Gene expression of cold shock and other stress-related genes in Vibrio vulnificus grown in pure culture under shellstock temperature control conditions.

Shellstock refrigeration after harvesting is recommended to prevent further increases in Vibrio vulnificus numbers in oysters, but it could potentially induce a cold shock response in this bacterium. V. vulnificus was incubated at 35, 25, 20, and 15 degrees C and then subjected to 7.2 and 4 degrees C for 1 week. A cold-adaptation response that enhanced cell culturability was observed when cells were incubated at 15 degrees C prior to cold shock at 7.2 degrees C. In vitro cold shock gene expression was analyzed by reverse transcriptase PCR (RT-PCR). The expression of cold shock genes csp1 and csp5 (homologous genes to cspA and cspV) remained constant, despite cold shock. However, the transcript of csp3 was constitutively expressed before and after cold shock, with a few exceptions. The synthesis of csp3 mRNA in V. vulnificus C7184Tr (an avirulent strain) was induced only after 15 degrees C incubation and cold shock at 4 degrees C. The expression of csp4 was repressed after cold shock. Our data showed that the csp(s) tested in this study are not cold inducible. The transcripts of two oxidative stress-related genes, oxyR and katG, showed different induction patterns among strains after cold shock, suggesting that V. vulnificus cells encountered oxidative stress during cold shock.

Towards the ictalurid catfish transcriptome: generation and analysis of 31,215 catfish ESTs.

BACKGROUND: EST sequencing is one of the most efficient means for gene discovery and molecular marker development, and can be additionally utilized in both comparative genome analysis and evaluation of gene duplications. While much progress has been made in catfish genomics, large-scale EST resources have been lacking. The objectives of this project were to construct primary cDNA libraries, to conduct initial EST sequencing to generate catfish EST resources, and to obtain baseline information about highly expressed genes in various catfish organs to provide a guide for the production of normalized and subtracted cDNA libraries for large-scale transcriptome analysis in catfish. RESULTS: A total of 17 cDNA libraries were constructed including 12 from channel catfish (Ictalurus punctatus) and 5 from blue catfish (I. furcatus). A total of 31,215 ESTs, with average length of 778 bp, were generated including 20,451 from the channel catfish and 10,764 from blue catfish. Cluster analysis indicated that 73% of channel catfish and 67% of blue catfish ESTs were unique within the project. Over 53% and 50% of the channel catfish and blue catfish ESTs, respectively, had significant similarities to known genes. All ESTs have been deposited in GenBank. Evaluation of the catfish EST resources demonstrated their potential for molecular marker development, comparative genome analysis, and evaluation of ancient and recent gene duplications. Subtraction of abundantly expressed genes in a variety of catfish tissues, identified here, will allow the production of low-redundancy libraries for in-depth sequencing. CONCLUSION: The sequencing of 31,215 ESTs from channel catfish and blue catfish has significantly increased the EST resources in catfish. The EST resources should provide the potential for microarray development, polymorphic marker identification, mapping, and comparative genome analysis.

Catfish CC chemokines: genomic clustering, duplications, and expression after bacterial infection with Edwardsiella ictaluri.

Chemokines are a family of structurally related chemotactic cytokines that regulate the migration of leukocytes, under both physiological and inflammatory conditions. CC chemokines represent the largest subfamily of chemokines with 28 genes in mammals. Sequence conservation of chemokines between teleost fish and higher vertebrates is low and duplication and divergence may have occurred at a significantly faster rate than in other genes. One feature of CC chemokine genes known to be conserved is genomic clustering. CC chemokines are highly clustered within the genomes of human, mouse, and chicken. To exploit knowledge from comparative genome analysis between catfish and higher vertebrates, here we mapped to bacterial artificial chromosome (BAC) clones 26 previously identified catfish (Ictalurus sp.) chemokine cDNAs. Through a combination of hybridization and fluorescent fingerprinting, 18 fingerprinted contigs were assembled from BACs containing catfish CC chemokine genes. The catfish CC chemokine genes were found to be not only highly clustered in the catfish genome, but also extensively duplicated at various levels. Comparisons of the syntenic relationships of CC chemokines may help to explain the modes of duplication and divergence that resulted in the present repertoire of vertebrate CC chemokines. Here we have also analyzed the expression of the transcripts of the 26 catfish CC chemokines in head kidney and spleen in response to bacterial infection of Edwardsiella ictaluri, an economically devastating catfish pathogen. Such information should pinpoint research efforts on the CC chemokines most likely involved in inflammatory responses.