A gene cluster that regulates both heterocyst differentiation and pattern formation in Anabaena sp strain PCC 7120

Wild-type Anabaena sp. strain PCC 7120, a filamentous nitrogen-fixing cyanobacterium, produces single heterocysts at semi-regular intervals. asr0100 (patU5) and alr0101 (patU3) are homologous to the 5' and 3' portions of patU of Nostoc punctiforme. alr0099 (hetZ) overlaps the 5' end of patU5. hetZ, patU5 and patU3 were all upregulated, or expressed specifically, in proheterocysts and heterocysts. Mutants of hetZ showed delayed or no heterocyst differentiation. In contrast, a patU3 mutation produced a multiple contiguous heterocyst (Mch) phenotype and restored the formation of otherwise lost intercalary heterocysts in a patA background. Decreasing the expression of patU3 greatly increased the frequency of heterocysts in a mini-patS strain. Two promoter regions and two principal, corresponding transcripts were detected in the hetZ-patU5-patU3 region. Transcription of hetZ was upregulated in a hetZ mutant and downregulated in a patU3 mutant. When mutants hetZ::C.K2 and hetZ::Tn5-1087b were nitrogen-deprived, P-hetC-gfp was very weakly expressed, and in hetZ::Tn5-1087b, P-hetR-gfp was relatively strongly expressed in cells that had neither a regular pattern nor altered morphology. We conclude that the hetZ-patU5-patU3 cluster plays an important role in co-ordination of heterocyst differentiation and pattern formation. The presence of homologous clusters in filamentous genera without heterocysts is suggestive of a more general role.

Phylogenetic relationships of Cyprinidae (Teleostei : Cypriniformes) inferred from the partial S6K1 gene sequences and implication of indel sites in intron 1

The family Cyprinidae is widely distributed in East Asia, and has the important phylogenetic significance in the fish evolution. In this study, the 5' end partial sequences (containing exon 1, exon 2 and indel 1) of S6K1 gene were obtained from 30 representative species in Cyprinidae and outgroup using PCR amplification and sequencing. The phylogenetic relationships of Cyprinidae were reconstructed with neighbor joining (NJ), maximum parsimony (MP), maximum likelihood (ML), and Bayesian methods. Myxocyprinus asiaticus (Catostomidae) was assigned to the outgroup taxon. Similar phylogenetic relationships within the family Cyprinidae were achieved with the four analyses. Leuciscini and Barbini were monophyletic lineages respectively with the high nodal supports. Leuciscini comprises Hypophthalmichthyinae, Xenocyprinae, Cultrinae, Gobioninae, Acheilognathinae and East Asian species of Leuciscinae and Danioninae. Monophyly of East Asian clade was supported with high nodal support. Barbini comprises Schizothoracinae, Barbinae, Cyprininae and Labeoninae. The monophyletic lineage consisting of Danio rerio, D. myersi, and Rasbora trilineata was basal in the tree. In addition, the large fragment indels in intron 1 were analyzed to improve the understanding of Cyprinidae relationships. The results showed that the large fragment indels were correlated with the relations among species. Some conserved regions in intron 1 were thought to be involved in the functional regulation. However, no correlation was found between sequence variations and species characteristic size.

Site-directed gene integration in transgenic zebrafish mediated by cre recombinase using a combination of mutant Lox sites

With current gene-transfer techniques in fish, insertion of DNA into the genome occurs randomly and in many instances at multiple sites. Associated position effects, copy number differences, and multiple gene interactions make gene expression experiments difficult to interpret and fish phenotype less predictable. To meet different fish engineering needs, we describe here a gene targeting model in zebrafish. At first, four target zebrafish lines, each harboring a single genomic lox71 target site, were generated by zebrafish transgenesis. The zygotes of transgenic zebrafish lines were coinjected with capped Cre mRNA and a knockin vector pZklox66RFP. Site-specific integration event happened from one target zebrafish line. In this line two integrant zebrafish were obtained from more than 80,000 targeted embryos (integrating efficiency about 10(-4) to 10(-5)) and confirmed to have a sole copy of the integrating DNA at the target genome site. Genomic polymerase chain reaction analysis and DNA sequencing verified the correct gene target events where lox71 and lox66 have accurately recombined into double mutant lox72 and wild-type loxP. Each integrant zebrafish chosen for analysis harbored the transgene rfp at the designated egfp concatenates. Although the Cre-mediated recombination is site specific, it is dependent on a randomly placed target site. That is, a genomic target cannot be preselected for integration based solely on its sequence. Conclusively, an rfp reporter gene was successfully inserted into the egfp target locus of zebrafish genome by Cre-lox-mediated recombination. This site-directed knockin system using the lox71/lox66 combination should be a promising gene-targeting platform serving various purposes in fish genetic engineering.

Characterization and expression analysis of Paralichthys olivaceus voltage-dependent anion channel (VDAC) gene in response to virus infection

Voltage-dependent anion channel (VDAC, also known as mitochondrial porin) is acknowledged to play an important role in stress-induced mammalian apoptosis. In this study, Paralichthys olivaceus VDAC (PoVDAC) gene was identified as a virally induced gene from Scophthalmus Maximus Rhabdovirus (SMRV)-infected flounder embryonic cells (FEC). The full length of PoVDAC cDNA is 1380 bp with an open reading frame of 852 bp encoding a 283 amino acid protein. The deduced PoVDAC contains one alpha-helix, 13 transmembrane beta-strands and one eukaryotic mitochondrial porin signature motif. Constitutive expression of PoVDAC was confirmed in all tested tissues by real-time PCR. Further expression analysis revealed PoVDAC mRNA was upregulated by viral infection. We prepared fish antiserum against recombinant VDAC proteins and detected the PoVDAC in heart lysates from flounder as a 32 kDa band on western blot. Overexpression of PoVDAC in fish cells induced apoptosis. Immunofluoresence localization indicated that the significant distribution changes of PoVDAC have occurred in virus-induced apoptotic cells. This is the first report on the inductive expression of VDAC by viral infection, suggesting that PoVDAC might be mediated flounder antiviral immune response through induction of apoptosis. (c) 2007 Elsevier Ltd. All rights reserved.

Molecular cloning, promoter analysis and induced expression of the complement component C9 gene in the grass carp Ctenopharyngodon idella

Complement-mediated killing of pathogens through lytic pathway is an important effector mechanism of innate immune response. C9 is the ninth member of complement components, creating the membrane attack complex (MAC). In the present study, a putative cDNA sequence encoding the 650 amino acids of C9 and its genomic organization were identified in grass carp Ctenopharyngodon idella. The deduced amino acid sequence of grass carp C9 (gcC9) showed 48% and 38.5% identity to Japanese flounder and human C9, respectively. Domain search revealed that gcC9 contains a LDL receptor domain, an EGF precursor domain, a MACPF domain and two TSP domain located in the N-terminal and C-terminal, respectively. Phylogenetic analysis demonstrated that gcC9 is clustered in a same clade with Japanese flounder, pufferfish and rainbow trout C9. The gcC9 gene consists of 11 exons with 10 introns, spacing over approximately 7 kb of genomic sequence. Analysis of gcC9 promoter region revealed the presence of a TATA box and some putative transcription factor such as C/EBP, HSF, NF-AT, CHOP-C, HNF-3B, GATA-2, IK-2, EVI- 1, AP-1, CP2 and OCT-1 binding sites. The first intron region contains C/EBPb, HFH-1 and Oct-1 binding sites. RT-PCR and Western blotting analysis demonstrated that the mRNA and protein of gcC9 gene have similar expression patterns, being constitutively expressed in all organs examined of healthy fish, with the highest level in hepatopancreas. By real-time quantitative RT-PCR analysis, gcC9 transcripts were significantly up-regulated in head kidney, spleen, hepatopancreas and down-regulated in intestine from inactivated fish bacterial pathogen Flavobacterium columnare-stimulated fish, demonstrating the role of C9 in immune response. (c) 2007 Elsevier B.V. All rights reserved.

Rapid growth cost in “all-fish” growth hormone gene transgenic carp: Reduced critical swimming speed

Evidence has accumulated that there is a trade-off between benefits and costs associated with rapid growth. A trade-off between growth rates and critical. swimming speed (U-crit) had been also reported to be common in teleost fish. We hypothesize that growth acceleration in the F-3 generation of "all-fish" growth hormone gene (GH) transgenic common carp (Cyprinus carpio L.) would reduce the swimming abilities. Growth and swimming performance between transgenic fish and non-transgenic controls were) compared. The results showed that transgenic fish had a mean body weight 1.4-1.9-fold heavier, and a mean specific growth rate (SGR) value 6%-10% higher than the controls. Transgenic fish, however, had a mean absolute U-crit (cm/s) value 22% or mean relative Ucrit (BL/s) value 24% lower than the controls. It suggested that fast-growing "all-fish" GH-transgenic carp were inferior swimmers. It is also supported that there was a trade-off between growth rates and swimming performance, i.e. faster-growing individuals had lower critical swimming speed.

Identification and characterization of two homologues of interferon-stimulated gene ISG15 in crucian carp

ISG15 is one of the most strongly induced genes upon viral infection, interferon (IFN) stimulation, and lipopolysaccharide, (LPS) stimulation, and only one copy has been found in mammals so far. Here two fish ISG15 genes, termed CaISG15-1 and CaISG15-2, have been cloned and sequenced from UV-inactivated GCHV (grass carp haemorrhagic virus)-infected and IFN-produced CAB cells (crucian carp Carassius auratus blastulae embryonic cells) by suppression subtractive hybridization. The full-length cDNA sequences of two crucian carp ISG15 encode a 155-amino-acid protein and a 161-amino-acid protein, both of which show 78.9% identity overall and possess the characteristic structures of mammalian ISG15 proteins including two tandem ubiquitin-like domains and the C-terminal canonical LRLRGG motif. In CAB cells treated with different stimuli including active virus, UV-inactivated GCHV and IFN containing supernatant (ICS), the expression of both CaISG15-1 and CaISG15-2 was up-regulated but displayed different kinetics. Poly I:C and LPS were also able to induce an increase in mRNA for both genes. In CAB cells responsive to active GCHV, UV-inactivated GCHV, CAB ICS, Poly 1:12 and LPS, CaISG15-1 was upregulated more significantly than CaISG15-2. These results suggest that there are two ISG15 homologues in crucian carp, both of which might play distinct roles in innate immunity against viral and bacterial infection. (c) 2006 Elsevier Ltd. All rights reserved.

Intelectin gene from the grass carp Ctenopharyngodon idella: cDNA cloning, tissue expression, and immunohistochemical localization

The cDNA encoding grass carp intelectin was isolated from a head kidney cDNA library, and termed gcIntL. The deduced amino acid sequence of gcIntL consists of 318 amino acids, and about 55% identical and 74% similar to human intelectin, which is a new type of lectin recognizing galactofuranose, and plays a role in the recognition of bacteria-specific components in animal hosts. The gcIntL gene consists of seven exons and six introns, spacing over approximately 3 kb of genomic sequence. Phylogenetic analysis clearly demonstrated that the gcIntL formed a clade with Danio rerio intelectin and 35 kDa serum lectin. By real-time quantitative RT-PCR analysis, gcIntL transcripts were significantly induced in head kidney, trunk kidney, spleen, and intestine from LPS-stimulated fish. RT-PCR and Western blotting analysis demonstrated that the mRNA and protein of gcIntL gene have the same expression pattern, and both were detected in brain, gill, intestine, head kidney, trunk kidney, spleen, and heart. Furthermore, gcIntL protein could be detected in gill, intestine, trunk kidney, head kidney, spleen, heart, and brain including medulla oblongata and optic lobe, as determined by immunohistochemistry. This is the first report of intelectin expression pattern in fish, and of recombinant gcIntL and polyclonal antibody against gcIntL. (c) 2006 Elsevier Ltd. All rights reserved.

Gene structure of an antimicrobial peptide from mandarin fish, Siniperca chuatsi (Basilewsky), suggests that moronecidins and pleurocidins belong in one family: the piscidins

The gene of piscidin, an antimicrobial peptide, has been cloned from the mandarin fish, Siniperca chuatsi. From the first transcription initiation site, the mandarin fish piscidin gene extends 1693 nucleotides to the end of the 3' untranslated region and contains four exons and three introns. A predicted 79-residue prepropeptide consists of three domains: a signal peptide (22 aa), a mature peptide (22 aa) and a C-terminal prodomain (35 aa). The shortage of XQQ motif in the prodomain of mandarin fish piscidin and the similar gene structure between moronecidins (piscidins) and pleurocidins may indicate that they are derived from the same ancestor gene. We thus suggest that piscidin should be used as a terminology for these antimicrobial peptides in the future. The mandarin fish piscidin mRNA was abundant in intestine, spleen, pronephros and kidney analysed by real-time polymerase chain reaction. After stimulation with lipopoly saccharides (LPS), a marked increase in transcripts was observed in most tissues, indicating that piscidin is not only a constitutively expressed molecule, but also has an increased response to bacterial infection. The synthetic, amidated mandarin fish piscidin exhibited different antimicrobial activity against different fish bacterial pathogens, especially against species of Aeromonas, which may to certain extent reflect the pathogenicity of these bacteria.

Identification of a gene, ccr-1 (sll1242), required for chill-light tolerance and growth at 15 degrees C in Synechocystis sp PCC 6803

Synechocystis sp. PCC 6803 exposed to chill (5 degrees C)-light (100 mu mol photons m(-2) s(-1)) stress loses its ability to reinitiate growth. From a random insertion mutant library of Synechocystis sp. PCC 6803, a sll1242 mutant showing increased sensitivity to chill plus light was isolated. Mutant reconstruction and complementation with the wild-type gene confirmed the role of sll1242 in maintaining chill-light tolerance. At 15 degrees C, the autotrophic and mixotrophic growth of the mutant were both inhibited, paralleled by decreased photosynthetic activity. The expression of sll1242 was upregulated in Synechocystis sp. PCC 6803 after transfer from 30 to 15 degrees C at a photosynthetic photon flux density of 30 mu mol photons m(-2) S-1. sll1242, named ccr (cyanobacterial cold resistance gene)- 1, may be required for cold acclimation of cyanobacteria in light.

Cloning, characterization and promoter analysis of common carp hairy/Enhancer-of-split-related gene, her6

Some members of hairy/Enhancer-of-split-related gene (HES) family have important effects on axial mesoderm segmentation and the establishment and maintenance of the somite fringe. In fishes. the her6 gene, a member of the HES family, is the homologue Of heS1 in mammals and chicken. In this study, the her6 gene and its full-length cDNA from the common carp (Cyprinus carpio) were isolated and characterized. The genomic sequence of common carp her6 is approximately 1.7 kb. with four exons and three introns, and the full-length cDNA of 1314 bp encodes a Putative polypeptide of 271 amino acids. To analyse the promoter sequence of common carp her6. sequences of various lengths upstream from the transcription initiation site of her6 were fused to enhanced green fluorescent. protein gene (eGFP) and introduced into zebrafish embryos by microinjection to generate transgenic embryos. Our results show that the upstream sequence of 500 bp can direct highly efficient and tissue-specific expression of eGFP in zebrafish embryos. whereas a fragment of 200 bp containing the TATA box and a partial suppressor of hairless paired site sequence (SPS) is not sufficient to drive eGFP expression in zebrafish embryos.

Non-monophyly of fish in the Sinipercidae (Perciformes) as inferred from cytochrome b gene

The sinipercids represent a group of 12 species of freshwater percoid fish, including nine in Siniperca and three species in Coreoperca. Despite several classification attempts and a preliminary molecular phylogeny, the phylogenetic relationships and systematic position of sinipercids remained still unsolved. The complete cytochrome b gene sequences from nine sinipercid species four non-sinipercid fish species were cloned, and a total of 12 cyt b sequences from 10 species of sinipercids and 11 cyt b sequences from 10 species of non-sinipercid fish also in Perciformes were included in the phylogenetic analysis. As expected, the two genera Siniperca and Coreoperca within sinipercids are recovered as monophyletic. However, nine species representing Moronidae, Serranidae, Centropomidae, Acropomatidae, Emmelichtyidae, Siganidae and Centrarchidae included in the present study are all nested between Coreoperca and Siniperca, which provides marked evidence for a non-monophyly of sinipercid fishes. Coreoperca appears to be closest to Centrachus representing the family Centrarchidae. Coreoperca whiteheadi and C. herzi are sibling species, which together are closely related to C. kawamebari. In the Siniperca, the node between S. roulei and the remaining species is the most ancestral, followed by that of S. fortis. S. chuatsi and S. kneri are sibling species, sister to S. obscura. However, the sinipercids do not seem to have a very clear phylogenetic history, for different methods of phylogenetic reconstruction result in different tree topologies, and the only conclusive result in favor of a paraphyletic origin of the two sinipercid genera is the parametric bootstrap test. The paraphyly of Sinipercidae may suggest that the "synapomorphs" such as cycloid scales, upon which this family is based, were independently derived at least twice within sinipercid fishes, and further study should be carried out to include the other two Siniperca species and to incorporate other genes.

Gene structure and transcription of IRF-1 and IRF-7 in the mandarin fish Siniperca chuatsi

The genes of IRF-1 and IRF-7 have been cloned from the mandarin fish (Siniperca chuatsi). The IRF-1 gene has 4919 nucleotides (nt) and contains 10exons and 9introns, with an open reading frame (ORF) of 903 ntencoding301 aa. The IRF-7 gene has 6057 nt and also contains 10exons and 9introns, with an ORF of 1308 nt encoding 436 aa. The IRF-1 and IRF-7 genes have only one copy each in the genome. The transcription of IRF-1 and IRF-7 in different organs was analyzed by real-time PCR, and both molecules were constitutively expressed. The IRF-I and IRF-7 mRNAs were abundant in gill, spleen, kidney and pronephros. The temporal transcriptional changes for IRF-1, IRF-7 and Mx were investigated within 48 h after poly I: C stimulation in liver, gill, spleen and pronephros. An increased transcription was detected for IRF-1 and IRF-7 12 h post-stimulation, being earlier than the transcription of Mx protein; however, IRF-1 and IRF-7 transcription decreased while the Mx protein was stable at 48 h post-stimulation. (c) 2007 Published by Elsevier B.V.

Phylogenetic relationships of the Chinese sisorid catfishes: a nuclear intron versus mitochondrial gene approach

Several recent molecular phylogenetic studies of the sisorid catfishes (Sisoridae) have challenged some aspects of their traditional taxonomy and cladistic hypotheses of their phylogeny. However, disagreement with respect to relationships within this family in these studies highlights the need for additional data and analyses. Here we subjected 15 taxa representing 12 sisorids genera to comprehensive phylogenetic analyses using the second intron of low-copy nuclear S7 ribosomal protein (rpS7) gene and the mitochondrial 16S rRNA gene segments both individually and in combination. The competing sisorid topologies were then tested by using the approximately unbiased (AU) test and the Shimodaira-Hasegawa (SH) test. Our results support previously suggested polyphyly of Pareuchiloglanis. The genus Pseudecheneis is likely to be nested in the glyptosternoids and Glaridoglanis might be basal to the tribe Glyptosternini. However, justified by AU and SH test, the sister-group relationship between Pseudecheneis and the monophyletic glyptosternoids cannot be rejected based on the second intron of rpS7 gene and combined data analyses. It follows that both gene segments are not suitable for resolving the phylogenetic relationships within the sisorid catfishes. Overall, the second intron of rpS7 gene yielded poor phylogenetic performance when compared to 16S rRNA gene, the evolutionary hypothesis of which virtually agreed with the combined data analyses tree. This phenomenon can be explained by the insufficient length and fast saturation of substitutions in the second intron of rpS7 gene, due to substitution patterns such as frequent indels (insertion/deletion events) of bases in the sequences during the evolution.