Transgene expressions in seabass (Lates calcarifer) following muscular injection of plasmid DNA: a strategy for vaccine development?

Muscular injection has become one of the direct methods for transferring foreign DNA into organisms. The technique has been recently introduced in the development of vaccines and gene therapy. Vaccine development, in particular, would be desirable in managing viral diseases in farmed fish. In this study, the technique was performed on seabass (Lates calcarifer) and was found that the foreign gene could be transferred successfully through injection into the muscles.

Identification of larval sea basses (Centropristis spp.) using ribosomal DNA-specific molecular assays

The identification of sea bass (Centropristis) larvae to species is difficult because of similar morphological characters, spawning times, and overlapping species ranges. Black sea bass (Centropristis striata) is an important fishery species and is currently considered to be overfished south of Cape Hatteras, North Carolina. We describe methods for identifying three species of sea bass larvae using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) assays based on species-specific amplification of rDNA internal transcribed spacer regions. The assays were tested against DNA of ten other co-occurring reef fish species to ensure the assay's specificity. Centropristis larvae were collected on three cruises during cross-shelf transects and were used to validate the assays. Seventy-six Centropristis larva were assayed and 69 (91%) were identified successfully. DNA was not amplified from 5% of the larvae and identification was inconclusive for 3% of the larvae. Those assays can be used to identify sea bass eggs and larvae and will help to assess spawning locations, spawning times, and larval dispersal.

Differenzierung roher und geräucherter Aale durch Protein- und DNA-Analyse

Raw or smoked eel was analysed by isoelectric focusing of sarcoplasmic proteins. For raw fish specific protein patterns were obtained for A. anguilla/A. rostrata, A. japonica and A. australis, but in case of smoked fish differentiation was only possible between Atlantic and Pacific species. Differentiation of raw or smoked eel was possible by PCR-SSCP, but patterns of ssDNA showed some intra-specific variability depending on the type of amplicon.

Toxische Wirkung von Schadstoffen auf Fischzellen. Bestimmung von DNA-Strangbrüchen mit dem Comet-Assay

The alkaline comet assay is a method of detecting DNA strand breaks and alkali labile sites in individual cells. The method was used to detect DNA strand breaks in isolated blood cells (leukocytes) of carp (Cyprius carpio). DNA damage have been induced by exposure of the cells to sediment extract. Therefore comet assay can be applied as in vitro bioassay for investigations on toxicity of marine sediments.

Fischartbestimmung von Caviar durch Protein- und DNA-Analyse

Deutscher Caviar, made from roe of lumpfish or capelin, gives species specific patterns in protein electrophoresis. The same techniques can be used to differentiate caviar from salmon and trout. The differentiation of sturgeon caviar (beluga, osietra, sevruga) is possible by isoelectric focusing, but not by SDS-PAGE. PCR-based methods of DNA-analysis for identification of the origin of sturgeon caviar are under development.

Fish species identification in Canned Tuna by DNA Analysis (PCR-SSCP)

DNA in canned tuna is degraded into short fragments of a rew hundred base pairs. The polymerase chain reaction (PCR) was used to amplify short sequences of mitochondrial DNA, which were denatured and analysed by polyacrylamide gel electrophoresis (native PAGE) for detection of single strand conformation polymorphisms. Species specific patterns of DNA bands were obtained for a number of tuna and bonito species. DE: In Thunfischkonserven liegt die DNA in Form kurzkettiger Fragmente von wenigen Hundert Basenpaaren Länge vor. Mit Hilfe der Polymerase-Kettenreaktion (PCR) wurden kurze Sequenzen der mitochondrialen DNA vervielfältigt. Anschließend wurde die gebildete DNA in Einzelsträngen überführt, die durch eine native Polyacrylamidgel-Elektrophorese (PAGE) aufgetrennt wurde. Für eine Reihe von Thunfischen und Boniten ergaben die Einzelstränge artspezifische Bandenmuster, die auf unterschiedliche Konformationen der DNA-Stränge der einzelnen Fischarten zurückzuführen sind.

Differenzierung von Pangasius (Pangasius hypophthalmus) und Asiatischem Rotflossenwels (Hemibagrus wyckioides) durch Protein- und DNA-Analyse

In the last years farmed Pangasius (Tra-Pangasius, Pangasius hypophthalmus) from Vietnam has reached a considerable market share, whereas aquaculture of Asian Redtail Catfish (Hemibagrus wyckioides) is in its infancy. Recently it has been detected by food control authorities in Hamburg, that Pangasius fillets have been mislabelled and sold as fillets produced from Asian Redtail catfish. The necessity to improve the analytical methods for differentiation of Pangasius and Redtail Catfish prompted us to evaluate the suitability of isoelectric focusing (IEF) and DNA-analysis for identification of the two species. IEF of water soluble proteins was found to be a fast, reliable and economical method for differentiation of raw fillets of Pangasius and Redtail Catfish, as long as reference material is available. PCR-based DNA analysis was performed as follows: (i) amplification of a 464 bp segment of the cytochrome b gene; (ii) sequencing of the PCR product; (iii) comparison of the sequence with entries in GenBank using BLAST. The sequences of both species differed considerably, allowing the unequivocal differentiation between P. hypophthalmus and H. wyckioides. Kurzfassung Pangasius (Schlankwels, Tra-Pangasius, Pangasius hypophthalmus) hat sich innerhalb weniger Jahre zu einem bedeutenden Zuchtfisch entwickelt, während die Aquakultur des Asiatischen Rotflossenwelses (Hemibagrus wyckioides) in Vietnam noch in einem relativ kleinen Maßstab stattfindet. Kürzlich wurde von der Lebensmittelüberwachung in Hamburg nachgewiesen, dass im Handel erhältliche Filets mit der Deklaration „Rotflossenwels“ aus Pangasius hergestellt worden waren. Vor diesem Hintergrund wurden zwei Methoden auf ihre Eignung zur Differenzierung von Pangasius und Rotflossenwels geprüft. Es zeigte sich, dass sowohl die isoelektrische Fokussierung (IEF) wasserlöslicher Proteine als auch die PCR-basierte DNA-Analyse zur Unterscheidung beider Arten gut geeignet ist. Die IEF stellt eine schnelle und kostengünstige Untersuchungsmethode dar, die allerdings Referenzmaterial benötigt. Mit Hilfe der PCR (Polymerase-Kettenreaktion) wurde ein Abschnitt des Cytochrom b-Gens vervielfältigt und sequenziert. Die Sequenzen von P. hypophthalmus und H. wyckioides wiesen beträchtliche Unterschiede auf. Es wird diskutiert, wie sich durch Vergleich dieser Sequenzen mit Einträgen in Gendatenbanken unbekannte Proben beider Arten sicher zuordnen lassen.

Genetic engineering of microorganisms: free release into the environment

Genetic engineering now makes possible the insertion of DNA from many organisms into other prokaryotic, eukaryotic and viral hosts. This technology has been used to construct a variety of such genetically engineered microorganisms (GEMs). The possibility of accidental or deliberate release of GEMs into the natural environment has recently raised much public concern. The prospect of deliberate release of these microorganisms has prompted an increased need to understand the processes of survival, expression, transfer and rearrangement of recombinant DNA molecules in microbial communities. The methodology which is being developed to investigate these processes will greatly enhance our ability to study microbial population ecology.