjects

Robert W Chapman

Publications and source records attributed to Robert W Chapman.

16 recordsLinked to original sources

PubMed2006

Insights into the immune transcriptome of the shrimp Litopenaeus vannamei: tissue-specific expression profiles and transcriptomic responses to immune challenge.

Infectious disease constitutes a major obstacle to the sustainability of shrimp aquaculture worldwide and a significant threat to natural populations of shrimp and other crustacea. The study of the shrimp immune system, including the response to viral infection, has been hampered by a relative lack of molecular genetic information and of tools suitable for high-throughput assessment of gene expression. In this report, the generation of a cDNA microarray encompassing 2,469 putative unigenes expressed in gills, circulating hemocytes, and hepatopancreas of Litopenaeus vannamei is described. The unigenes printed on the microarray were derived from the analyses of 7,021 expressed sequence tags obtained from standard cDNA libraries as well as from libraries generated by suppression subtractive hybridization, after challenging shrimp with a variety of immune stimuli. The general utility of the cDNA microarray was demonstrated by interrogating the array with labeled RNA from four different shrimp tissues (gills, hemocytes, hepatopancreas, and muscle) and by analyzing the transcriptomic response of shrimp to a lethal challenge with white spot syndrome virus. Our results indicate that white spot syndrome virus infection upregulates (in the hepatopancreas) genes encoding known and potential antimicrobial effectors, while some genes involved in protection from oxidative stress were found to be downregulated by the virus.

Animals↗

PubMed2006

Double-stranded RNA and antiviral immunity in marine shrimp: inducible host mechanisms and evidence for the evolution of viral counter-responses.

Double-stranded RNA (dsRNA) is a common virus-associated molecular pattern and a potent inducer of antiviral responses in many organisms. While it is clear that the specific RNA interference (RNAi) response, a phenomenon triggered by dsRNA, serves antiviral functions in invertebrates, innate (non-specific) antiviral immune reactions induced by dsRNA (e.g. the Interferon response) have long been thought to be restricted to vertebrates. Recent work in an underappreciated experimental model, the penaeid shrimp, is challenging these traditional distinctions, by demonstrating the existence of both innate (non sequence-specific) and RNAi-related (sequence-specific) antiviral phenomena in crustacea. Here we discuss the evidence for this bivalent role of dsRNA in the initiation of antiviral responses in shrimp, and present new data that suggest that the antiviral functions of the shrimp RNAi machinery have imposed selective pressures on an evolving viral pathogen. These findings open the door for the discovery of novel mechanisms of innate immunity, and provide a basis for the future development of strategies to control viral diseases in the commercially important penaeid shrimp.

Animals↗

PubMed2006

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.

Animals↗

PubMed2006

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).

Animals↗

PubMed2006

Regulation of metallothionein genes in the American oyster (Crassostrea virginica): ontogeny and differential expression in response to different stressors.

Metallothioneins (MTs) are typically low molecular weight (6-7 kDa), metal-binding proteins with characteristic repeating cysteine motifs (Cys-X-Cys or Cys-Xn-Cys) and a prolate ellipsoid shape containing single alpha- and beta-domains. While functionally diverse, they play important roles in metals homeostasis, detoxification and the stress response. The present study, combined with previous observations (e.g., Jenny et al., Eur. J. Biochem. 2005; 271:1702-1712) defines an unprecedented diversity of MT primary structure and domain organization in the American oyster, Crassostrea virginica. Two novel molluscan MT families are described. One of these (CvMT-III) is characterized by the presence of two beta-domains and the absence of alpha-domains. This family exhibits constitutive expression during larval development and is the dominant CvMT isoform expressed in larvae. CvMT-III displays low basal levels of expression in adult tissues and only moderate responsiveness to metal challenges in both larvae and adults. A second novel MT isoform (CvMT-IV) was isolated from hemocytes by subtractive hybridization techniques following a 4-hour immune challenge with heat-killed bacteria (Vibrio, Bacillus, Micrococcus spp. mixture). Based on conservation of the cysteine motifs, this isoform appears to be a sub-family related to the molluscan alphabeta-domain MTs. A series of amino acid substitutions has resulted in four additional cysteines which give rise to a Cys-Cys motif and three Cys-Cys-Cys motifs. Northern blot analyses demonstrate that CvMT-IV is down-regulated upon sterile wounding and immune challenge, displays moderate expression in larvae and adults and differential gene induction in response to metals exposure.

Amino Acid Sequence↗

PubMed2006

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.

Amino Acid Sequence↗

PubMed2005

Marine genomics: a clearing-house for genomic and transcriptomic data of marine organisms.

BACKGROUND: The Marine Genomics project is a functional genomics initiative developed to provide a pipeline for the curation of Expressed Sequence Tags (ESTs) and gene expression microarray data for marine organisms. It provides a unique clearing-house for marine specific EST and microarray data and is currently available at http://www.marinegenomics.org. DESCRIPTION: The Marine Genomics pipeline automates the processing, maintenance, storage and analysis of EST and microarray data for an increasing number of marine species. It currently contains 19 species databases (over 46,000 EST sequences) that are maintained by registered users from local and remote locations in Europe and South America in addition to the USA. A collection of analysis tools are implemented. These include a pipeline upload tool for EST FASTA file, sequence trace file and microarray data, an annotative text search, automated sequence trimming, sequence quality control (QA/QC) editing, sequence BLAST capabilities and a tool for interactive submission to GenBank. Another feature of this resource is the integration with a scientific computing analysis environment implemented by MATLAB. CONCLUSION: The conglomeration of multiple marine organisms with integrated analysis tools enables users to focus on the comprehensive descriptions of transcriptomic responses to typical marine stresses. This cross species data comparison and integration enables users to contain their research within a marine-oriented data management and analysis environment.

Animals↗

PubMed2005

Double-stranded RNA induces sequence-specific antiviral silencing in addition to nonspecific immunity in a marine shrimp: convergence of RNA interference and innate immunity in the invertebrate antiviral response?

Double-stranded RNA (dsRNA) is a common by-product of viral infections and a potent inducer of innate antiviral immune responses in vertebrates. In the marine shrimp Litopenaeus vannamei, innate antiviral immunity is also induced by dsRNA in a sequence-independent manner. In this study, the hypothesis that dsRNA can evoke not only innate antiviral immunity but also a sequence-specific antiviral response in shrimp was tested. It was found that viral sequence-specific dsRNA affords potent antiviral immunity in vivo, implying the involvement of RNA interference (RNAi)-like mechanisms in the antiviral response of the shrimp. Consistent with the activation of RNAi by virus-specific dsRNA, endogenous shrimp genes could be silenced in a systemic fashion by the administration of cognate long dsRNA. While innate antiviral immunity, sequence-dependent antiviral protection, and gene silencing could all be induced by injection of long dsRNA molecules, injection of short interfering RNAs failed to induce similar responses, suggesting a size requirement for extracellular dsRNA to engage antiviral mechanisms and gene silencing. We propose a model of antiviral immunity in shrimp by which viral dsRNA engages not only innate immune pathways but also an RNAi-like mechanism to induce potent antiviral responses in vivo.

Animals↗

PubMed2004

Sampling and evaluation of white spot syndrome virus in commercially important Atlantic penaeid shrimp stocks.

In 1997, white spot syndrome virus (WSSV) was discovered in shrimp culture facilities in South Carolina, USA. This disease was known to cause devastating mortalities in cultured populations in Southeast Asia and prompted concern for the health of wild populations in the USA. Our study surveyed wild shrimp populations for the presence of WSSV by utilizing molecular diagnostics and bioassay techniques. A total of 1150 individuals (586 Litopenaeus setiferus, 477 Farfantepenaeus aztecus and 87 F. dourarum) were examined for the presence of WSSV DNA by PCR. A total of 32 individuals tested positive and were used in a bioassay to examine the transmission of disease to healthy individuals of the culture species L. vannamei. DNA sequencing of PCR products from a positive individual confirmed that the positive individuals carried WSSV DNA. Significant mortalities were seen in test shrimp injected with tissue extracts from heavily infected wild shrimp. These data confirm the existence of WSSV in wild shrimp stocks along the Atlantic Coast and that the virus can cause mortalities in cultured stocks.

Animals↗

PubMed2004

Molecular characterization and expression of vitellogenin receptor from white perch (Morone americana).

A full-length (4021 base pair [bp]) cDNA encoding a polypeptide (844 amino acids) with a predicted mass of 93 kDa and other characteristic structural features of a vertebrate vitellogenin receptor (VgR) was isolated from a white perch (Morone americana) ovarian cDNA library. Northern blotting performed using a specific digoxygenin-labeled VgR cDNA probe revealed a distinct approximately 4.1 kilobase (kb) hybridization signal in an mRNA preparation obtained from previtellogenic perch ovaries. The deduced amino acid sequence of the perch VgR was 89% and 82% identical, respectively, to that of the tilapia and rainbow trout. Because it possessed an eight-repeat ligand-binding domain (LR8) but lacked an O-linked sugar domain (-), the perch VgR was identified as a non-O-linked form of VgR (LR8-). Unlike the case in other vertebrates investigated, including tilapia and trout, no species of mRNA encoding an O-linked form of VgR (LR8+) could be detected when perch ovarian or liver mRNA reverse transcripts or cDNA libraries were screened by PCR using primer sets flanking the putative O-linked sugar domain. These novel findings call into question the assumptions that an LR8+ splice variant of the VgR always is dominantly present in somatic tissues and exists at lower levels in ovarian tissues to sequester lipoproteins distinct from Vg. A SYBR-green-based real-time reverse transcription-polymerase chain reaction assay was developed and used to quantitatively measure VgR expression in gonadal and somatic tissues, for the first time in any vertebrate. The main site of perch VgR mRNA expression was the ovary and the highest level of VgR mRNA expression was in ovaries whose largest follicles contained previtellogenic oocytes. Expression of VgR mRNA decreased with oocyte growth during vitellogenesis and was very limited in ovulated eggs. These quantitative results verify the concept that growing oocytes must extensively recycle LR8- forms of the VgR.

Alternative Splicing↗

PubMed2004

Diversity of metallothioneins in the American oyster, Crassostrea virginica, revealed by transcriptomic and proteomic approaches.

Metallothioneins are typically low relative molecular mass (6000-7000), sulfhydryl-rich metal-binding proteins with characteristic repeating cysteine motifs (Cys-X-Cys or Cys-X(n)-Cys) and a prolate ellipsoid shape containing single alpha- and beta-domains. While functionally diverse, they play important roles in the homeostasis, detoxification and stress response of metals. The originally reported metallothionein of the American oyster, Crassostrea virginica showed the canonical molluscan alphabeta-domain structure. Oyster metallothioneins have been characterized as cDNA and as expressed proteins, and here it is shown that the previously reported metallothionein is a prototypical member of a subfamily (designated as CvMT-I) of alphabeta-domain metallothioneins. A second extensive subfamily of oyster metallothioneins (designated as CvMT-II) has apparently arisen from (a) a stop mutation that truncates the protein after the alpha-domain, and (b) a subsequent series of duplication and recombination events that have led to the development of metallothionein isoforms containing one to four alpha-domains and that lack a beta-domain. Analysis of metallothioneins revealed that certain CvMT-I isoforms showed preferential association either with cadmium or with copper and zinc, even after exposure to cadmium. These data extend our knowledge of the evolutionary diversification of metallothioneins, and indicate differences in metal-binding preferences between isoforms within the same family.

Animals↗

PubMed2003

Population genetic analysis of white shrimp, Litopenaeus setiferus, using microsatellite genetic markers.

The white shrimp (Litopenaeus setiferus) is a commercially and recreationally valuable species, yet little is known of its population structure or genetic diversity. White shrimp are distributed along the Atlantic coast of the United States and from the west coast of Florida to the Bay of Campeche, Mexico. In this study, shrimp were collected from North Carolina, South Carolina (four separate collections were taken from 1995 to 1999), Georgia, the Atlantic and Gulf coasts of Florida, Louisiana, Texas and Mexico. DNA was isolated from these individuals, and genetic variation was assessed at six microsatellite loci. These loci were, for the most part, highly polymorphic with an average expected heterozygosity of 0.68. Deviations from Hardy-Weinberg proportions were observed over all samples, but experimental results suggested the presence of null alleles, which confounded a biological interpretation of this result. Pairwise tests of the similarity of allele frequency distributions and distance measure analyses showed broad-scale genetic homogeneity superimposed over occasional indications of random geographical and temporal differentiation. FST and RST estimates over all loci and samples were 0.002 or less and indicated little population structure. Weak but significant genetic differentiation was evident only between pooled western Atlantic and pooled Gulf of Mexico samples. Within the Gulf of Mexico or within the western Atlantic, the large-scale genetic homogeneity observed may be a consequence of genetic mixing resulting from pelagic larvae and adult migrations, while the random local genetic differentiation may be a result of genetic sampling or experimental sampling error. The weak differentiation between shrimp from the Gulf of Mexico and the western Atlantic can be explained by a relatively recent separation of these two populations and/or small amounts of ongoing gene flow.

Animals↗

PubMed2002

Diversity of the penaeidin antimicrobial peptides in two shrimp species.

Penaeidins, a unique family of antimicrobial peptides (AMPs) with both proline and cysteine-rich domains, were initially identified in the hemolymph of the Pacific white shrimp, Litopenaeus vannamei. Described here are the results of an investigation of penaeidin diversity in individual shrimp from two species, L. vannamei and L. setiferus (Atlantic white shrimp). We report the discovery of a novel penaeidin class, designated penaeidin 4 present in both L. vannamei and L. setiferus, and that all penaeidin classes were expressed in a single individual. In addition, nearly all penaeidins, regardless of class, shared an identical leader sequence while differing dramatically in the remainder of the peptide. Several new class 3 isoforms were identified, as well as sequence variants of Lv3a, which differ in the 3' untranslated region. Penaeidin sequence variability (especially of class 3), within and between individuals, is not interpretable as simple allelic polymorphism and may reflect alternate transcriptional mechanisms. Penaeidins are encoded by a small number of genetic loci and are not likely representatives of a large gene family produced by whole gene duplication, but rather may be products of a multi-component locus. Based on phylogenetic analysis, penaeidins fall into three classes where 1 and 2 are combined while classes 3 and 4 remain distinct. Phylogenetic analysis indicates that all classes of penaeidin were likely present in both species prior to speciation.

Amino Acid Sequence↗

PubMed2002

Potential indicators of stress response identified by expressed sequence tag analysis of hemocytes and embryos from the American oyster, Crassostrea virginica.

A pilot program was initiated to identify genes from the American oyster, Crassostrea virginica, that are potentially involved in the stress response for use as bioindicators of exposure to environmental pollutants and to toxic and infectious agents. A PCR-based method was used to construct cDNA libraries from pooled embryos and the hemocytes of a single individual. A total of 998 randomly selected clones (expressed sequence tags, ESTs) were sequenced. Approximately 40% of the ESTs are novel sequences. Several potential biomarkers identified include an antimicrobial peptide, recognition molecules (lectin receptors), proteinases and proteinase inhibitors, and a novel metallothionein. Diversity analysis shows that 363 and 286 unique genes were identified from the hemocyte and embryo libraries, respectively, indicating that full-scale EST collection is a valuable approach for the discovery of new genes of potential significance in the molluscan stress response.

Journal Article↗

PubMed2002

Crustins, homologues of an 11.5-kDa antibacterial peptide, from two species of penaeid shrimp, Litopenaeus vannamei and Litopenaeus setiferus.

The response of crustaceans to pathogens is believed to depend solely on innate, nonadaptive immune mechanisms, including phagocytosis, encapsulation, clotting, and a variety of soluble antimicrobial activities. Arthropod antimicrobial peptides, while characterized primarily from insects, also have been isolated from crustaceans. Expressed sequence tag analysis of hemocyte complementary DNA libraries from 2 species of shrimp, Litopenaeus vannamei and Litopenaeus setiferus, revealed transcripts with strong sequence similarity to an 11.5-kDa antibacterial peptide (crustin Cm1) found in Carcinus maenas. Crustins were also observed to contain motifs common to proteinase inhibitors. Analysis of these cDNA libraries yielded at least 3 different isoforms of this peptide in L. vannamei (crustin Lv1-Lv3) and 3 in L. setiferus (crustin Ls1-Ls3). Further analysis of a second L. vannamei cDNA library revealed the presence of 3 more possible isoforms (crustin Lv4-Lv6), which differed from those seen in the first L. vannamei cDNA library. Genomic Southern blot analysis revealed a complex family of crustin-related sequences. However, full-length crustin appears to be encoded by a much more restricted subset of sequences within this family.

Journal Article↗

PubMed2002

Spatial homogeneity and temporal heterogeneity of red drum (Sciaenops ocellatus) microsatellites: effective population sizes and management implications.

The red drum (Sciaenops ocellatus) is one of a number of species that occupy estuarine waters as juveniles and migrate to open ocean waters as adults. This species has experienced dramatic declines in population numbers over the past 2 decades, which has prompted increasing fishery restriction. In addition, hatchery augmentation has been initiated by several states to increase the abundance of juveniles in local areas. In South Carolina hatchery-reared fish have made significant (20%) contributions to the juvenile population on very local scales. As hatchery-reared fish are typically produced by a small number of individuals, the genetic consequences of augmentation programs are of concern. In this article we assess genetic variation at 5 microsatellite loci in S. ocellatus. The data indicate little geographic differentiation among samples collected along the Atlantic Coast of the United States, but substantial differences among year classes taken from South Carolina. The gene frequency differences among year classes were used to estimate the effective population size (Nc) of S. ocellatus in South Carolina and suggested that Ne was less than 300 from 1990 to 1993 and increased to about 1000 in 1994 and 1995. Whether this increase reflects the effectiveness of management regulations or simply a random fluctuation in S. ocellatus populations is not clear. The data suggest that a limited number of individuals produce the bulk of a given year class and support the sweepstakes hypothesis. Given the small Ne and estimates of the contribution of hatchery-reared fish to the wild stock, it is suggested that programs have the potential to increase, rather than decrease; Ne in the wild.

Journal Article↗