Effects of adrenergic agonists on the extrahepatic expression of vitellogenin Ao1 in heart and brain of the Chinese rare minnow (Gobiocypris rarus)

Teleost vitellogenins (VTGs) are large multidomain apolipoproteins and traditionally considered as the estrogen responsive precursors of the major egg yolk proteins. We identified five clones encoding VTGs, about 16% of the random EST clones from our constructed cDNA library from Chinese rare minnow liver tissue treated with 17 beta-estradiol (E2). Full-length vtgAo1 has been obtained based on the sequence information of four partial cDNA inserts by RACE. The inducibility of the vtgAo1 expression in liver by E2 was confirmed by RT-PCR. The presence of vtgAo1 transcripts have been observed primarily in liver. However. a significant level of the vtgAo1 was found in an unexpected location, heart, particularly in atrial cells by RT-PCR and whole mount in situ hybridization analyses. The vtgAo1 mRNA expression in heart and liver tissue could be suppressed by both alpha-adrenergic agonist, phenylephrine (PE) and beta-adrenergic agonist, isoproterenol (ISO). The expression of VTG in the heart observed in the present studies suggested it may provide protection from surplus intracellular lipids in fish cardiomyocytes as triglyceride transport proteins do in mammals. The results also indicated that the production of teleost vtg in vivo can be regulated by riot only estrogenic agents, but adrenergic signals as well. (c) 2008 Elsevier B.V. All rights reserved.

Identification of differential transcript profiles between mutual crossbred embryos of zebrafish (Danio rerio) and Chinese rare minnow (Gobiocypris rarus) by cDNA-AFLP

The crosstalk between naive nucleus and maternal factors deposited in egg cytoplasm before zygotic genome activation is crucial for early development. In this study, we utilized two laboratory fishes, zebrafish (Danio rerio) and Chinese rare minnow and Chinese rare minnow (Gobiocypris rarus), to obtain mutual crossbred embroys and examine the interaction between nucleus and egg cytoplasm from different species. Although these two types of crossbred embryos originated from common nuclei, various developmental capacities were gained due to different origins of the egg cytoplasm. Using cDNA amplified fragment length polymorphism (cDNA-AFLP), We Compared transcript profiles between the mutual crossbred embryos at two developmental stages (50%- and 90%-epiholy). Three thousand cDNA fragments were generated in four cDNA pools with 64 primer combinations. All differently displayed transcript-derived fragments (TDFs) were screened by (lot blot hybridization, and the selected sequences were further analyzed by semi-quantitative RT-PCR and quantitative real-time RT-PCR. Compared with ZR embryos, 12 genes were up-regulated and 12 were down-regulated in RZ embryos. The gene fragments were sequenced and subjected to BLASTN analysis. The sequences encoded various proteins which functioned at various levels of proliferation, growth, and development. One gene (ZR6), dramatically down-regulated in RZ embryos, was chosen for loss-of-function study; the knockdown of ZR6 gave rise to the phenotype resembling that of RZ embryos. (c) 2008 Elsevier Inc. All rights reserved.

Identification of differentially expressed immune-relevant genes in Chinese soft-shelled turtle (Trionyx sinensis) infected with Aeromonas hydrophila

Expressed sequence tag (EST) analysis is an efficient tool for gene discovery and profiling gene expression. Aeromonas hydrophila, a ubiquitous waterborne bacterium, is one of the most frequent pathogens isolated from diseased aquatic organisms. In order to understand the molecular mechanism of anti-bacteria immune response in reptile, we have investigated the differentially expressed genes in Chinese soft-shelled turtle (Trionyx sinensis) experimentally infected with A. hydrophila by suppression subtractive hybridization (SSH). Forty-two genes were identified from more than 200 clones, of which 25 genes are found for the first time in reptiles, and classified into 6 categories: 18 in defense/immunity. 4 in catalysis, 2 in retrotransposon; 2 in cell signal transduction, 5 in cell metabolism, 10 in protein expression, and 1 in cell structure. Of the 42 differentially expressed genes, 6 genes, IL-8, serum amyloid A (SAA), CD9, CD59, activating transcription factor 4 (ATF4) and cathepsin L genes, were further observed to be up-regulated in the infected turtles by virtual Northern hybridization and RT-PCR assays. (C) 2008 Elsevier B.V. All rights reserved.

Avian influenza virus infection induces differential expression of genes in chicken kidney

The pathogenic process of highly pathogenic avian influenza virus (HPAIV) infection is poorly understood. To explore the differential expression of kidney genes as a result of HPAIV infection, two cDNA libraries were constructed from uninfected and infected kidneys by suppression subtractive hybridization (SSH). Fifteen genes including IFN-stimulated genes (ISG12), lymphocyte antigen 6 complex locus E gene (LY6E), matrix Gla protein gene (MGP), lysozyme gene, haemopoiesis related membrane protein I gene, KIAA1259, MGC68696, G6pe-prov protein gene (G6PC), MGC4504, alcohol dehydrogenase gene (ADH), glutathione S-transferase gene (GST), sodium-dependent high-affinity dicarboxylate transporter gene (SDCT), Synaptotagmin XV (SytXV) and two novel genes were found significantly up-regulated or dramatically suppressed. Differential expression of these genes was further identified by Northern blot. Functional analysis indicated that the regulation of their expression might contribute to the pathogenic process of HPAIV infection. In contrast, the increased expression of three IFN-stimulated genes named ISG12, LY6E, and haemopoiesis related membrane protein 1 gene might reflect host defense responses. Further study showed that ISG12 protein failed to directly interact with NS1 protein of HPAIV which expressed simultaneously in the organs where HPAIV replication occurred, by use of BacterioMatch two-hybrid system. Therefore, our findings may provide new insights into understanding the molecular mechanism underlying the pathophysiological process of HPAIV infection in chicken. (c) 2007 Elsevier Ltd. All rights reserved.

Structure, organization and expression of common carp (Cyprinus carpio L.) SLP-76 gene

SLP-76 is an important member of the SLP-76 family of adapters, and it plays a key role in TCR signaling and T cell function. Partial cDNA sequence of SLP-76 of common carp (Cyprinus carpio L.) was isolated from thymus cDNA Library by the method of suppression subtractive hybridization (SSH). Subsequently, the full length cDNA of carp SLP-76 was obtained by means of 3' RACE and 5' RACE, respectively. The full Length cDNA of carp SLP-76 was 2007 bp, consisting of a T-terminal untranslated region (UTR) of 285 bp, a T-terminal. UTR of 240 bp, and an open reading frame of 1482 bp. Sequence comparison showed that the deduced amino acid sequence of carp SLP-76 had an overall similarity of 34-73% to that of other species homotogues, and it was composed of an NH2-terminal domain, a central proline-rich domain, and a C-terminal SH2 domain. Amino acid sequence analysis indicated the existence of a Gads binding site R-X-X-K, a 10-aa-long sequence which binds to the SH3 domain of LCK in vitro, and three conserved tyrosine-containing sequence in the NH2-terminal domain. Then we used PCR to obtain a genomic DNA which covers the entire coding region of carp SLP-76. In the 9.2 k-long genomic sequence, twenty one exons and twenty introns were identified. RT-PCR results showed that carp SLP-76 was expressed predominantly in hematopoietic tissues, and was upregulated in thymus tissue of four-month carp compared to one-year old carp. RT-PCR and virtual northern hybridization results showed that carp SLP-76 was also upregulated in thymus tissue of GH transgenic carp at the age of four-months. These results suggest that the expression level of SLP-76 gene may be related to thymocyte development in teleosts. (c) 2007 Published by Elsevier Ltd.

Molecular cloning and characterization of common carp (Cyprinus carpio L.) TCR gamma and CD3 gamma/delta chains

Partial cDNA sequences of TCR gamma and CD3 gamma/delta were isolated from the thymus of common carp (Cyprinus carpio L.) by the method of suppression subtractive hybridization (SSH). Subsequently the full length cDNAs of carp TCR gamma and CD3 gamma/delta were obtained by means of 3' RACE and 5' RACE, respectively. The full length of carp TCR gamma chain is 1368 bp and encodes 326 amino acids including a signal peptide region of 19 amino acids and a transmembrane region of 23 amino acids at the C-terminal region from aa 291 to 313. The V region of carp TCR gamma contains 109 amino acids, the core motif FGXG in J segment was also found in carp TCR gamma. The C region of carp TCR gamma contains the characteristic CX6PX6WX45C motif. The CP region of carp TCR C gamma contains 37 amino acids. The full length of carp CD3 gamma/delta is 790 bp and encodes 175 amino acids including a signal peptide region of 17 amino acids and a transmembrane region of 23 amino acids from aa 93 to 115. Similar to other known CD3 gamma/delta s, four cysteine residues in the extracellular domain and an immunoreceptor tyrosine-based activation motif ITAM (YxxL/Ix6-8YxxL/I) in the intracellular domain are also included in carp CD3 gamma/delta. Differing from other known CD3 gamma/delta s, carp CD3 gamma/delta tacks the CXXCXE motif in the extracellular domain. RTPCR analysis demonstrated that the expression of TCR gamma gene was mainly in the thymus and gill of 6-month carp, but in 18-month carp, TCR gamma gene was detected in all the examined tissues. The expression of CD3 gamma/delta gene was detected in all examined tissues of 6 and 18-month carp; among them, the highest expression level was in the thymus of 6-month carp. In situ hybridization showed that CD3 gamma/delta-expressing cells were widely distributed in the head kidney, spleen and kidney of carp, whereas in the thymus, they were densely distributed in the lymphoid outer zone and scattered in the epithelioid inner zone. (c) 2007 Published by Etsevier Ltd.

Identification and characterization of a novel gene differentially expressed in zebrafish cross-subfamily cloned embryos

m Background: Cross-species nuclear transfer has been shown to be a potent approach to retain the genetic viability of a certain species near extinction. However, most embryos produced by cross-species nuclear transfer were compromised because that they were unable to develop to later stages. Gene expression analysis of cross-species cloned embryos will yield new insights into the regulatory mechanisms involved in cross-species nuclear transfer and embryonic development. Results: A novel gene, K31, was identified as an up-regulated gene in fish cross-subfamily cloned embryos using SSH approach and RACE method. K31 complete cDNA sequence is 1106 base pairs (bp) in length, with a 342 bp open reading frame (ORF) encoding a putative protein of 113 amino acids (aa). Comparative analysis revealed no homologous known gene in zebrafish and other species database. K31 protein contains a putative transmembrane helix and five putative phosphorylation sites but without a signal peptide. Expression pattern analysis by real time RT-PCR and whole-mount in situ hybridization (WISH) shows that it has the characteristics of constitutively expressed gene. Sub-cellular localization assay shows that K31 protein can not penetrate the nuclei. Interestingly, over-expression of K31 gene can cause lethality in the epithelioma papulosum cyprinid (EPC) cells in cell culture, which gave hint to the inefficient reprogramming events occurred in cloned embryos. Conclusion: Taken together, our findings indicated that K31 gene is a novel gene differentially expressed in fish cross-subfamily cloned embryos and over-expression of K31 gene can cause lethality of cultured fish cells. To our knowledge, this is the first report on the determination of novel genes involved in nucleo-cytoplasmic interaction of fish cross-subfamily cloned embryos.

Cloning and characterization of interferon stimulated genes Viperin and ISG15, and their promoters from snakehead Channa argus

By suppression subtractive hybridization, rapid amplification of cDNA ends and gene walking methods, interferon stimulated genes (ISGs), Viperin and ISG15, and their promoters have been cloned and characterized from snakehead Channa argus. The Viperin cDNA was found to be 1474 nt and contain an open reading frame (ORF) of 1059 nt that translates into a putative peptide of 352 amino acid (aa). The putative peptide of Viperin shows high identity to that in teleosts and mammals except for the N-terminal 70 aa. The ISG15 cDNA was found to be 758 nt and contain an ORF of 468 nt that translates into a putative peptide of 155 aa. The putative peptide of ISG15 is composed of two tandem repeats of ubiquitin-like (UBL) domains, and a canonical conjugation motif (LRGG) at C-terminal. Viperin and ISG15 promoter regions were characterized by the presence of interferon stimulating response elements (ISRE) and gamma-IFN activation sites (GAS). ISRE is a feature of IFN-induced gene promoter and partially overlaps interferon regulatory factor (IRF) 1 and IRF2 recognition sites. GAS is responsible for the gamma-IFN mediated transcription. One conserved site for NF-kappa B was found in the promoter region of Viperin. This is the first report of conservative binding motif for NF-kappa B in accordance with the consensus sequence (GGGRN-NYYCC) among teleost ISG promoters. Moreover, there were also TATA, CAAT and Sp1 transcription factor sites in Viperin and ISG15 promoters. In 5' untranslated region (UTR), snakehead ISG15 gene contains a single intron, which differs from Viperin gene. The transcripts of Vipeirn and ISG15 mRNA were mainly expressed in head kidney, posterior kidney, spleen and gill. The expression levels in liver were found to increase obviously in response to induction by IFN-inducer poly I : C.

Investigations into the perplexing interrelationship of the Genus Takifugu Abe, 1949 (Tetraodontiformes, Tetraodontidae)

The phylogenetic relationships within the genus Takifugu Abe, 1949 (Tetraodontiformes, Tetraodontidae) remain unresolved. Because of the use of Takifugu as model organisms, the resolution of these relationships is crucial for the interpretation of evolutionary trends in biology. Pufferfishes of this genus are comprised of a comparatively small number of species and are mainly distributed along the coastal region of the western part of the Sea of Japan and the coastline of China. Mitochondrial gene sequences were employed to test the phylogenetic hypotheses within the genus. Seventeen species of the genus were examined. Molecular phylogenetic trees were constructed using the maximum parsimony, neighbor-joining, maximum likelihood and Bayesian methods. Our hypothesis of internal relationships within the genus differs from previous hypotheses. Our results indicate that (1) the genus Takifugu is a monophyletic assemblage; (2) the genus is divided into 6 subgroups based on the molecular data; and (3) there is low genetic diversity among the species within this genus. In addition, speciation within Takifugu appears to be driven by hybridization and isolation by distribution. Our results also suggested that the taxonomy in the genus should be clarified based on both molecular and morphological data.

Molecular cloning and characterization of carp (Cyprinus carpio L.) CD8 beta and CD4-like genes

Partial cDNA sequences of both CD8 beta and CD4-like (CD4L) genes of common carp (Cyprinus carpio L.) were isolated from thymus cDNA library by the method of suppression subtractive hybridization (SSH). Subsequently the full length cDNAs of carp CD8 and CD4L were obtained by means of 3' RACE and 5' RACE, respectively. The full length cDNA of carp CD8 is 1164 bp and encodes 207 amino acids including a signal peptide region of 24 amino acids, a transmembrane region of 23 amino acids from aa 167 to aa189 and an immunoglobulin V-set from aa 19 to aa 141. Similar to other species CD8 beta s,carp CD8 beta also lacks p56(lck) domain in the cytoplasmic region. The full length cDNA of carp CD4L is 2001 bp and encodes 458 amino acids including four immunoglobulin (Ig)-like domains in the extracellular region, a transmembrane region of 23 amino acids at the C-terminal region from aa 402 to aa 424 and a cytoplasmic tail. Similar to mammalian, avian CD4s and fugu CD4L, carp CD4L also has the conserved p56(lck) tyrosine kinase motif (C-X-C) in the cytoplasmic region. RT-PCR analysis demonstrated that carp CD8 beta and CD4L genes were both expressed predominantly in thymus. The results from this study can be used to understand the evolution of both the CD8 beta and CD4 molecules which can be used as markers for cytotoxic and helper T cells in carp. (c) 2007 Published by Elsevier Ltd.

Identification of differentially expressed genes from the cross-subfamily cloned embryos derived from zebrafish nuclei and rare minnow enucleated eggs

Cross-species nuclear transfer (NT) has been used to retain the genetic viability of a species near extinction. However, unlike intra-species NT, most embryos produced by cross-species NT were unable to develop to later stages due to incompatible nucleocytoplasmic interactions between the donor nuclei and the recipient cytoplasm from different species. To study the early nucleocytoplasmic interaction in cross-species NT, two laboratory fish species (zebrafish and rare minnow) from different subfamilies were used to generate cross-subfamily NT embryos in the present study. Suppression subtractive hybridization (SSH) was performed to screen out differentially expressed genes from the forward and reverse subtractive cDNA libraries. After dot blot and real-time PCR analysis, 80 of 500 randomly selective sequences were proven to be differentially expressed in the cloned embryos. Among them, 45 sequences shared high homology with 28 zebrafish known genes, and 35 sequences were corresponding to 22 novel expressed sequence tags (ESTs). Based on functional clustering and literature mining analysis, up-and down-regulated genes in the cross-subfamily cloned embryos were mostly relevant to transcription and translation initiation, cell cycle regulation, protein binding, etc. To our knowledge, this is the first report on the determination of genes involved in the early development of cross-species NT embryos of fish. (C) 2007 Elsevier Inc. All rights reserved.

Knock down of gfp and no tail expression in zebrafish embryo by in vivo-transcribed short hairpin RNA with T7 plasmid system

A short-hairpin RNA (shRNA) expression system, based on T7 RNA polymerase (T7RP) directed transcription machinery, has been developed and used to generate a knock down effect in zebrafish embryos by targeting green fluorescent protein (gfp) and no tail (ntl) mRNA. The vector pCMVT7R harboring T7RP driven by CMV promoter was introduced into zebrafish embryos and the germline transmitted transgenic individuals were screened out for subsequent RNAi application. The shRNA transcription vectors pT7shRNA were constructed and validated by in vivo transcription assay. When pT7shGFP vector was injected into the transgenic embryos stably expressing T7RP, gfp relative expression level showed a decrease of 68% by analysis of fluorescence real time RT-PCR. As a control, injection of chemical synthesized siRNA resulted in expression level of 40% lower than the control when the injection dose was as high as 2 mu g/mu l. More importantly, injection of pT7shNTL vector in zebrafish embryos expressing T7RP led to partial absence of endogenous ntl transcripts in 30% of the injected embryos when detected by whole mount in situ hybridization. Herein, the T7 transcription system could be used to drive the expression of shRNA in zebrafish embryos and result in gene knock down effect, suggesting a potential role for its application in RNAi studies in zebrafish embryos.

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.

Identification of genome organization in the unusual allotetraploid form of Carassius auratus gibelio

The unusual allotetraploid form with unequal contribution of chromosome sets was discovered from the gynogenetic offspring of Carassius auratus gibelio stimulated by red common carp sperm. In this study, genomic in situ hybridization (GISH) and fluorescent in situ hybridization (FISH) with 45S rDNA probe are used. The GISH results lead to the identification of species-specific chromosomes, which permits to demonstrate the origin and genome organization in the allotetraploid form. Moreover, chromosome localization of 45S rDNA and co-localizations of 45S rDNA and Cyprinus carpio genomic DNA further confirm that one extra 45S rDNA positive chromosome in the allotetraploid form originates from the paternal haploid genome of C carpio, and other 5 45S rDNA-containing chromosomes are from the maternal genome of Carassius auratus gibelio. And, the correlation between 45 rDNA and the nucleolar organizer regions (NORs) is confirmed by silver nitrate staining. The data provide direct experiment evidence that the allotetraploid actually contains three chromosome sets of Carassius auratus gibelio and one chromosome set of C carpio, and will be a useful genetic material for both basic research and breeding practice. (c) 2006 Elsevier B.V. All rights reserved.

Identification of novel SINEs from Cyprinidae and their evolutionary significance

Short interspersed nuclear elements (SINEs) are widespread among eukaryotic genomes. They are repetitive DNA sequences that have been amplified by retrotransposition. In this study, a class of SINEs were isolated from the Opsariichthys bidens genome, and named Opsar. Sequence analysis confirmed that Opsar is a new class of typical SINEs derived from tRNA molecules. With the tRNA-derived region of Opsar and through BLASTN search, we further identified Zb-SINEs from the zebrafish genome, which includes two groups: Zb-SINE-A and Zb-SINE-B. The Zb-SINE-A group comprises subfamilies of -Al--A5, and the Zb-SINE-B group is a dimer of the tRNA(Ala)-derived region and shares a similar dimeric composition to Alu. Zb-SINEs are composed of three distinct regions: a 5 end tRNA-derived region, a tRNA-unrelated region and a 3 end AT-rich region. The flanking regions are AT rich. The average length of Zb-SINEs elements is about 340 6p. Zb-SINEs account for as much as 0.1% of the whole zebrafish genome. About 70% of the Zb-SINEs are on chromosomes 11, 18, and 19. These Zb-SINEs were characterized by PCR and dot hybridization. The distribution pattern of Zb-SINEs in genome strongly supports the master genes model. The tRNA-derived regions of Opsar and Zb-SINEs were compared with the tRNA(Ala) gene, and they showed 76% similarity, indicating that Opsar and Zb-SINEs originated from an inactive tRNA(Ala) sequence or a tRNA(Ala)-like sequence. In view of the evolutionary status of zebrafish in the Cyprinidae, we deduced that Zb-SINEs were a very old class of interspersed sequences.