Transient expression assays with the proximal promoter of a newly characterized actin gene from the oyster Crassostrea gigas.

We undertook the characterization of an actin gene and its proximal promoter in the oyster Crassostrea gigas. A complete actin cDNA was identified, sequenced and its amino acid sequence deduced. Comparative analysis showed a high homology with actin of other species and that this gene is closer to the cytoplasmic form of actins than to the muscle type. A probe derived from the 5'-untranslated region of the cDNA was then used to isolate the actin gene from a genomic library. The gene was sequenced and shown to contain a single 643 bp intron. A 1670 bp fragment upstream from the open reading frame was isolated and sequenced. This upstream region displays typical features of actins such as a serum response element (CarG box). This fragment was cloned into the promoterless vector pGL3-basic and the resulting construct was transfected into cells of dissociated oyster heart primary cultures. Its capacity to express the luciferase in this in vitro homologous system was monitored and showed high expression levels. This is the first complete actin sequence reported so far for the oyster C. gigas and its promoter is the first available among bivalves.

Promoters from Drosophila heat shock protein and cytomegalovirus drive transient expression of luciferase introduced by particle bombardment into embryos of the oyster Crassostrea gigas.

Using high velocity particle bombardment, we transferred a reporter gene into early stages of the oyster Crassostrea gigas and showed the expression of the introduced genes in these embryos at later stages of development. We tested two promoters: (1) the heat shock protein 70 promoter of Drosophila; (2) the cytomegalovirus early promoter, both linked to the luciferase reporter gene. The hsp 70-luc (pDrluc) construct allowed an expression level up to 55-fold higher than the control in a heat inducible fashion. The CMV-luc (pCMVL) construct constitutively gave a 4-fold higher expression than the control. This confirms the suitability of particle bombardment for transfecting genes into eggs, zygotes and trochopores of bivalves and demonstrates the functionality of two heterologous expression vectors in C. gigas.

Infection of cultured embryo cells of the pacific oyster, Crassostrea gigas, by pantropic retroviral vectors.

The inability to stably introduce and express foreign genes has hampered basic research in molluscan species. We cultured cells from dissociated embryos of the Pacific oyster, Crassostrea gigas, and infected these primary cultures with pantropic retroviral vectors containing the envelope glycoprotein of vesicular stomatitis virus. Luciferase transgene expression mediated by different heterologous promoters was demonstrated for at least 9 d after infection of the cells. Surprisingly, the promoter reproducibly mediating the highest level of luciferase expression was the retroviral promoter (U3 region of long terminal repeat) from the Moloney murine leukemia virus. The infection efficiency using a low multiplicity of infection (0.05) was estimated by quantitative polymerase chain reaction to be between 0.1-0.5%. This system will facilitate studies of gene expression and regulation and should be widely applicable to other molluscan species.

Nucleotide and deduced amino acid sequences of Biomphalaria glabrata actin cDNA.

The complete nucleotide sequence of Biomphalaria glabrata actin has been cloned by PCR amplification and screening of a cDNA library of Biomphalaria glabrata. The comparison of the deduced amino acid sequence with other actins suggests that a cytoskeletal form of the protein has been cloned.

Comparative proteomics reveals proteins impacted by nitrogen deprivation in wild-type and high lipid-accumulating mutant strains of Tisochrysis lutea.

Understanding microalgal lipid accumulation under nitrogen starvation is of major interest for biomass feedstock, food and biofuel production. Using a domesticated oleaginous algae Tisochrysis lutea, we performed the first comparative proteomic analysis on the wild type strain and a selected lipid over-accumulating mutant. 2-DE analysis was made on these strains cultured in two metabolic conditions, with and without nitrogen deprivation, which revealed significant differences in proteomes according to both strain and nitrogen availability. Mass spectrometry allowed us to identify 37 proteins that were differentially expressed between the two strains, and 17 proteins regulated by nitrogen starvation concomitantly with lipid accumulation. The proteins identified are known to be involved in various metabolic pathways including lipid, carbohydrate, amino acid, energy and pigment metabolisms, photosynthesis, protein translation, stress response and cell division. Four candidates were selected for possible implication in the over-accumulation of lipids during nitrogen starvation. These include the plastid beta-ketoacyl-ACP reductase protein, the coccolith scale associated protein and two glycoside hydrolases involved in biosynthesis of fatty acids, carbon homeostasis and carbohydrate catabolism, respectively. This proteomic study confirms the impact of nitrogen starvation on overall metabolism and provides new perspectives to study the lipid over-accumulation in the prymnesiophyte haptophyte T. lutea. BIOLOGICAL SIGNIFICANCE: This paper study consists of the first proteomic analysis on Tisochrysis lutea, a non-model marine microalga of interest for aquaculture and lipids production. Comparative proteomics revealed proteins putatively involved in the up-accumulation of neutral lipids in a mutant strain during nitrogen starvation. The results are of great importance for future works to improve lipid accumulation in microalgae of biotechnological interest for biofuel production. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Transient expression of luciferase reporter gene after lipofection in oyster (Crassostrea gigas) primary cell cultures.

Transient expression of the luciferase gene, under transcriptional control of several heterologous promoters, was obtained in heart primary cell cultures of the Pacific oyster, Crassostrea gigas. Drosophila heat shock protein 70 promoter (hsp70), cytomegalovirus, and simian virus early promoters, controlling the luciferase gene, were transfected into the cell cultures using liposomes. Two culture media were used to establish primary cell cultures and tested as transfection media. Parameters such as the quantity of DNA and the ratio of DNA to liposome were analyzed to define the best transfection conditions. In oysters, the Drosophila inducible hsp70 promoter behaved in a way similar to that observed in other animal species. Moreover, for this study, hsp70 was more efficient than the cytomegalovirus and simian virus promoters.

Microinjection of bivalve eggs: application in genetics.

New strategies for embryonic manipulation have been developed in recent years through plant and animal research. However, research on marine invertebrate embryos has suffered from a lack of basic tools, such as microinjection. Here we present a technique developed for microinjecting eggs and embryos of the oyster Crassostrea gigas and the mussel Mytilus edulis. In experimental trials, approximately 40% of microinjected embryos survived. This technique was used to microinject beta-galactosidase, for which specific detection techniques were developed. A reporter construct (CMV-beta) based on a promoter of cytomegalovirus linked to the beta-galactosidase-encoding gene was then microinjected, and the expression level of this construct was monitored. The suitability of this technique is discussed in terms of its application to the manipulation of bivalve mollusks in pathology and genetics.

Future of biotechnology-based control of disease in marine invertebrates.

Infectious disease is the single most devastating problem in mollusc and shrimp aquaculture. Pathogens causing the greatest problems have been identified as viruses, prokaryotes, and protozoans. Two approaches employing methods of biotechnology have been proposed to prevent, manage, and control mollusc and shrimp diseases. The first is development of a diagnostic scheme for detection and identification of pathogens, using molecular probes. This offers the opportunity for prophylactic measures to be taken. Molecular probes have been prepared for the major pathogens of molluscs, but in the case of shrimp pathogens, only a few are available. Monoclonal antibodies have also been prepared and are used in immunodiagnosis, e.g., immunofluorescence detection. Such diagnostic tools are relatively new to aquaculture, but have enormous potential. A second approach to the control of disease in marine invertebrates, notably shrimp, involves use of genetically transformed strains resistant to specific pathogens. Pathogen-resistant transgenic animals have been developed, but such research has only just begun for molluscs and shrimp. Transfection methods applied to mollusc and shrimp embryos have been successful, with preliminary data showing efficiency of heterologous promoters in controlling expression of reporter genes. Other transformation systems also show promise, including transposable elements and densoviruses.