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 upregulated 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 I:C 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.     

Subcellular localization and functional characterization of a fish IRF9 from crucian carp Carassius auratus

Mammalian interferon (IFN) regulatory factor 9 (IRF-9) has long been recognized as the DNA sequence recognition subunit of IFN-stimulated gene factor 3 (ISGF3) complex, which is critical for type I IFN to induce the expression of IFN-stimulated genes (ISGs) against viral infection. Recent studies have shown that fish IFN exerts antiviral effects by induction of a number of ISGs and also of itself; however, little is known about the role of fish IRF9 in IFN signaling. Here we identify a fish IRF9 orthologue (CaIRF9) from IFN-producing cell line, crucian carp Carassius auratus blastulae embryonic (CAB) cells. Analysis of subcellular distribution of CaIRF9-green fluorescent protein indicates that CaIRF9 is constitutively present in the nucleus, which is driven by two nuclear localization signals (NLS), one locating within DNA-binding domain (DBD) of CaIRF9 and the other immediately behind DBD, although human IRF9 contains only one NLS analogous to the former of CaIRF9. Overexpression of CaIRF9 together with CaSTAT2 not only activates ISRE-containing promoter but also upregulates the expression of fish ISGs. Strikingly, CaIRF9 together with CaSTAT2 also exhibits an ability to activate crucian carp IFN promoter, and blockade of cellular CaIRF9 attenuates IFN itself-induced activation of crucian carp IFN promoter. Taken together, these data suggest that crucian carp IFN induces the expression of ISGs and also of itself possibly by the JAK-STAT signaling pathway that is conserved from fish to mammals.     

鲫Nub1基因的克隆及特征分析

Nub1(NEDD8 Ultimate Buster-1)是近年来发现的一种干扰素诱导表达基因,过量表达该基因能抑制细胞生长。研究通过RACE-PCR方法从产生干扰素的鲫囊胚培养细胞(Carassius auratus blastulae embryonic cells,CAB)中克隆出鲫Nub1基因。鲫Nub1全长cDNA为2298bp,编码一个由589个氨基酸残基组成的蛋白。推导的鲫NUB1蛋白具有哺乳类同源蛋白保守的结构域,包括N端的UBL结构域和C端两个UBA结构域,与已知的哺乳类包括人和小鼠、鸟类和两栖类的同源蛋白具有41%-45%的相似性。诱导表达分析显示PolyI:C和LPS均能诱导CAB细胞Nub1mRNA的上调,表明Nub1基因在鱼类的抗病免疫反应中发挥某种重要作用。       

银鲫与彩鲫卵母细胞cDNA文库构建及周期蛋白A1的cDNA克隆

分别取行天然雌核发育繁殖的银鲫和两性生殖的彩鲫的卵母细胞为材料,提取总RNA,分离mRNA,进而反转录合成cDNA并定向插入λgtll Sfi-Not克隆载体,经体外包装构建了银鲫与彩鲫卵母细胞的表达型cDNA文库。测试结果表明库容量分别达到3.1×106(银鲫)和1.6×106(彩鲫)。进一步人工合成Cyclin A1保守引物,采用PCR扩增文库的方法,克隆了银鲫(1616bp)与彩鲫(1626bp)的Cyclin A1全长cDNA。序列分析结果表明:两种鱼编码区长度均为1173bp,起始于一个包含在脊椎动物起始密码子ANNATG基元内ATG的单一开放读码框,编码391个氨基酸;5'-端非编码区长度也同为70bp,3-'端非编码区长度略有不同,银鲫为373bp,而彩鲫则为383bp;二者3'-端均带有AATAAA的Poly(A)加尾信号以及24bp(银鲫)和27bp(彩鲫)的Poly(A)尾巴。比较银鲫、彩鲫和金鱼与人、爪蟾Cyclin A1氨基酸序列同源性的结果表明,Cyclin A1在人、爪蟾与鱼类之间具有较高同源性;而在银鲫、彩鲫和金鱼之间,Cyclin A1仅在周期蛋白框外存在5个氨基酸的差异,且这些差异均是由个别碱基的变异造成的。       

Inhibition of interferon regulatory factor 7 (IRF7)-mediated interferon signal transduction by the Kaposi's sarcoma-associated herpesvirus viral IRF homolog vIRF3

Upon viral infection, the major defense mounted by the host immune system is activation of the interferon (IFN)-mediated antiviral pathway that is mediated by IFN regulatory factors (IRFs). In order to complete their life cycle, viruses must modulate the host IFN-mediated immune response. Kaposi's sarcoma-associated herpesvirus (KSHV), a human tumor-inducing herpesvirus, has developed a unique mechanism for antagonizing cellular IFN-mediated antiviral activity by incorporating viral homologs of the cellular IRFs, called vIRFs. Here, we report a novel immune evasion mechanism of KSHV vIRF3 to block cellular IRF7-mediated innate immunity in response to viral infection. KSHV vIRF3 specifically interacts with either the DNA binding domain or the central IRF association domain of IRF7, and this interaction leads to the inhibition of IRF7 DNA binding activity and, therefore, suppression of alpha interferon (IFN-alpha) production and IFN-mediated immunity. Remarkably, the central 40 amino acids of vIRF3, containing the double alpha helix motifs, are sufficient not only for binding to IRF7, but also for inhibiting IRF7 DNA binding activity. Consequently, the expression of the double alpha helix motif-containing peptide effectively suppresses IRF7-mediated IFN-alpha production. This demonstrates a remarkably efficient means of viral avoidance of host antiviral activity.     

斑马鱼一个IFIT 家族基因的鉴定及启动子分析

IFIT 家族由一类受干扰素诱导表达并具有TPR 结构域的蛋白组成, 但是在鱼类关于IFIT 基因的研究还很少。研究利用哺乳类IFIT 家族基因IFI56 的序列搜索斑马鱼基因组数据库鉴定出一个未知基因, 该基因具有哺乳类IFIT 家族保守的基因组结构, 编码蛋白具有保守的TPR 结构域, 暂命名为IFIT-A。RT-PCR 分析表明, Poly I:C 能够诱导IFIT-A 基因转录水平上调。与哺乳类IFIT 家族基因相似, 斑马鱼IFIT-A 启动子存在ISG 基因特有的典型ISRE 结构域。荧光素酶活性实验揭示Poly I:C 和重组IFN 蛋白能激活斑马鱼IFIT-A 启动子活性。此外, 过量表达IFN 调控因子IRF3 和IRF7 能诱导斑马鱼IFIT-A 启动子活性。实验结果证明IFIT-A基因是斑马鱼IFIT 家族成员, IRF3 和IRF7 在其诱导表中具有重要调控作用。       

Molecular cloning, characterization and expression analysis of QM gene from grass carp (Ctenopharyngodon idellus) homologous to Wilms' tumor suppressor

QM, a novel gene that was originally identified as a tumor suppressor, has been cloned from species encompassing members of higher vertebrate, plant and fungal kingdoms, but it is not well documented in fish. In present study, a gene homologous to QM was obtained from grass carp (Ctenopharyngodon idellus) head kidney and spleen cDNA library. The full-length grass carp QM (GcQM) cDNA of 759 bp contains a short 5' UTR of 22 bp, a 3' UTR of 89 bp and an open reading frame of 648 nucleotides that translates into a 215-amino acid peptide with a molecular weight of 24.5 kDa. The predicted GcQM contains a series of functional motifs that belong to the QM family signature conserved among different species. Multiple alignment analysis reveals that GcQM shares an overall identity of 62.4% approximately 97.7% with other members of QM family. The fish QM has a closest genetic relationship to chicken homologue Jif-1. The GcQM expresses constitutively in spleen, heart and brain, and significantly up-regulated by Aeromonas hydrophila and grass carp haemorrhagic virus (GCHV) in head kidney, spleen and liver. The results suggest that grass carp QM homolog is an inflammatory stress inducible gene associated with anti-bacterial and viral defense, and it plays an important role in immune defense.     

The interferon system and interferon regulatory factor transcription factors -- studies from gene knockout mice

Interferon regulatory factors (IRFs) were initially identified as regulators of IFN-alpha/beta genes and to date nine members have been determined in human and mouse. They share a conserved DNA-binding domain in the N-terminal portion that recognizes similar DNA sequences. Despite their similar DNA binding specificity, the IRFs show diverse functions in response to extra cellular stimuli. Although the study of IRFs was started with respect to regulation of the IFN-alpha/beta gene expression, recent studies have revealed other aspects of IRF functions. In this review, we summarize our current knowledge of the functions of IRF family members revealed by our gene targeting study in mice, focusing on the regulation of the IFN system.     

Differential screening and characterization analysis of the egg envelope glycoprotein ZP3 cDNAs between gynogenetic and gonochoristic crucian carp

INTRODUCTIONFertilization in animals is the trigger that ini-tiates development, and results in a series of well-choreographed interactions between molecules lo-cated on the surfaces of egg and sperm[1, 2]. Somecell surface proteins, such as zolla pellucida gly-coprotein ZPI, ZPZ and ZP3, acrosomal proteinbindin, acrosin and lysin, and sperm plasma mem-brane protein a- and p--fertilin, have been identi-fied to mediate the interaction process[2-8]. Al-though studies on the regulative fa…     

Evidence for recombination of mitochondrial DNA in triploid crucian carp

In this study, we report the complete mitochondrial DNA (mtDNA) sequences of the allotetraploid and triploid crucian carp and compare the complete mtDNA sequences between the triploid crucian carp and its female parent Japanese crucian carp and between the triploid crucian carp and its male parent allotetraploid. Our results indicate that the complete mtDNA nucleotide identity (98%) between the triploid crucian carp and its male parent allotetraploid was higher than that (93%) between the triploid crucian carp and its female parent Japanese crucian carp. Moreover, the presence of a pattern of identity and difference at synonymous sites of mitochondrial genomes between the triploid crucian carp and its parents provides direct evidence that triploid crucian carp possessed the recombination mtDNA fragment (12,759 bp) derived from the paternal fish. These results suggest that mtDNA recombination was derived from the fusion of the maternal and paternal mtDNAs. Compared with the haploid egg with one set of genome from the Japanese crucian carp, the diploid sperm with two sets of genomes from the allotetraploid could more easily make its mtDNA fuse with the mtDNA of the haploid egg. In addition, the triple hybrid nature of the triploid crucian carp probably allowed its better mtDNA recombination. In summary, our results provide the first evidence of mtDNA combination in polyploid fish.