Cloning and characterization of cytochrome c oxidase subunit I (COXI) in Gobiocypris rarus

In study of gene expression profile in cloned embryos which derived from D. rerio embryonic nuclei and G. rarus enucleated eggs, cytochrome c oxidase subunit I (COXI) of G. rarus, exhibiting difference at expression level between cloned embryos and zebrafish embryo, was cloned. Its full cDNA length is 1654 bp and contains a 1551 bp open reading frame, encoding a 5.64 kDa protein of 516 amino acids. The alignment result shows that mitochondrion tRNA(ser) is co-transcripted with COXI, which just was the 3'-UTR of COXI. Molecular phylogenic analysis based on COXI indicates G. rarus should belong to Gobioninae, which was not in agreement with previous study according to morphological taxonomy. Comparison of DNA with cDNA shows that RNA editing phenomenon does not occur in the COXI of G. rarus.

Lysis of Aphanizomenon flos-aquae (Cyanobacterium) by a bacterium Bacillus cereus

A phytoplankton-lytic (PL) bacterium, Bacillus cereus, capable of lysing the bloom-forming cyanobacterium. Aphanizomenon flos-aquae was isolated from Lake Dianchi of Yunnan province, China. This bacterium showed lytic activities against a wide range of cyanobacteria/algae, including A. flos-aquae, Microcystis viridis, Microcystis wesenbergi, Microcystis aeruginosa, Chlorella ellipsoidea, Oscillatoria tenuis, Nostoc punctiforme, Anabaena flos-aquae, Spirulina maxima, and Selenastrum capricornutum. Chlorophyll a contents, phycocyanin contents, and photosynthetic activities of the A. flos-aquae decreased evidently in an infected culture for a period. Bacterium B. cereus attacked rapidly A. flos-aquae cells by cell-to-cell contact mechanism. It was shown that the lysis of A. flos-aquae began with the breach of the cyanobacterial cell wall, and the cyanobacterial cell appeared abnormal in the presence of the PL bacterium. Moreover, transmission electron microscope examinations revealed that a close contact between the bacterium and the cyanobacterium was necessary for lysis. Some slime extrusions produced from B. cereus assisted the bacterial cells to be in close association with and lyse the cyanobacterial cells. These findings suggested that this bacterium could play an important role in controlling the Aphanizomenon blooms in freshwaters. (c) 2006 Elsevier Inc. All rights reserved.

Isolation and identification of a novel WSSV nucleocapsid protein by cDNA phage display using an scFv antibody

In a previous study, a scFv phage display library against white spot syndrome virus (WSSV) was constructed and yielded a clone designated A I with conformational specificity against native but not denatured viral antigen. Although the clone A1 has been used successfully as a diagnostic antibody, its precise target antigen has not been elucidated. A different strategy was adopted involving the construction of a second T7 phage display library utilizing mRNA isolated from shrimp infected with WSSV. Following RT-PCR and T7 phage library construction, phages displaying the candidate epitope were selected with A I scFv. Since successive enrichment steps were not associated with an increased titer of the phages, enrichment after successive tests was confirmed by PCR resulting in the prefer-red selection of a specific DNA sequence encoding a novel nucleocapsid protein WSSV388. Immune electron microscopy revealed that WSSV388 is located on the nucleocapsid. This result demonstrated that unknown antigen could be identified by phage display using the epitope conformation dependent scFv. (c) 2006 Elsevier B.V. All rights reserved.

Nitrate and phosphate supplementation to increase toxin production by the marine dinoflagellate Alexandrium tamarense

Alexandrium tamarense toxins have great value in biotechnology research as well as important in connection with shellfish poisoning. The influence of nitrate or nitrate and phosphate supplementation on cell biomass and toxin content were investigated in batch cultures. When cultures at low nitrate (88.2 mu M NaNO3) Were supplemented with 793.8 mu M NaNO3 at day 10 the cell density and cellular toxin contents were increased by 6-29% and 20-76%, respectively, compared with controls, and maximal values were 43,600 cells/ml (day 38) and 0.91 pg/cell (day 31). Supplementation with nitrate at day 14 or with nitrate and phosphate at day 10/14 to the cultures did not increase the cell density compared with the non-supplemented middle nitrate or high phosphate (108 mu M NaH2PO4) cultures, respectively, but increased the cellular toxin contents by an average of 52%. The results showed that supplementation with nitrate or with nitrate and phosphate at different growth phases of the cultures increased toxin yield by an average of 46%. Supplementation with nitrate at selected times to maintain continuous low level of nitrate might contribute to the effective increase of toxin yield of A. tamarense. (c) 2005 Elsevier Ltd. All rights reserved.

Improvement in growth and toxin production of Alexandrium tamarense by two-step culture method

The growth and toxin content of the dinoflagellate Alexandrium tamarense ATHK was markedly affected by culture methods. In early growth phase at lower cell density static or mild agitation methods were beneficial to growth, but continuous agitation or aeration, to some extent, had an adverse effect on cell growth. Static culture in 2 L Erlenmeyer flasks had the highest growth rate (0.38 d(-1)) but smaller cell size compared with other culture conditions. Cells grown under aerated conditions possessed low nitrogen and phosphorus cell yields, namely high N and P cell-quota. At day 18, cells grown in continuous agitated and 1 h aerated culture entered the late stationary phase and their cellular toxin contents were higher (0.67 and 0.54 pg cell(-1)) compared with cells grown by other culture methods (0.27-0.49 pg cell(-1)). The highest cell density and cellular toxin content were 17190 cells mL(-1) and 1.26 pg cell(-1) respectively in an airlift photobioreactor with two-step culture. The results indicate that A. tamarense could be grown successfully in airlift photobioreactor by a two-step culture method, which involved cultivating the cells statically for 4 days and then aerating the medium. This provides an efficient way to enhance cell and toxin yield of A. tamarense.

Seasonal pattern of reproduction of Hizikia fusiformis (Sargassaceae, Phaeophyta) from Nanao Island, Shantou, China

The maturation pattern of sexual reproduction in Hizikia fusiformis (Harvey) Okamura (Sargassaceae, Phaeaophyta) was examined in 2003 at Yunao Bay, Nanao Island, Shantou, China. Maturation began in mid-April (seawater temperature 19-21 degrees C), reached the peak in mid-May (maturation rate ca. 70%, and seawater temperature 23.5-25 degrees C) and finished in late-June (seawater temperature 27.5-30 degrees C). The Hizikia plants continued to gain the length from the beginning of maturation season to reach a maximum mean length of 34.8 cm in mid-May, after which the mean length was reduced drastically due to the senescence and rupture of the larger plants in size. The major portion of the mature plants belonged to the larger plants between April and May, but to the smaller ones in June. It is suggested that the plant must achieve a critical size before reproductive maturation occurred. There was a positive relationship between the number of receptacles (NR), as well as the reproductive allocation (RA), and the plant size of Hizikia population, with the recorded maximum values of NR and RA being 1220 and 64.3% respectively, for a single plant.

Upstream regulatory region of zebrafish lunatic fringe: Isolation and promoter analysis

Lunatic fringe (Lfng), one modulator of Notch signaling, plays an essential part in demarcation of tissues boundaries during animal early development, especially somitogenesis. To characterize the promoter of zebrafish 1fng and generate somite-specific transgenic zebrafish, we isolated the upstream regulatory region of zebrafish 1fng by blast search at the Ensembl genome database (http://www. ensembl.org) and analyzed the promoter activity using green fluorescent protein (GFP) as a reporter. Promoter activity assay in zebrafish shows that the 0.2-kb fragment containing GC-box, CAAT-box, and TATA-box can direct tissue-specific GFP expression, while the 0.4-kb and 1.2-kb fragments with further upstream sequence included drive GFP expression more efficiently. We produced 1fngEGFP-transgenic founders showing somite-specific expression of GFP and consequently generated a hemizygous 1fngEGFP-transgenic line. The eggs from 1fngEGFP-transgenic female zebrafish show strong GFP expression, which is consistent to the reverse-transcription polymerase chain reaction PCR (RT-PCR) detection of 1fng transcripts in the fertilized eggs. This reveals that zebrafish 1fng is a maternal factor existing in matured eggs, suggesting that fish somitogcnesis may be influenced by maternal factors.

Response of growth and fatty acid compositions of Nannochloropsis sp to environmental factors under elevated CO2 concentration

Nannochloropsis sp. was grown with different levels of nitrate, phosphate, salinity and temperature with CO2 at 2,800 mu l l(-1). Increased levels of NaNO3 and KH2PO4 raised protein and polyunsaturated fatty acids (PUFAs) contents but decreased carbohydrate, total lipid and total fatty acids (TFA) contents. Nannochloropsis sp. grew well at salinities from 22 to 49 g l(-1), and lowering salinity enhanced TFA and PUFAs contents. TFA contents increased with the increasing temperature but PUFAs contents decreased. The highest eicosapentaenoic acid (EPA, 20:5 omega 3) content based on the dry mass was above 3% under low N (150 mu M NaNO3) or high N (3000 mu M NaNO3) condition. Excessive nitrate, low salinity and temperature are thus favorable factors for improving EPA yields in Nannochloropsis sp.

Integration of double-fluorescence expression vectors into zebrafish genome for the selection of site-directed knockout/knockin

Production of zebrafish by modifying endogenous growth hormone (GH) gene through homologous recombination is described here. We first constructed the targeting vectors pGHT1.7k and pGHT2.8k, which were used for the knockout/knockin of the endogenous GH gene of zebrafish, and injected these two vectors into the embryos of zebrafish. Overall, the rate of targeted integration with the characteristic of germ line transmission in zebrafish was 1.7x10(-6). In one experimental patch, the integrating efficiency of pGHT2.8k was higher than that of pGHT1.7k, but the lethal effect of pGHT2.8k was stronger than that of pGHT1.7k. The clones with the correct integration of target genes were identified by a simple screening procedure based on green fluorescent protein (GFP) and RFP dual selection, which corresponded to homologous recombination and random insertion, respectively. The potential homologous recombination zebrafish was further bred to produce a heterozygous F-1 generation, selected based on the presence of GFP. The potential targeted integration of exogenous GH genes into a zebrafish genome at the P-0 generation was further verified by polymerase chain reaction and Southern blot analysis. Approximately 2.5% of potential founder knockout and knockin zebrafish had the characteristic of germ line transmission. In this study, we developed an efficient method for producing the targeted gene modification in zebrafish for future studies on genetic modifications and gene functions using this model organism.

Electron microscopic examination of the viromatrix of Rana grylio virus in a fish cell line

Rana grylio virus (RGV), a Ranavirus belonging to the family Iridoviridae, assembles in the viromatrix which is a factory for viral genome replication and particle assembly. Ultrastructural studies of the viromatrix will clarify the pathway of assembly. The viromatrix and quantitative changes in RGV infected epithelipma papulosum cyprini (EPC) cells, one of fish cell lines, were studied by electron microscopy. It was shown that viromatrices were adjacent to the nucleus, and the electron density was lower than that of the surrounding cytoplasm. The viromatrix contained virus particles with different forms, electron-dense materials and amorphous structures which included tubules and membranous materials. Tubules were often observed in direct continuity with empty capsids. Several bundles of intermediate filaments were seen alongside the viromatrix and crystalline aggregates. Large clusters of mitochondria occurred in proximity to viromatrix. A total of 990 cells profiles were examined. The results showed that 394 cells contained viromatrix: 89.3% contained one, and 10.7% contained two to four viromatrices. The number of viromatrices increased gradually and reached a peak at 16 h p.i. The viromatrix area at 24 h p.i. increased up to 7.4 +/- 0.69 mu m(2) which was three-times lower than that at 6 h p.i. The number of empty capsids within viromatrix was generally more than that of "full" particles at different time points, and there was a strong positive correlation between them. (c) 2005 Elsevier B.V. All rights reserved.

A perspective on fish gonad manipulation for biotechnical applications

The gonad is an essential organ for generating sperm and ova in vertebrates. This review describes several pilot studies on gonad gene manipulation and development in fish. With antisense RNA techniques, we suppressed the gonad development, and thus the fertility, of an antisense gonadotropin-releasing hormone (sGnRH) transgenic common carp. Then, using a tissue-specific exogenous gene excision strategy with sexual compensation, we knocked out the gonad-specific transgene. Under the control of the rainbow trout protamine promoter, the transgenic fish expressed the reporter gene eGFP specifically in the spermary. These results indicate that the fish gonad is a new model organ that can improve contemporary biotechnology experiments. Herein we discuss the potential of fish gonad manipulation for resolving important biosafety problems regarding transgenic fish generation and producing the new transgenic animal bioreactor.

Population genetic structure of an endemic cyprinid fish, Ancherythroculter nigrocauda, in the upper reaches of the Yangtze River and its implication for conservation

Ancherythroculter nigrocauda is a cyprinid fish endemic to the upper reaches of the Yangtze River, which has been reported to have 2 or 3 chambers to its air bladder. Morphological studies showed no differences between individuals with different types of air bladder, but did demonstrate geographical differences from different sources. After the completion of the Three Gorges Dam, it was expected that the population of this species would decrease, but artificial breeding and stocking is under consideration to protect this species from extinction. In the present study, mtDNA cytochrome b gene sequences were determined and analyzed for A. nigrocauda samples of different morphotypes and sources to identify their genetic differentiations, and thereby guide plans for the artificial propagation and conservation of this species. Haplotype diversity index values (h) and nucleotide diversity values (pi) for all the populations were found to be high indicating their high level genetic diversity. An analysis of molecular variance identified no differentiation among the studied populations. Therefore, we suggested that the individuals of different morphological types and geographical sources belong to the same species. To maintain its high level genetic diversity, it mill he necessary to use large and diverse sources of parental fish for artificial reproduction.

Growth promotion of vegetative gametophytes of Undaria pinnatifida by blue light

Through an acclimation period of 10 days, compared to white light, the maximal net photosynthetic rates were significantly higher for gametophytes of Undaria pinnatifida cultivated under blue light (400-500 nm), and were lower under red light (600-700 nm). Chlorophyll c and the carotenoid content of gametophytes were similar under blue light and red light but were much lower under white light. The growth rate of female gametophytes under blue light was higher than that under other lights, and the growth rate of male gametophytes showed little variation with respect to blue and white light. Male and female gametophytes were mixed together to form sporophytes under white, blue and red light. After approximately 5 days, 50% gametophytes became fertile under blue and white light, but remained vegetative under red light after 10 days.

Cloning and analysis of 16 Rab genes from macronuclear DNA of Euplotes octocarinatus

Rab proteins belong to the largest family of the Ras superfamily of small GTPase that play an important role in intracellular vesicular traffic. So far, almost 60 members of Rab family have been identified in mammalian cells. To further study the diversity and function of Rab protein in evolution, unicellular protozoa ciliates, Euplotes octocarinatus, were used in this study, Rab genes were screened by PCR method from macronuclear DNA of E. octocarinatus. Sixteen Rab genes were obtained. They share 87.6 - 99.5% identities. Highly conserved GTP-binding domains were found. There are some hot regions that diverse sharply in these genes as well.

Effects of solar UV radiation on morphology and photosynthesis of filamentous cyanobacterium Arthrospira platensis

To study the impact of solar UV radiation (UVR) (280 to 400 nm) on the filamentous cyanobacterium Arthrospira (Spirulina) platensis, we examined the morphological changes and photosynthetic performance using an indoor-grown strain (which had not been exposed to sunlight for decades) and an outdoor-grown strain (which had been grown under sunlight for decades) while they were cultured with three solar radiation treatments: PAB (photosynthetically active radiation [PAR] plus UVR; 280 to 700 nm), PA (PAR plus UV-A; 320 to 700 nm), and P (PAR only; 400 to 700 nm). Solar UVR broke the spiral filaments of A. platensis exposed to full solar radiation in short-term low-cell-density cultures. This breakage was observed after 2 h for the indoor strain but after 4 to 6 h for the outdoor strain. Filament breakage also occurred in the cultures exposed to PAR alone; however, the extent of breakage was less than that observed for filaments exposed to full solar radiation. The spiral filaments broke and compressed when high-cell-density cultures were exposed to full solar radiation during long-term experiments. When UV-B was screened off, the filaments initially broke, but they elongated and became loosely arranged later (i.e., there were fewer spirals per unit of filament length). When UVR was filtered out, the spiral structure hardly broke or became looser. Photosynthetic 0, evolution in the presence of UVR was significantly suppressed in the indoor strain compared to the outdoor strain. UVR-induced inhibition increased with exposure time, and it was significantly lower in the outdoor strain. The concentration of UV-absorbing compounds was low in both strains, and there was no significant change in the amount regardless of the radiation treatment, suggesting that these compounds were not effectively used as protection against solar UVR. Self-shading, on the other hand, produced by compression of the spirals over adaptive time scales, seems to play an important role in protecting this species against deleterious UVR. Our findings suggest that the increase in UV-B irradiance due to ozone depletion not only might affect photosynthesis but also might alter the morphological development of filamentous cyanobacteria during acclimation or over adaptive time scales.