Cloning of two different 5' untranslated exons of bovine acidic fibroblast growth factor by the single strand ligation to single-stranded cDNA methodology.

In an attempt to characterize the 5' UTR of the aFGF mRNAs we used the new anchored PCR methodology, single strand ligation to ss-cDNAs (SLIC). In bovine brain and retina, two kinds of aFGF cDNA clones were isolated. They contained two alternative exons located 34 bp upstream to the translation initiation codon ATG. Taking into account the number of clones specific for each exon, the two mRNAs are expressed with the same ratio in both tissues. One of these bovine 5' UTR exons (136 bp) showed 81% identity to a human 5' UTR exon, the second one (323 bp) was 70% identical to the second human 5' UTR exon with a central region of 90 nucleotides showing 41% identity. The conservation of the splicing positions for these 5' UTR alternate exons in both bovine and human species, suggests that the overall structure of the aFGF gene is conserved in mammals. Furthermore, the conservation of the nucleotide sequences and of the localization of these 5' UTR exons suggests that these non-coding regions may be involved in the control of aFGF gene expression.     

Cloning and analysis of non-specific cytotoxic cell receptor (NCCRP)-1 from common carp Cyprinus carpio L

Nonspecific cytotoxic cell receptor protein (NCCRP-1) provides an important function in target cell recognition and activation of cytotoxicity. NCCRP-1 has been cloned from common carp Cyprinus carpio L. from fish barbel by EST analysis. The isolated gene is composed of 945 bp with a 79 bp 5' UTR, 714 bp open reading frame and 152 bp 3' UTR. The predicted NCCRP-1 gene is composed of 237 amino acid residues and its predicted signal peptide is 19 amino acid residues in length. This gene has conservation of all the related domains characteristic to the NCCRP-1 gene in fish. Phylogenetic and genomic analyses showed that carp NCCRP-1 was similar to other fish orthologues. The expression of NCCRP-1 gene was constitutive in both lymphoid and non-lymphoid tissues. Furthermore, by semi-quantitative RT-PCR studies, we showed that NCCRP-1 gene expression is increased in anterior kidney challenged with Aeromonas hydrophila.     

草鱼CD81基因的克隆及功能分析

为研究白细胞表面分化抗原81(CD81)的功能, 对草鱼CD81进行了克隆, CD81全长共1376 bp, 其中5'非翻译区87 bp, 3'非翻译区581 bp, 开放阅读框为708 bp, 包括8个外显子, 7个内含子, 编码235个氨基酸。实验采用实时荧光定量PCR的方法检测了CD81在健康草鱼不同组织中的表达情况及草鱼出血病病毒(GCRV)攻毒前后的表达变化情况。结果显示草鱼CD81在所有被检测组织中均有表达, 在头肾中表达量最高。在GCRV攻毒前后草鱼鳃、脾、肝、肠及头肾5个组织中的CD81表达量均有明显变化。同时, 采用绿色荧光蛋白(GFP)来示踪CD81的亚细胞表达部位, 激光共聚焦显微镜显示, 同人类一样, 草鱼CD81定位于细胞膜上。       

Molecular cDNA cloning and analysis of the organization and expression of the IL-1beta gene in the Nile tilapia, Oreochromis niloticus

The full-length cDNA sequence of interleukin-1beta (IL-1beta) from the Nile tilapia, Oreochromis niloticus, was determined by using PCR with primers designed from known fish IL-1beta sequences followed by elongation of the 5' and 3' ends using Rapid Amplification of cDNA Ends (RACE). The cDNA contains a 92-bp 5' untranslated region (UTR), a single open reading frame (ORF) of 732 bp that translates into a 243-amino acid molecule, a 341-bp 3' UTR with four cytokine RNA instability motifs (ATTTA), and a polyadenylation signal (AATAAA) at 15 nucleotides upstream of the poly(A) tail. The organization of the genomic IL-1beta based on the cDNA sequence appeared to be 4 introns and 5 exons. In comparison with known IL-1beta amino acid sequences, including human, catshark, trout, turbot, carp, sea bream, sea bass and goldfish, the amino acid sequence deduced from the cDNA sequence of Nile tilapia showed different levels of identity ranging from 25.32% to 66.80% and homology ranging from 41.88% to 82.19%. Although the entire cDNA sequence of Nile tilapia IL-1beta showed from 49.45% to 67.05% identity to those of other reported IL-1beta cDNAs, each exon also showed different levels of identity to the corresponding exons of other reported IL-1beta cDNAs. The highest nucleotide sequence identity for exon 1 and exons 2-5 of Nile tilapia IL-1beta was found in the corresponding exons of sea bream and sea bass, respectively. After in vitro stimulation with lipopolysaccharide (LPS), we found an increased level of IL-1beta expression in head kidney cells compared to that of unstimulated cells. However, this difference was no longer apparent after 4 h of stimulation, at which time the levels were similar in stimulated and unstimulated cells. Head kidney cells stimulated in vivo by an intraperitoneal injection of LPS showed a peak level of IL-1beta expression after 1 day and a decreased level after 3 days. At 7 days after stimulation, we were hardly able to detect IL-1beta expression.     

Cloning, characterization and expression analysis of two Tetraodon nigroviridis interleukin-16 isoform genes

Interleukin-16 (IL-16) is an important pro-inflammatory cytokine that functions as a chemoattractant factor and is well characterized in human and other mammals, but is largely unknown in fish. In the present study, two isoforms of pro-IL-16 homologues were cloned and characterized from pufferfish Tetraodon nigroviridis. The full-length T. nigroviridis pro-IL-16 isoform 1 cDNA exhibits 2453 bp in size including 291 bp 5'UTR (untranslated region), 1704 bp ORF (open reading frame) and 458 bp 3'UTR, while pro-IL-16 isoform 2 cDNA exhibits a 3801 bp ORF and a 458 bp 3'UTR. Bioinformatics analysis demonstrated that the pro-IL-16 isoform 1 with a predicted mass of 60.6 kDa contained two PDZ (postsynaptic density/disc large/zona occludens-1) domains, whereas the 138.2 kDa pro-IL-16 isoform 2 had two additional PDZ domains in its N-terminal extension. RT-PCR results revealed that ,almost in all examined organs and tissues, the mRNA of both pro-IL-16 isoforms can be detected, except in intestine and gill, where the isoform 2 mRNA is absent. The two putative precursor proteins showed 30.0-33.0% identity to various mammalian and avian homologues. This is the first report of such genes in teleostean fish and we hope the molecular characterization of these two pro-IL-16 isoforms will provide insights into the study of both evolution of IL-16 precursor proteins and the immune system as a whole.     

Interleukin-1beta isolated from a marine fish reveals up-regulated expression in macrophages following activation with lipopolysaccharide and lymphokines.

The gilthead seabream IL-1beta gene consists of five exons/four introns. The complete coding sequence contains a 102 bp 5' untranslated region (UTR), a single open reading frame of 762 bp which translates into a 253 amino acid molecule, and a 407 bp 3'UTR with a polyadenylation signal 14 nucleotides upstream of the poly(A)tail. The seabream sequence has the highest degree of nucleotide (61.7%) and amino acid (53%) identity with the trout IL-1beta sequences. The IL-1beta message was detected by RT-PCR in head-kidney, blood, spleen, liver, gill and peritoneal exudate of both non-infected and Vibrio anguillarum-challenged fish. More importantly, IL-1beta was highly expressed by purified macrophage monolayers and was up-regulated by lipopolysaccharide and lymphocyte-derived macrophage-activating factor stimulation.     

Molecular cloning of interleukin 1beta from rainbow trout Oncorhynchus mykiss reveals no evidence of an ice cut site.

The complete coding sequence of rainbow trout IL-1beta has been obtained. The gene contains a short 5' UTR (97 bp), a 780 bp open reading frame and a 466 bp 3' UTR, which includes a polyadenylation signal, 7 ATTTA motifs and an 18 bp poly A tail. The predicted amino acid sequence (260 amino acids) contains 3 potential glycosylation sites, with a predicted molecular weight of 29 kDa, and shows between 49 and 56% amino acid similarity to mammalian IL-1betas and 57% similarity to carp IL-1beta. Greatest homology was apparent within the secondary structure of the gene, with few of the amino acids known to bind to the IL-1 receptor being conserved. No ICE cut site was apparent but multiple alignment with mammalian sequences allowed a putative mature peptide of 166 amino acids to be identified, in which Ala(95)would be the amino terminus. Northern blot analysis showed that whilst no IL-1beta expression was detectable in head kidney leukocytes immediately after isolation, expression could be induced by stimulation with LPS for 4 h in culture. Similarly, with isolated head kidney macrophages expression was significantly increased following stimulation with LPS.     

Subfunctionalization of expression and peptide domains following the ancient duplication of the proopiomelanocortin gene in teleost fishes

The proopiomelanocortin gene (POMC) encodes several bioactive peptides, including adrenocorticotropin hormone, alpha-, beta-, and gamma-melanocyte-stimulating hormone, and the opioid peptide beta-endorphin, which play key roles in vertebrate physiology. In the human, mouse, and chicken genomes, there is only one POMC gene. By searching public genome projects, we have found that Tetraodon (Tetraodon nigroviridis), Fugu (Takifugu rubripes), and zebrafish (Danio rerio) possess two POMC genes, which we called POMCalpha and POMCbeta, and we present phylogenetic and mapping evidence that these paralogue genes originated in the whole-genome duplication specific to the teleost lineage over 300 MYA. In addition, we present evidence for two types of subfunction partitioning between the paralogues. First, in situ hybridization experiments indicate that the expression domains of the ancestral POMC gene have been subfunctionalized in Tetraodon, with POMCalpha expressed in the nucleus lateralis tuberis of the hypothalamus, as well as in the rostral pars distalis and pars intermedia (PI) of the pituitary, whereas POMCbeta is expressed in the preoptic area of the brain and weakly in the pituitary PI. Second, POMCbeta genes have a beta-endorphin segment that lacks the consensus opioid signal and seems to be under neutral evolution in tetraodontids, whereas POMCalpha genes possess well-conserved peptide regions. Thus, POMC paralogues have experienced subfunctionalization of both expression and peptide domains during teleost evolution. The study of regulatory regions of fish POMC genes might shed light on the mechanisms of enhancer partitioning between duplicate genes, as well as the roles of POMC-derived peptides in fish physiology.     

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.