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.

Complete genome sequence of lymphocystis disease virus isolated from China

Lymphocystis diseases in fish throughout the world have been extensively described. Here we report the complete genome sequence of lymphocystis disease virus isolated in China (LCDV-C), an LCDV isolated from cultured flounder (Paralichthys olivaceus) with lymphocystis disease in China. The LCDV-C genome is 186,250 bp, with a base composition of 27.25% G+C. Computer-assisted analysis revealed 240 potential open reading frames (ORFs) and 176 nonoverlapping putative viral genes, which encode polypeptides ranging from 40 to 1,193 amino acids. The percent coding density is 67%, and the average length of each ORF is 702 bp. A search of the GenBank database using the 176 individual putative genes revealed 103 homologues to the corresponding ORFs of LCDV-1 and 73 potential genes that were not found in LCDV-1 and other iridoviruses. Among the 73 genes, there are 8 genes that contain conserved domains of cellular genes and 65 novel genes that do not show any significant homology with the sequences in public databases. Although a certain extent of similarity between putative gene products of LCDV-C and corresponding proteins of LCDV-1 was revealed, no colinearity was detected when their ORF arrangements and coding strategies were compared to each other, suggesting that a high degree of genetic rearrangements between them has occurred. And a large number of tandem and overlapping repeated sequences were observed in the LCDV-C genome. The deduced amino acid sequence of the major capsid protein (MCP) presents the highest identity to those of LCDV-1 and other iridoviruses among the LCDV-C gene products. Furthermore, a phylogenetic tree was constructed based on the multiple alignments of nine MCP amino acid sequences. Interestingly, LCDV-C and LCDV-1 were clustered together, but their amino acid identity is much less than that in other clusters. The unexpected levels of divergence between their genomes in size, gene organization, and gene product identity suggest that LCDV-C and LCDV-1 shouldn't belong to a same species and that LCDV-C should be considered a species different from LCDV-1.

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.

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.

Genome mapping in aquatic animals: Progress and future perspectives

The genome of aquatic animals is poorly understood and information from different taxonomic groups is sketchy. While there have been intensive genomic studies on some fish models, investigations on other fishes and invertebrates have been scarce. Yet there are recently some coordinated studies on genome mapping in a number of aquaculture animals of economic importance. This review summarizes information available on genome mapping of the important fish models and aquaculture animals. The future perspectives of this field of studies are discussed.

Genome segment S8 of grass carp hemorrhage virus encodes a virion protein

The complete nucleotide sequence of the genome segment S8 of grass carp hemorrhage virus (GCHV) was determined from cDNA corresponding to the viral genomic RNA. It is 1,287 nucleotides in length and contains a large open reading frame that could encode a protein of 409 amino acids with a predicted molecular mass of 44 kD. The S8 was expressed using the pET fusion protein vector and detected by Western blotting analysis using the chicken egg IgY against intact GCHV particles, indicating that S8 encodes a virion protein. Amino acid sequence comparisons revealed that the protein encoded by S8 is closely related to protein alpha2 of mammalian reovirus, suggesting that the deduced protein of S8 is an inner capsid protein. Copyright (C) 2001 S. Karger AG, Basel.

Complete nucleotide sequence of the S10 genome segment of grass carp reovirus (GCRV)

Hemorrhagic disease, caused by the grass carp reovirus (GCRV), is one of the major diseases of grass carp in China. Little is known about the structure and function of the gene segments of this reovirus. The S10 genome segment of GCRV was cloned and the complete nucleotide sequence is reported here. The S10 is 909 nucleotides long and contains a large open reading frame (ORF) encoding a protein of 276 amino acids with a deduced molecular weight of approximately 29.7 kDa. Comparisons of the deduced amino acid sequence of GCRV S10 with those of other reoviruses revealed no significant homologies. However, GCRV S10 shared a putative zinc-finger sequence and a similar distribution of hydrophilic motifs with the outer capsid proteins encoded by Coho salmon aquareovirus (SCSV) S10, striped bass reovirus (SBRV) S10, and mammalian reovirus (MRV) S4. It was predicted that this segment gene encodes an outer capsid protein.

Sequence of genome segments 1, 2, and 3 of the grass carp reovirus (Genus Aquareovirus, family Reoviridae)

The genome segments 1, 2, and 3 of the grass carp reovirus (GCRV), a tentative species assigned to genus Aquareouirus, family Reouiridae, were sequenced. The respective segments 1, 2, and 3 were 3949, 3877, and 3702 nucleotides long. Conserved moths 5' (GUUAUUU) and 3' (UUCAUC) were found at the ends of each segment. Each segment contains a single ORF and the negative strand does not permit identification of consistent ORFs. Sequence analysis revealed that VP2 is the viral polymerase, while VPI might represent the viral guanyly/methyl transferase (involved in the capping process of RNA transcripts) and VP3 the NTPase/helicase (involved in the transcription and capping of viral RNAs), The highest amino acid identities (26-41%) were found with orthoreovirus proteins. Further genomic characterization should provide insight about the genetic relationships between GCRV, aquareoviruses, and orthoreoviruses, It should also permit to precise the taxonomic status of these different viruses. (C) 2000 Academic Press.

Genome-wide survey of putative serine/threonine protein kinases in cyanobacteria

Background: Serine/threonine kinases (STKs) have been found in an increasing number of prokaryotes, showing important roles in signal transduction that supplement the well known role of two-component system. Cyanobacteria are photoautotrophic prokaryotes able to grow in a wide range of ecological environments, and their signal transduction systems are important in adaptation to the environment. Sequence information from several cyanobacterial genomes offers a unique opportunity to conduct a comprehensive comparative analysis of this kinase family. In this study, we extracted information regarding Ser/Thr kinases from 21 species of sequenced cyanobacteria and investigated their diversity, conservation, domain structure, and evolution. Results: 286 putative STK homologues were identified. STKs are absent in four Prochlorococcus strains and one marine Synechococcus strain and abundant in filamentous nitrogen-fixing cyanobacteria. Motifs and invariant amino acids typical in eukaryotic STKs were conserved well in these proteins, and six more cyanobacteria- or bacteria-specific conserved residues were found. These STK proteins were classified into three major families according to their domain structures. Fourteen types and a total of 131 additional domains were identified, some of which are reported to participate in the recognition of signals or substrates. Cyanobacterial STKs show rather complicated phylogenetic relationships that correspond poorly with phylogenies based on 16S rRNA and those based on additional domains. Conclusion: The number of STK genes in different cyanobacteria is the result of the genome size, ecophysiology, and physiological properties of the organism. Similar conserved motifs and amino acids indicate that cyanobacterial STKs make use of a similar catalytic mechanism as eukaryotic STKs. Gene gain-and-loss is significant during STK evolution, along with domain shuffling and insertion. This study has established an overall framework of sequence-structure-function interactions for the STK gene family, which may facilitate further studies of the role of STKs in various organisms.

Genome-wide survey of putative serine/threonine protein kinases in cyanobacteria

Background: Serine/threonine kinases (STKs) have been found in an increasing number of prokaryotes, showing important roles in signal transduction that supplement the well known role of two-component system. Cyanobacteria are photoautotrophic prokaryotes able to grow in a wide range of ecological environments, and their signal transduction systems are important in adaptation to the environment. Sequence information from several cyanobacterial genomes offers a unique opportunity to conduct a comprehensive comparative analysis of this kinase family. In this study, we extracted information regarding Ser/Thr kinases from 21 species of sequenced cyanobacteria and investigated their diversity, conservation, domain structure, and evolution. Results: 286 putative STK homologues were identified. STKs are absent in four Prochlorococcus strains and one marine Synechococcus strain and abundant in filamentous nitrogen-fixing cyanobacteria. Motifs and invariant amino acids typical in eukaryotic STKs were conserved well in these proteins, and six more cyanobacteria- or bacteria-specific conserved residues were found. These STK proteins were classified into three major families according to their domain structures. Fourteen types and a total of 131 additional domains were identified, some of which are reported to participate in the recognition of signals or substrates. Cyanobacterial STKs show rather complicated phylogenetic relationships that correspond poorly with phylogenies based on 16S rRNA and those based on additional domains. Conclusion: The number of STK genes in different cyanobacteria is the result of the genome size, ecophysiology, and physiological properties of the organism. Similar conserved motifs and amino acids indicate that cyanobacterial STKs make use of a similar catalytic mechanism as eukaryotic STKs. Gene gain-and-loss is significant during STK evolution, along with domain shuffling and insertion. This study has established an overall framework of sequence-structure-function interactions for the STK gene family, which may facilitate further studies of the role of STKs in various organisms.

Genome-wide analysis of restriction-modification system in unicellular and filamentous cyanobacteria

Cyanobacteria are an ancient group of gram-negative bacteria with strong genome size variation ranging from 1.6 to 9.1 Mb. Here, we first retrieved all the putative restriction-modification (RM) genes in the draft genome of Spirulina and then performed a range of comparative and bioinformatic analyses on RM genes from unicellular and filamentous cyanobacterial genomes. We have identified 6 gene clusters containing putative Type I RMs and 11 putative Type II RMs or the solitary methyltransferases (MTases). RT-PCR analysis reveals that 6 of 18 MTases are not expressed in Spirulina, whereas one hsdM gene, with a mutated cognate hsdS, was detected to be expressed. Our results indicate that the number of RM genes in filamentous cyanobacteria is significantly higher than in unicellular species, and this expansion of RM systems in filamentous cyanobacteria may be related to their wide range of ecological tolerance. Furthermore, a coevolutionary pattern is found between hsdM and hsdR, with a large number of site pairs positively or negatively correlated, indicating the functional importance of these pairing interactions between their tertiary structures. No evidence for positive selection is found for the majority of RMs, e. g., hsdM, hsdS, hsdR, and Type II restriction endonuclease gene families, while a group of MTases exhibit a remarkable signature of adaptive evolution. Sites and genes identified here to have been under positive selection would provide targets for further research on their structural and functional evaluations.