Presence of prophenoloxidase in the humoral fluid of amphioxus Branchiostoma belcheri tsingtauense

The presence of phenoloxidase (PO) activity in the humoral fluid of amphioxus Branchiostoma belcheri tsingtauense was electrophoretically and spectrophotometrically studied. The enzyme was present in the humoral fluid predominantly as an inactive proenzyme, prophenoloxidase (proPO). The optimum temperature for activation of the proPO ranged from 30 degrees C to 35 degrees C, and the enzyme exhibited optimum activity at pH between 7.0 and 7.5. ProPO in the humoral fluid was readily activated to active form PO by exogenous elicitors such as trypsin, zymosan and LPS. The activation of the proPO by exogenous elicitors was significantly enhanced in the presence of 10 mM Ca2+, but was susceptible to serine protease inhibitors like soybean trypsin inhibitor and p-nitrophenyl-p'-guanidinobenzoate. PAGE revealed a single band of PO activity in the humoral fluid with an apparent molecular mass of 150 kDa, which was resolved to three bands with molecular masses of 44, 46 and 72 kDa, respectively, after SDS-PAGE. This is the first report on the presence of the enzyme PO in amphioxus humoral fluid.     

Purification and characterisation of phenoloxidase from amphioxus Branchiostoma belcheri tsingtauense

Phenoloxidase (PO) from the humoral fluid of amphioxus B. belcheri tsingtauense was purified using a sequential combination of ammonium sulphate precipitation, Sephadex G-200 chromatography and DEAE Sepharose Fast Flow chromatography. In PAGE, the purified enzyme exhibited a single band of 150 kDa under non-reducing conditions, and was resolved to three bands with molecular masses of 72, 46 and 44 kDa, respectively, under reducing conditions, suggesting that the PO in amphioxus humoral fluid seems to be a heterotrimer of three polypeptides held together by disulphide bonds. The substrate specificity and inhibition characteristics both indicate that the PO isolated from amphioxus humoral fluid is a tyrosinase-type enzyme. In addition, mouse antisera against the purified PO were prepared, and their specificity was confirmed by Western blotting, facilitating the future determination of the origin of PO in the humoral fluid and the distribution of PO-synthesising tissues in amphioxus.     

雌雄文昌鱼同工酶的表型差异

本文应用聚丙烯酰胺凝胶电泳结合生化染色方法分析了雌雄文昌鱼中苹果酸酶、苹果酸脱氢酶、酸性磷酸酶和酯酶四种同工酶的酶谱。首次发现苹果酸酶、苹果酸脱氢酶和酸性磷酸酶表型在文昌鱼雌性和雄性个体之间存在差异 ,而在同一性别不同个体之间无差异。酯酶表型较复杂 ,不但在不同性别个体之间而且在同一性别不同个体之间都出现一定差异     

Electron microscopic study of the development of amphioxus,Branchiostoma belcheri tsingtauense: The gastrula

The gastrulae of amphioxus were investigated by means of scanning and transmission electron microscopy (SEM and TEM) during 7 arbitrary stages that were seen about 4 to 10 hr after fertilization. Throughout gastrulation, SEM revealed subtle differences in cells of the blastoporal lip. In fractured specimens at early and middle stages, two opposing zones different in shape, size, and connection of the component cells were found: one which consists of columnar smaller cells in close contact in animal region and the other which is composed of round or polygonal larger cells in looser association in vegetal region. The polar body was found unexpectedly on the concave vegetal surface of the early gastrula in about 25% of cases. This might be the result of migration of the polar body. A short cilium that later elongated was recognized on each cell at mid-gastrula stage. The cilia on the dorsal surface (the neural ectoderm) of the final-stage gastrula became shorter than those on the epidermal ectoderm. TEM of thin sections demonstrated that the cytoplasmic components of gastrula cells are essentially the same as those of cleavage cells. But, the homogeneous nucleus seen during cleavage changed into a heterogeneous structure in which a nucleolus and dense particles were seen. Until the late stage, regional characteristics of the gastrulae indicating definitively the anterior-posterior and dorso-ventral polarity were not detected in the present SEM and TEM study.     

An electron microscopic study of the development of amphioxus,Branchiostoma belcheri tsingtauense: Cleavage

Development of the Asian amphioxus, Branchiostoma belcheri tsingtauense, was investigated by scanning and transmission electron microscopy (SEM and TEM) from the fertilized egg through the blastula stage. The fertilized egg is spherical (mean diameter 115 μm after SEM preparation) and is covered with microvilli. Throughout cleavage, the second polar body remains attached to the animal pole. The cleavage type in this species is essentially radial, as revealed by SEM observations. At the third cleavage or 8-cell stage, and at later stages, a size difference between blastomeres in the animal and the vegetal halves is clearly discernible, but less marked than that reported for the European amphioxus, B. lanceolatum. During the period spanning the third to the fifth cleavage (8–32-cell) stages, blastomeres are arranged in tiers along the animal-vegetal axis. After the sixth cleavage, or 64-cell stage, the tiered arrangement of the blastomeres is no longer seen. At the 4-cell stage, the blastocoel or cleavage cavity is seen as an intercellular space, opening to the outside. The blastocoel remains open at the animal and the vegetal poles in later stages. Throughout early development, the cytoplasm of the blastomeres includes yolk granules, mitochondria, Golgi complexes, and rough and smooth endoplasmic reticulum. Chromatin in the interphase nucleus is not clearly demonstrated, and chromosomes in the mitotic phase are also extremely difficult to detect. As yet, regional differences have not been found in distribution and organization of cytoplasmic components with respect to prospective ectodermal, mesodermal, and endodermal areas in the fertilized egg and later cleaved embryos, although there are possibly fewer yolk granules in the region of the animal pole than in the vegetal polar zone.     

Concerted action between Ca2+ and hyperosmolality initiates sperm motility in amphioxus Branchiostoma belcheri tsingtauense

This study investigated the effects of different environmental conditions on the initiation and maintenance of sperm motility in amphioxus Branchiostoma belcheri tsingtauense. The findings were that: (1) hyperosmolality in the absence of Ca(2+) ions did not initiate amphioxus sperm motility; (2) addition of Ca(2+) into EGTA-containing Ca(2+)-free artificial sea water (ASW), in which no sperm were motile, restored sperm motility; (3) Ca(2+) failed to induce sperm motility under conditions of hypoosmolality; (4) K(+) channel blockers quinine and 4-aminopiridine did not suppress the initiation of sperm motility; and (5) changes in pH did not cause sperm motility in a solution isotonic to seawater without Ca(2+). In conclusion, we inferred that a concerted action between Ca(2+) and hyperosmolality was essential to initiate motility of amphioxus sperm, whereas K(+) and pH were indispensable to maintain motility.     

Expression of Bblhx3, a LIM-homeobox gene,in the development of amphioxus Branchiostoma belcheri tsingtauense

Amphioxus Bblhx3 was identified as a LIM-homeobox gene expressed in gastrulae. Structural analysis suggested that it is a member of lhx3 but not of lhx1 gene group. Whole mount in situ hybridization revealed that expression of Bblhx3 was initiated at the early gastrula stage and continued at least until 10-day larvae. Expression of Bblhx3 first appeared in the vegetal and future dorsal area in initial gastrulae and became restricted to the endoderm during gastrulation. In neurulae and early larvae, Bblhx3 was expressed in the developing neural tube, the notochord and preoral pit lineage. In 10-day larvae, Bblhx3 was expressed only in the preoral pit. This expression pattern is apparently distinct from that of vertebrate lhx3 genes that are not expressed during gastrulation.     

Ribosomal Protein Genes S23 and L35 from Amphioxus Branchiostoma belcheri tsingtauense： Identification and Copy Number

The complete cDNA and deduced amino acid sequences of the ribosomal proteins S23 （AmphiS23） and L35 （AmphiL35） from amphioxus Branchiostoma belcheri tsingtauense were identified in this study. AmphiS23 cDNA is 546 bp long and encodes a protein of 143 amino acids. It has a predicted molecular mass of 15,851 Da and a pI of 10.7. AmphiL35 cDNA comprises 473 bp, and codes for a protein of 123 amino acids with a predicted molecular mass of 14,543 Da and a pI of 10.8. AmphiS23 shares more than 83% identity with its homologues in the vertebrates and more than 84% identity with those in the invertebrates. AmphiL35 is more than 63% identical to its counterparts in the vertebrates and more than 52% identical to those in the invertebrates. Southern blot analysis demonstrated the existence of 1-2 copies of the S23 gene and 2-3 copies of the L35 gene in the genome of amphioxus B. belcheri tsingtauense. This is in sharp contrast to the presence of 6-13 copies of the S23 gene and 15-17 copies of the L35 gene in therat genome. It is clear that the housekeeping genes like S23 and L35 underwent a large-scale duplication in the vertebrate lineage, reinforcing the gene/genome duplication hypothesis.     

Ribosomal proteins L34 and S29 of amphioxus Branchiostoma belcheri tsingtauense: cDNAs cloning and gene copy number

The complete cDNA and deduced amino-acid sequences of ribosomal proteins L34 (AmphiL34) and S29 (AmphiS29) from the amphioxus Branchiostoma belcheri tsingtauense were identified in this study. The AmphiL34 cDNA is 435 nucleotides in length and encodes a 118 amino-acid protein with calculated molecular mass of 13.6 kDa. It shares 53.6-67.5% amino-acid sequence identity with its eukaryotic counterparts including human, mouse, rat, pig, frog, catfish, fruit fly, mosquito, armyworm, nematode and yeast. The AmphiS29 cDNA comprises 453 nucleotides and codes for a 56 amino-acid protein with a calculated molecular mass of 6.6 kDa. It shows 66.1-78.6% amino-acid sequence identity to eukaryotic S29 proteins from human, mouse, rat, pig, zebrafish, seahorse, fruit fly, nematode, sea hare and yeast. AmphiL34 contains a putative nucleolar localization signal, while AmphiS29 has a zinc finger-like domain. A phylogenetic tree deduced from the conserved sequences of AmphiL34 and AmphiS29 and other known counterparts indicates that the positions of AmphiL34/AmphiS29 are intermediate between the vertebrate and invertebrate L34/S29. Southern blot analysis demonstrates the presence of one copy of the L34 gene and 2-3 copies of the S29 gene in the genome of the amphioxus B. belcheri tsingtauense. This is in sharp contrast to the existence of 7-9 copies of the L34 gene and 14-17 copies of the S29 gene in the rat genome. These date suggest that housekeeping genes like AmphiL34 and AmphiS29 have undergone large-scale duplication in the chordate lineage.     

Sperm-derived sperm motility-initiating substance from amphioxus Branchiostoma belcheri

The sperm of amphioxus, Branchiostoma belcheri, were immotile when excised from the testis and suspended in seawater. The sperm became motile upon spawning in natural seawater, suggesting mechanisms that triggered sperm motility during spawning. When a male amphioxus that underwent spawning was transferred to a cup containing a small amount of natural seawater, and then the seawater containing the spawned sperm was centrifuged, the supernatant caused motility initiation in the immotile sperm from the testis. This sperm motility-initiating activity was also found in the supernatant of seawater in which immotile sperm from the testis were incubated overnight. These suggest that in the amphioxus, a sperm motility-initiating substance resides in the sperm, and upon spawning, the substance is transformed into a free and active form to activate the sperm. Partial purification of the substance revealed it as a small and heat-stable substance with maximum UV absorbance at 234 nm.     

青岛文昌鱼过氧化氢酶基因的克隆及生物信息学分析

过氧化氢酶(catalase,CAT)是一类广泛存在于动物、植物和微生物体内的末端氧化酶,是生物体内抗氧化酶体系的重要成员,因此本研究旨在克隆文昌鱼CAT基因并对其进行生物信息学分析。本研究以青岛文昌鱼(Branchiostoma belcheri tsingtauense)为材料,用RACE技术首次克隆了其CAT全长cDNA的基因序列,命名为AmphiCAT(GenBank登录号:KU058636);该序列总长为2640 bp,开放阅读框(open reading frame,ORF)为1533 bp,编码510个氨基酸,含有一个长为19个氨基酸序列的潜在活性位点和一个长为9个氨基酸序列的血红素配体信号,总分子量在线预测约为57.85 kD;经生物软件分析确定该蛋白质无信号肽序列,预测该蛋白质为非分泌性蛋白,属于单功能CAT的clade3分支;该基因的分子进化树表明青岛文昌鱼CAT同软体动物的亲缘关系较近。     

Genomic organization and evolution of actin genes in the amphioxus Branchiostoma belcheri and Branchiostoma floridae

We previously described the cDNA cloning and expression patterns of actin genes from amphioxus Branchiostoma floridae (Kusakabe, R., Kusakabe, T., Satoh, N., Holland, N.D., Holland, L.Z., 1997. Differential gene expression and intracellular mRNA localization of amphioxus actin isoforms throughout development: implications for conserved mechanisms of chordate development. Dev. Genes Evol. 207, 203–215). In the present paper, we report the characterization of cDNA clones for actin genes from a closely related species, Branchiostoma belcheri, and the exon–intron organization of B. floridae actin genes. Each of these two amphioxus species has two types of actin genes, muscle and cytoplasmic. The coding and non-coding regions of each type are well-conserved between the two species. A comparison of nucleotide sequences of muscle actin genes between the two species suggests that a gene conversion may have occurred between two B. floridae muscle actin genes BfMA1 and BfMA2. From the conserved positions of introns between actin genes of amphioxus and those of other deuterostomes, the evolution of deuterostome actin genes can be inferred. Thus, the presence of an intron at codon 328/329 in vertebrate muscle and cytoplasmic actin genes but not in any known actin gene in other deuterostomes suggests that a gene conversion may have occurred between muscle and cytoplasmic actin genes during the early evolution of the vertebrates after separation from other deuterostomes. A Southern blot analysis of genomic DNA revealed that the amphioxus genome contains multiple muscle and cytoplasmic actin genes. Some of these actin genes seem to have arisen from recent duplication and gene conversion. Our findings suggest that the multiple genes encoding muscle and cytoplasmic actin isoforms arose independently in each of the three chordate lineages and that gene duplications and gene conversions established the extant actin multigene family during the evolution of chordates.     

Characterization and expression of p23 gene in the amphioxus Branchiostoma belcheri

The cDNA AmphiP23, encoding an amphioxus p23, was identified from the gut cDNA library of amphioxus Branchiostoma belcheri. It contains a 513 bp open reading frame corresponding to a deduced protein of 170 amino acids. Phylogenetic analysis shows that vertebrate and invertebrate p23/p23-like proteins are each grouped together, with AmphiP23 falling at the base of vertebrate p23/p23-like clade, suggesting that the divergence of vertebrate and invertebrate p23 genes probably occurs prior to the split of invertebrate/vertebrate from a common ancestor around 550 million years ago. Northern blotting reveals a ubiquitous expression pattern of AmphiP23 in all adult tissues examined, while whole mount in situ hybridization demonstrates a tissue- and stage-specific expression pattern of AmphiP23 in developing embryos and larvae. Presumably, the ubiquitous expression pattern of AmphiP23 in adult amphioxus represents the ancestral type of p23 gene prior to its split to human paralogs p23 and tsp23, while the tissue- and stage-specific expression pattern during early embryonic development implicates a role of AmphiP23 in anterior/posterior patterning.     

Cloning, expression, purification, and characterization of AmphiTip30, a member of short-chain dehydrogenases/reductases family from the amphioxus Branchiostoma belcheri tsingtauense

In this paper we describe the cloning, expression and identification study of the TIP30 gene from amphioxus (Branchiostoma belcheri). The amphioxus TIP30 cDNA is comprised of 1499 bp and is translated in one open-reading frame to give a protein of 237 amino acids, with a predicted 23 amino acids signal peptide, a 147 bp 5'-UTR and a 638 bp 3'-UTR. A multiple alignment of TIP30 from amphioxus with other known TIP30 sequences shows the conservation of most amino acid residues involved in the peculiar structural domains found within TIP30's. Phylogenetic analysis places AmphiTIP30 at the base of the phylogenetic tree, suggesting that AmphiTIP30 is the archetype of the vertebrate TIP30 genes. We express the amphioxus TIP30 gene in Escherichia coli. driven by T7 promoter. The recombinant amphioxus TIP30 protein was purified by HisTrap affinity column. Subsequently, the binding constant and enzyme activity was mensurated. Western blot and immunohistochemistry analysis confirmed that amphioxus has a native molecular mass of approximately 26 kDa, and TIP30 was strongly expressed in ovary. Finally, the initial function of TIP30 is discussed.