Cloning and expression analysis in mature individuals of two chicken type-II GnRH (cGnRH-II) genes in common carp (Cyprinus carpio)

Gonadotropin-releasing hormone (GnRH) is a conservative neurodecapeptide family, which plays a crucial role in regulating the gonad development and in controlling the final sexual maturation in vertebrate. Two differing cGnRH-II cDNAs of common carp, namely cGnRH-II cDNA1 and cDNA2, were firstly cloned from the brain by rapid amplification of cDNA end (RACE) and reverse transcription- polymerase chain reaction (RT-PCR). The length of cGnRH-II cDNA1 and cDNA2 was 622 and 578 base pairs (bp), respectively. The cGnRH-II precursors encoded by two cDNAs consisted of 86 amino acids, including a signal peptide, cGnRH-II decapeptide and a GnRH-associated peptide (GAP) linked by a Gly-Lys-Arg proteolytic site. The results of intron trapping and Southern blot showed that two differing cGnRH-II genes in common carp genome were further identified, and that two genes might exist as a single copy. The multi-gene coding of common carp cGnRH-II gene offered novel evidence for gene duplication hypothesis. Using semi-quantitative RT-PCR, expression and relative expression levels of cGnRH-II genes were detected in five dissected brain regions, pituitary and gonad of common carp. With the exception of no mRNA2 in ovary, two cGnRH-II genes could be expressed in all the detected tissues. However, expression levels showed an apparent difference in different brain regions, pituitary and gonad. According to the expression characterization of cGnRH-II genes in brain areas, it was presumed that cGnRH-II might mainly work as the neurotransmitter and neuromodulator and also operate in the regulation for the GnRH releasing. Then, the expression of cGnRH-II genes in pituitary and gonad suggested that cGnRH-II might act as the autocrine or paracrine regulator.

Cloning of biologically active genomes from a Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus isolate by using a bacterial artificial chromosome

Purification of genotypes from baculovirus isolates provides understanding of the diversity of baculoviruses and may lead to the development of better pesticides. Here, we report the cloning of different genotypes from an isolate of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) by using a bacterial artificial chromosome (BAC). A transfer vector (pHZB10) was constructed which contained an Escherichia coli mini-F replicon cassette within the upstream and downstream arms of HaSNPV polyhedrin gene. Hz2e5 cells were co-transfected with wild-type HaSNPV DNA and pHZB10 to generate recombinant viruses by homologous recombination. The DNA of budded viruses (BVs) was used to transform E. coli. One of the bacmid colonies, HaBacHZ8, has restriction enzyme digestion profiles similar to an in vivo cloned strain HaSNPV-G4, the genome of which has been completely sequenced. For testing the oral infectivity, the polyhedrin gene of HaSNPV was reintroduced into HaBacHZ8 to generate the recombinant bacmid HaBacDF6. The results of one-step growth curves, electron microscopic examination, protein expression analysis and bioassays indicated that HaBacDF6 replicated as well as HaSNPV-G4 in vitro and in vivo. The biologically functional HaSNPV bacmids obtained in this research will facilitate future studies on the function genomics and genetic modification of HaSNPV. (C) 2003 Elsevier B.V. All rights reserved.

Two unisexual artificial polyploid clones constructed by genome addition of common carp (Cyprinus carp) and crucian carp (Carassius auratus)

A polyploid hybrid fish with natural gynogenesis can prevent segregation and maintain their hybrid vigor in their progenies. Supposing the reproduction mode of induced polyploid fish being natural gynogenesis, allopolyploid hybrid between common carp and crucian carp into allopolyploid was performed. The purpose of this paper is to describe a lineage from sexual diploid carp transforming into allotriploid and allotetraploid unisexual clones by genome addition. The diploid hybrid between common carp and crucian carp reproduces an unreduced nucleus consisting of two parental genomes. This unreduced female pronucleus will fuse with male pronucleus and form allotriploid zygote after penetration of related species sperms. Allotriploid embryos grow normally, and part of female allotriploid can produce unreduced mature ova with three genomes. Mature ova of most allotriploid females are provided with natural gynogenetic trait and their nuclei do not fuse with any entrance sperm. All female offspring are produced by gynogenesis of allotriploid egg under activation of penetrating sperms. These offspring maintain morphological traits of their allotriploid maternal and form an allotetraploid unisexual clone by gynogenetic reproduction mode. However, female nuclei of rare allotriploid female can fuse with penetrating male pronuclei and result in the appearance of allotetraploid individuals by means of genome addition. All allotetraploid females can reproduce unreduced mature eggs containing four genomes. Therefore, mature eggs of allotetraploid maintain gynogenetic trait and allotetraploid unisexual clone is produced under activation of related species sperms.

Growth hormone gene transfer in common carp

The first successful case of transgenic fish was achieved in 1984. It is in a model system that the integration and expression of recombinant human growth hormone (hGH) in host red common carp (Cyprinus carpio, red var.) have been thoroughly studied. Recently, the integration sites have been recovered and characterized. Compared with non-transgenic peers, hGH-transgenic fish are prior in dietary utilization and growth performance. In view of bio-safety and bio-ethics, an "all-fish" construct CAgcGH, grass carp growth hormone fused with common carp P-actin promoter, has been generated and transferred into Yellow River carp (C carpio, local strain in Yellow River) fertilized eggs. Under middle-scale trial, CAgcGH-transgenics show higher growth rate and food conversion efficiency than the controls, which is consistent to laboratory findings. To avoid the potential impact of transgenic fish on the environment, a sterile strain of transgenic triploid fish has been successfully produced. The "all-fish" transgenic common carp is also approved safe enough as daily food, according to a test based on the pathological principles of new medicines issued by the Ministry of Health of China. The "all-fish" transgenic common carp with growth enhancement is now ready for market, but looking for governmental authorization. (C) 2003 Editions scientifiques et medicales Elsevier SAS and Ifremer/IRD/Inra/Cemagref. All rights reserved.

Transgene for growth hormone in common carp (Cyprinus carpio L.) promotes thymus development

The transgenic carp were produced by micro-injection of CAgcGHc into the fertilized eggs. Observation of the thymus development between the transgenics and non-transgenic controls was carried out. The thymus of one-year-old transgenics F1 showed a great increase in both size and weight. The unilateral thymus of the transgenics weighed from 190 to 295 mg with average 218.6 mg, whereas the unilateral thymus of the controls weighed 20-81 mg with average 42.5 mg; i.e. the thymus weight in the transgenics was 5.14 fold over that in the controls. The index of thymus/body weight in the transgenics was 2.97 fold over the controls. Light microscopy observation indicated that the thymus of the transgenics; well developed with the thickened outer region and compactly arranged thymocytes, while the thymus in the controls were degenerating with the thinned outer region, scattered thymocytes and groups of fatty cells. Further analysis with the electron microscopy revealed that pro-liferous cells in the transgenics; were mainly small lymphocytes and no pathological changes were found. The results confirmed that the "All-fish" GH-transgene promotes thymus development and thymocyte proliferation, and retards thymus degeneration. The study has laid a foundation for further analysis of the immunobiological function in GH-transgenic carp.

Responses of antioxidant systems in the hepatocytes of common carp (Cyprinus carpio L.) to the toxicity of microcystin-LR

The freshwater, bloom-forming cyanobacterium (blue-green alga) Microcystis aeruginosa produces a peptide hepatotoxin, which causes the damage of animal liver. Recently, toxic Microcystis blooms frequently occur in the eutrophic Dianchi Lake (300 km(2) and located in the South-Westem of China). Microcystin-LR from Microcystis in Dianchi was isolated and purified by high performance liquid chromatography (HPLC) and its toxicity to mouse and fish liver was studied (Li et al., 2001). In this study, six biochemical parameters (reactive oxygen species, glutathione, superoxide dismutase, catalase, glutathione peroxide and glutathione S-transferase) were determined in common carp hepatocytes when the cells were exposed to 10 mug microcystin-LR per litre. The results showed that reactive oxygen species (ROS) contents increased by more than one-time compared with the control after 6 h exposure to the toxin. In contrast, glutathione (GSH) levels in the hepatocytes exposed to microcystin-LR decreased by 47% compared with the control. The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxide (GSH-Px) increased significantly after 6 h exposure to microcystin-LR, but glutathione S-transferase (GST) activity showed no difference from the control. These results suggested that the toxicity of microcystin-LR caused the increase of ROS contents and the depletion of GSH in hepatocytes exposed to the toxin and these changes led to oxidant shock in hepatocytes. Increases of SOD, CAT and GSH-Px activities revealed that these three kinds of antioxidant enzymes might play important roles in eliminating the excessive ROS. This paper also examined the possible toxicity mechanism of microcystin-LR on the fish hepatocytes and the results were similar to those with mouse hepatocytes. (C) 2003 Elsevier Science Ltd. All rights reserved.

PCR-RFLP analysis of mitochondrial DNA ND5/6 region among 3 subspecies of common carp (Cyprinus carpio L.) and its application to genetic discrimination of subspecies

Mitochondrial DNA ND5/6 region was studied by PCR-RFLP analysis among ten representative strains belonging to three subspecies (Cyprinus carpio carpio, Cyprinus carpio haematopterus and Cyprinus carpio rubrofuscus) of common carp (Cyprinus carpio L.). A total of 2.4 kb fragment was amplified and subjected to restriction endonuclease analysis with nine restriction endonucleases subsequently. The results indicated that each subspecies owned one hyplotype and four restriction enzymes (Dde I, HaeIII, Taq I and Mbo I) produced diagnostic restriction sites which could be used for discriminating the three subspecies and as molecular genetic markers for assistant selective breeding of common carp.

Cross-species amplification in silver carp and bighead carp with microsatellite primers of common carp

About a third of microsatellite primers designed for common carp (Cyprinus carpio) was successfully amplified in silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis). These markers, inherited in Mendelian mode, are of potential applications in cypinid genetics.

Common evolutionary origin of aquareoviruses and orthoreoviruses revealed by genome characterization of Golden shiner reovirus, Grass carp reovirus, Striped bass reovirus and golden ide reovirus (genus Aquareovirus, family Reoviridae)

Full-length and partial genome sequences of four members of the genus Aquareovirus, family Reoviridae (Golden shiner reovirus, Grass carp reovirus, Striped bass reovirus and golden ide reovirus) were characterized. Based on sequence comparison, the unclassified Grass carp reovirus was shown to be a member of the species Aquareovirus C The status of golden ide reovirus, another unclassified aquareovirus, was also examined. Sequence analysis showed that it did not belong to the species Aquareovirus A or C, but assessment of its relationship to the species Aquareovirus B, D, E and F was hampered by the absence of genetic data from these species. In agreement with previous reports of ultrastructural resemblance between aquareoviruses and orthoreoviruses, genetic analysis revealed homology in the genes of the two groups. This homology concerned eight of the 11 segments of the aquareovirus genome (amino acid identity 17-42%), and similar genetic organization was observed in two other segments. The conserved terminal sequences in the genomes of members of the two groups were also similar. These data are undoubtedly an indication of the common evolutionary origin of these viruses. This clear genetic relatedness between members of distinct genera is unique within the family Reoviridae. Such a genetic relationship is usually observed between members of a single genus. However, the current taxonomic classification of aquareoviruses and orthoreoviruses in two different genera is supported by a number of characteristics, including their distinct G+C contents, unequal numbers of genome segments, absence of an antigenic relationship, different cytopathic effects and specific econiches.

Transgenes in F-4 pMThGH-transgenic common carp (Cyprinus carpio L.) are highly polymorphic

To gain information on the integration pattern of pMThGH-transgene, 50 transgenes were recovered from F-4 generation of pMThGH transgenic common carp (Cyprinus carpio L,) and 33 recovered genes were analyzed. The restriction maps of these recovered genes were constructed by digestion with five kinds of enzymes. These transgenes can be classified into 4 types according to their restriction maps. Only one type of transgenes maintains its original molecular form, whereas the other three types are very different from the original one and vary each other on both molecular weight and restriction maps. This implies that the sequences of most transgenes have been deleted and/or rearranged during integration and inheritance. The results of PCR amplification and Southern blot hybridization indicate that MThGH in Type I transgene keeps intact but most of its sequence has been deleted in other three types. All these results suggest that transgenes in F-4 generation of transgenic carp are highly polymorphic. Two DNA fragments concerning integration site of transgenes were cloned from recovered transgenes, and found to be homologous to the 5'UTR of beta -actin gene of common carp and mouse mRNA for receptor tyrosine kinase (RTK), respectively.

Whole-body amino acid pattern of F-4 human growth hormone gene-transgenic red common carp (Cyprinus carpio) fed diets with different protein levels

F-4 generation of human growth hormone (hGH) gene-transgenic red common carp, and the non-transgenic controls were fed for 8 weeks on purified diets with 20%, 30% or 40% protein. Analysis of whole-body amino acids showed that the proportions of lysine, leucine, phenylalanine, valine and alanine, as percentages of body protein, increased significantly, while those of arginine, glutamic acid and tyrosine decreased, with increases in dietary protein level in at least one strain of fish. Proportions of the other amino acids were unaffected by the diets. The proportions of lysine and arginine were significantly higher, while those of leucine and alanine were lower in the transgenics than in the controls in at least one diet group. Proportions of the other amino acids were unaffected by strain. The results suggest that the whole-body amino acid profile of transgenic carp, when expressed as proportions of body protein, was in general, similar to that of the non-transgenic controls. (C) 2000 Elsevier Science B.V. All rights reserved.

Quantum-well anisotropic forbidden transitions induced by a common-atom interface potential

A prominent effect of the interface potential (IP) [E. L. Ivchenko and A. Yu. Kaminski, Phys. Rev. B 54, 5852 (1996); O. Krebs and P. Voisin, Phys. Rev. Lett. 77, 1829 (1996)], the optical anisotropy of the forbidden transitions in quantum wells has been observed by reflectance-difference spectroscopy. Predictions by the heavy-light-hole coupling IP models are qualitatively consistent with all the observed features of the forbidden and the allowed transitions. The fact that the predicted value of the relative, transition strength, which depends on neither the IP strength nor the electric field, disagrees with the observed one indicates that coupling involving X and/or L bands may also be important. [S0163-1829(99)04227-7].