Tissue- and stage-specific expression of a fatty acid binding protein-like gene from amphioxus Branchiostoma belcheri

A cDNA clone encoding an amphioxus fatty acid binding protein-like (AmphiFABPL) protein was isolated from a gut cDNA library of Branchiostoma belcheri. It contained a 423 bp open reading frame corresponding to a deduced protein of 140 amino acids with a predicted molecular mass of approximately 15.9 kDa. Phylogenetic analysis showed that AmphiFABPL fell outside the vertebrate clade of fatty acid binding proteins (FABPs), being positioned at the base of the chordate lineage, and was almost equally homologous to various vertebrate FABPs, suggesting that it may be the archetype of vertebrate FABPs. Both northern blotting and in situ hybridization analyses demonstrated that AmphiFABPL was expressed in the hepatic caecum and hind-gut, and although at a much lower level, it was also present in the endostyle, ovary and testis. In addition, whole-mount in situ hybridization revealed that AmphiFABPL was initially expressed in the posterior two thirds of the primitive gut, including the mid-gut where the hepatic caecum will form later, in 2-day larvae. The expression pattern is closely similar to that of the L-FABP and I-FABP genes in vertebrates, supporting the hypothesis that the hepatic caecum in the amphioxus is homologous to the vertebrate liver.     

A trypsin homolog in amphioxus: expression, enzymatic activity and evolution

Trypsin has been documented in a variety of species including both vertebrates and invertebrates, but little is known about it in amphioxus, a model organism for insights into the origin and evolution of vertebrates. Here we identified a trypsin gene in Branchiostoma japonicum. The cDNA was 978 bp long with an ORF encoding a deduced protein of 272 amino acids. The deduced protein had an N-terminal signal peptide of 15 amino acids, a 16 activation peptide with the typical cleavage site Arg/Ile, a Tryp_SPc domain with the catalytic triad His⁷²-Asp¹¹⁸-Ser²¹⁵ and the S1 substrate binding residue Asp²⁰⁹, which are all characteristic of trypsinogens. The recombinant trypsin protein was able to hydrolyse the trypsin prototypic substrate BAEE, which was inhibited by the trypsin-specific inhibitor soybean trypsin inhibitor. Both northern blotting and tissue-section in situ hybridization demonstrated that trypsin gene was expressed in a tissue-specific manner, with most abundant levels in the hepatic caecum, mid-gut and ovary. And the whole mount in situ hybridization showed that it began to express in the middle third of the full-length primitive gut in 2-day larvae, where the hepatic caecum will form later during development. Phylogenetic analysis indicated that both amphioxus and ascidian trypsins are more closer to each other than to vertebrate trypsins, suggesting a continuous evolutionary divergence of vertebrate trypsins after split from protochordate/vertebrate common ancestor.     

Verification and tissue-specific expression of nifU-like gene from the amphioxus Branchiostoma belcheri

A nifU-like gene exhibiting similarity to nifU of nitrogen fixation gene cluster was identified for the first time from the gut cDNA library of amphioxus Branchiostoma belcheri. Both RT-PCR and Northern blotting as well as in situ hybridization histochemistry verified that the cDNA represents an amphioxus nifU-like gene rather than a microbial contaminant. The nifU-like gene encodes a protein of 164 amino acid residues including a highly conserved U-type motif (C-X26-C-X43-C), and shares 66-86% identity to NifU-like proteins from a variety of species including vertebrates, invertebrates and microbes. It is expressed in a tissue-specific manner in the digestive system including epipharyngeal groove, endostyle, hepatic caecum and hind-gut and in the gill, ovary and testis. Taken together, it is highly likely that NifU-like protein plays some tissue-dependent and critical role in amphioxus.     

Verification and tissue-specific expression of nifU-like gene from the amphioxus Branchiostoma belcheri.

A nifU-like gene exhibiting similarity to nifU of nitrogen fixation gene cluster was identified for the first time from the gut cDNA library of amphioxus Branchiostoma belcheri. Both RT-PCR and Northern blotting as well as in situ hybridization histochemistry verified that the cDNA represents an amphioxus nifU-like gene rather than a microbial contaminant. The nifU-like gene encodes a protein of 164 amino acid residues including a highly-conserved U-type motif (C-X(26)-C-X(43)-C), and shares 66-86% identity to NifU-like proteins from a variety of species including vertebrates, invertebrates and microbes. It is expressed in a tissue-specific manner in the digestive system including epipharyngeal groove, endostyle, hepatic caecum and hindgut and in the gill, ovary and testis. Taken together, it is highly likely that NifU-like protein plays some tissue-dependent and critical role in amphioxus.     

Presence and localization of antithrombin and its regulation after acute lipopolysaccharide exposure in amphioxus, with implications for the origin of vertebrate liver

Antithrombin (AT), which is mainly synthesized in the liver, is an acute-phase plasma protein in mammalian species. Here, we demonstrated that sheep anti-human AT antibody cross-reacted with the humoral fluids in amphioxus Branchiostoma belcheri tsingtauense as well as human serum. The concentration of AT in the humoral fluids in amphioxus decreased slightly at first and then increased after the acute challenge with lipopolysaccharide, while the level of total proteins remained unchanged. These suggest the presence of the same acute-phase response pattern in amphioxus, as observed in some mammalian species. Immunohistochemically, AT was localized in the hepatic diverticulum. It is clear that the hepatic diverticulum in amphioxus is homologous to the vertebrate liver with respect to AT synthesis. This lends support to the hypothesis originally suggested by Müller that the vertebrate liver evolved from the hepatic diverticulum of an amphioxus-like ancestor during early chordate evolution. This work was supported by the Natural Science Foundation of China (NSFC; 30470203) and the Ministry of Science and Technology (MOST) of China.     

Amphioxus allantoicase: molecular cloning, expression and enzymatic activity

Allantoicase, one of the purine metabolism enzymes, is progressively truncated during the chordate evolution, yet it is unknown when its activity became phylogenetically extinct. In this study, a cDNA encoding allantoicase was isolated from the gut cDNA library of amphioxus Branchiostoma belcheri tsingtauense. It is 2441 bp long, and contains an open reading frame encoding a protein of 392 amino acid residues. RT-PCR analysis showed that amphioxus allantoicase was strongly expressed in the hepatic caecum, and weakly expressed in other tissues including hind-gut, gill, muscle, notochord, testis and ovary. The parallel experiment was performed measuring the allantoicase activity in the same tissues revealed that its activity was high in the hepatic caecum, but low or undetectable in other tissues examined. These suggest that allantoicase remains in action in the primitive chordate amphioxus.     

Identification and tissue-specific expression of a promethin-like homolog in amphioxus <Emphasis Type="Italic">Branchiostoma belcheri</Emphasis>

Promethins have been shown to be present in the vertebrates examined so far, yet little is known to date about them in invertebrates. Here we isolated a cDNA encoding a promethin-like homolog from the gut cDNA library of the amphioxus Branchiostoma belcheri, a cephalochordate occupying a nodal position transient from invertebrates to vertebrates. It contained a 504 bp open reading frame corresponding to a protein of 167 amino acids. Primary structural examination showed that the deduced promethin-like homolog was a transmembrane protein with three potential transmembrane helices, resembling the vertebrate promethins. Phylogenetic analysis showed that B. belcheri promethin-like homolog was located at the base of the vertebrate counterparts, suggesting that it represents the archetype of vertebrate promethins. Both Northern blotting and in situ hybridization histochemistry revealed a tissue-specific expression pattern of promethin-like gene, like that of mammalian promethins. This is the first report on invertebrate promethin-like homolog, paving the way for further insights into the evolution and function of promethins.     

Characterization, expression and localization of S-adenosylhomocysteine hydrolase from amphioxus Branchiostoma belcheri tsingtaunese

A cDNA clone encoding AmphiSAHH [amphioxus SAHH (S-adenosylhomocysteine hydrolase)] protein was isolated from a cDNA library from the gut of Branchiostoma belcheri tsingtaunese. It contained a 1305 bp open reading frame corresponding to a deduced protein of 434 amino acid residues, with a predicted molecular mass of approx. 47.8 kDa. Phylogenetic analysis showed that AmphiSAHH and sea-urchin SAHH joined together and positioned at the base of the vertebrate SAHH clade, suggesting that both AmphiSAHH and sea-urchin SAHH might share some characteristics of the archetype of vertebrate SAHH proteins. The genomic DNA sequence of AmphiSAHH contained eight exons and seven introns, which was similar to B. floridae and sea-urchin SAHH exon/intron organization. Sequence comparison suggested the evolutionary appearance of the ten exon/nine intron organization of SAHH genes after the split of invertebrates and vertebrates, after which it has been highly conserved. AmphiSAHH has been successfully expressed in Escherichia coli and purified. Western blotting confirmed that the enzyme has a native molecular mass of approx. 48 kDa, and the catalytic activities and NAD(+)/NADH binding affinity of recombinant AmphiSAHH were measured. Immunohistochemistry analysis showed that SAHH was strongly expressed in hepatic caecum, gill, spermary and ovary of amphioxus.     

青岛文昌鱼S-腺苷高半胱氨酸水解酶基因的组织特异性表达

为了探索文昌鱼S-腺苷高半胱氨酸水解酶AdoHcyase基因在文昌鱼组织中的表达分布情况,利用组织原位杂交技术,以地高辛标记的反义RNA为探针,检测了文昌鱼AdoHcyase基因在组织中的表达分布特点.结果表明,AdoHcyase基因在雌性文昌鱼的卵巢、肝盲囊和后肠杂交信号十分强烈,在内柱、鳃等组织中也有微弱信号表达,...     

文昌鱼sfy1基因的克隆及其在早期发育中的表达

文昌鱼是公认现存最接近于脊椎动物的一种头索动物,具有与脊椎动物相似但简单得多的身体图式[1],因而是研究脊椎动物发育机制起源和进化的宝贵材料,也是发育生物学的经典实验模型之一.近年来,人们在对果蝇和脊椎动物发育分子机制的研究取得了一系列重大突破之后,利用发育调控基     

Identification and expression of a novel class of glutathione-S-transferase from amphioxus Branchiostoma belcheri with implications to the origin of vertebrate liver

Glutathione-S-transferases have been identified in all the living species examined so far, yet little is known to date about them in amphioxus, a model organism for insights into the origin and evolution of vertebrates. We have isolated a cDNA encoding an amphioxus (Branchiostoma belcheri) glutathione-S-transferase with a predicted molecular mass of approximately 26 kDa, from the gut cDNA library. The glutathione-S-transferase had 43.7-51.8% identity to most glutathione-S-transferases identified from aquatic organisms including fish and green alga, but it was much less identical (<27%) to other cytosolic glutathione-S-transferase classes. The phylogenetic analysis revealed that the glutathione-S-transferase was grouped together with most piscine and algal glutathione-S-transferases, separating from other cytosolic glutathione-S-transferase classes. Moreover, the glutathione-S-transferase had an exon-intron organization typical of zebrafish putative GST, red sea bream GSTR1 and plaice GSTA1 genes. The recombinant glutathione-S-transferase has been successfully expressed and purified, which showed a relatively high catalytic activity (3.37+/-0.1 unit/mg) toward 1-chloro-2, 4-dinitrobenzene and a moderate activity toward ethacrynic acid (0.41+/-0.01 unit/mg), although it had no detectable activity toward 1, 2-dichloro-4-nitrobenzene, 4-hydroxynonenal, 4-nitrobenzyl chloride and cumene hydroperoxide. In addition, we have revealed a tissue-specific expression pattern of the glutathione-S-transferase gene in B. belcheri, with the most abundant expression in the hepatic caecum. All these indicate that the amphioxus glutathione-S-transferase belongs to a novel rho-class of glutathione-S-transferases with a tissue-specific expression pattern. The relation between the glutathione-S-transferase expression in amphioxus hepatic caecum and the origin of vertebrate liver is also discussed.     

Characterization and expression profile of AmphiCD63 encoding a novel member of TM4SF proteins from amphioxus Branchiostoma belcheri tsingtauense.

The study on CD antigen genes remains lacking in the cephalochordate amphioxus to date. In this report, the cDNA encoding CD63 was identified for the first time from the gut cDNA library of amphioxus Branchiostoma belcheri tsingtauense. Primary structural examination showed that the protein encoded by the cDNA contained four potential transmembrane domains characteristic of transmembrane 4 superfamily (TM4SF) proteins and a conserved CCG motif in the putative major extracellular loop. BLAST search revealed that the cDNA is closely associated with other known CD63 antigen genes, and it was thus designated AmphiCD63. Phylogenetic analysis indicated that AmphiCD63 is extremely close to vertebrate CD63, CD151 and CD53, suggesting they may have been evolved from a common ancestral gene. RT-PCR analysis exhibited that AmphiCD63 mRNA was abundant in muscle, ovary, foregut including hepatic caecum and hindgut, while it was present at considerably lower levels in notochord and gill and absent in testis.     

Expression and regulation by thyroid hormone (TH) of zebrafish IGF-I gene and amphioxus IGFl gene with implication of the origin of TH/IGF signaling pathway

Thyroid hormone (TH)/insulin-like growth factor (IGF) signaling pathway has been identified in all the vertebrates, but its evolutionary origin remains elusive. In this study we examined the expression profiles in vitro as well as in vivo of the IGF-I gene of fish Danio rerio (vertebrate) and the IGF-like gene (IGFl) of amphioxus Branchiostoma japonicum (protochordate) following T₃ treatment. Our results showed that T₃ was able to enhance hepatic IGF-I/IGFl gene expression in vitro in both zebrafish and amphioxus in a dose-dependent manner. This T₃-induced hepatic expression of IGF-I/IGFl genes in both species was significantly inhibited by the T₃-specific inhibitor DEA, indicating the specificity of IGF-I/IGFl gene regulation by T₃. At 100 nM T₃, in both the long (42 h) and short (8 h) time course experiments, the IGF-I/IGFl gene expression profiles following T₃ treatment in the tissue cultures of both species exhibited closely similar pattern and trend. Moreover, exposure of zebrafish and amphioxus to T₃in vivo for 72 h induced a significant increase in the expression of IGF-I/IGFl genes in both the liver and the hepatic caecum. These data together suggest that amphioxus and zebrafish both share a similar regulatory mechanism of IGF gene expression in response to T₃, providing an evidence for the presence of a vertebrate-like TH/IGF signaling pathway in the protochordate amphioxus.     

Identification and expression of amphioxus beta-microseminoprotein (MSP)-like gene encoding an ancient and rapidly evolving protein in chordates

The cDNA encoding beta-microseminoprotein-like (beta-MSPL) was identified from the gut cDNA library of amphioxus. It contains a 336 bp open reading frame corresponding to a deduced protein of 111 amino acids and has eight cysteines conserved and located at the same positions as those in the vertebrate beta-MSPs. At amino acid level, it shares 12-20% similarity to the vertebrate beta-MSPs, and seems lacking the signal peptide at the N-terminus. This not only confirms that beta-MSP is a rapidly evolving protein during phylogeny, but also provides further data on the degree of diversity between species of this protein. RT-PCR and Northern blotting show that amphioxus beta-MSPL is expressed in all tissues examined, suggesting that beta-MSPL plays a fundamental role. However, in situ hybridization reveals that positive hybridization signals were present in all blastomeres of the embryos from 4-cell to gastrula stages, while its expression is restricted exclusively to notochord, somites and primitive gut in neurulae and larvae, and disappears in the ectoderm including the neural tube differentiated from the ectoderm. This suggests that beta-MSPL is possibly involved in the differentiation of ectoderm during embryonic development of cephalochordate amphioxus though it is ubiquitously expressed in embryos prior to gastrula stage and in the adult animal.     

Characterization, evolution and tissue-specific expression of AmphiCalbin, a novel gene encoding EF-hand calcium-binding protein in amphioxus Branchiostoma belcheri

An amphioxus full-length cDNA, AmphiCalbin, encoding a novel EF-hand calcium-binding protein （EFCaBP）, was isolated from the gut cDNA library of amphioxus Branchiostoma belcheri. It consists of 1321 bp with a 636 bp open reading frame encoding a protein of 211 amino acids with a molecular mass of approximately 24.5 kDa. The phylogenetic analysis offers two interesting inferences. First, AmphiCalbin clusters with a group of unnamed EFCaBPs that are differentiated from other identified EFCaBPs. Second, AmphiCalbin falls at the base of the vertebrate unnamed EFCaBPs clade, probably representing their prototype. This is also corroborated by the fact that AmphiCalbin has an exon-intron organization identical to that of vertebrate unnamed EFCaBP genes. Both tissue-section in situ hybridization and whole-mount in situ hybridization prove a tissue-specific expression pattern of AmphiCalbin, with high levels of expression in the digestive system and gonads. It is proposed that AmphiCalbin might play a role in the digestive system and gonads. These observations lay the foundation for further understanding of the function of the unnamed EFCaBPs.     

ycaCR基因在文昌鱼早期胚胎表达研究

文昌鱼作为现存的与脊椎动物最接近的无脊椎动物,一直被作为研究生物进化和胚胎发育的典型材料.利用整体原位杂交方法对从文昌鱼肠cDNA文库克隆到的ycaCR基因进行基因的胚胎表达模式研究,结果显示该基因在早期胚胎发育阶段没有表达,在2天幼虫的原始消化道表达,暗示ycaCR基因可能在原始消化道内发挥生物学作用.