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cDNA cloning, nucleotide sequence and expression of the gene for arylsulfatase in the sea urchin (Hemicentrotus pulcherrimus) embryo 总被引:17,自引:0,他引:17
H Sasaki K Yamada K Akasaka H Kawasaki K Suzuki A Saito M Sato H Shimada 《European journal of biochemistry》1988,177(1):9-13
Arylsulfatase is known to be synthesized in large amounts at the early gastrula stage of sea urchin development. We determined the amino acid sequence of a portion of the purified sea urchin embryonic arylsulfatase, and then isolated a cDNA clone for arylsulfatase by screening a sea urchin plutei lambda gt10 cDNA library with an oligodeoxynucleotide probe synthesized according to the determined amino acid sequence. The longest cDNA clones were selected and the nucleotide sequence determined. The cDNA is 2422 nucleotides long and encodes 551 amino acids. The deduced amino acid sequence has not sequence similarity with any of the peptides registered in NBRF peptide databank. Northern blot analysis revealed that the arylsulfatase cDNA hybridizes to a 2.9-kb mRNA. This mRNA exists in the unfertilized egg in small amounts, but markedly increases after the blastula stage preceding the increase of the arylsulfatase activity. 相似文献
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Structure and tissue-specific developmental expression of a sea urchin arylsulfatase gene 总被引:10,自引:0,他引:10
Arylsulfatases are a group of enzymes that remove sulfate moieties from a diverse set of substrates including glycoproteins, steroids, and cerebrosides. We have isolated recombinant cDNA clones corresponding to an arylsulfatase (SpARS) message that encodes an abundant protein of pluteus larvae of the sea urchin Strongylocentrotus purpuratus. Although vertebrate arylsulfatases have broad tissue distributions, in situ hybridization with a probe for SpARS shows that the sea urchin message accumulates in the embryo only in the single cell type of aboral ectoderm and its precursors. The message is first detectable by RNase protection assays around hatching blastula stage and accumulates through pluteus larva stage. The open reading frame of cDNA clones is 1701 nt long and encodes a deduced protein with a predicted molecular mass of 61 kDa. Analysis of corresponding genomic DNA clones reveals that the pre-mRNA contains six exons. Consistent with the fact that arylsulfatase enzyme activity is extracellular, this polypeptide has a hydrophobic leader sequence and three potential glycosylation sites. Furthermore, hybridization in situ shows that in blastulae arylsulfatase message is preferentially concentrated around nuclei at the basal sides of cells. The S. purpuratus sequence is very similar to that recently reported for the same enzyme from Hemicentrotus pulcherrimus and 30% of the amino acid residues are also identical to those of both human arylsulfatase C (steroid sulfatase) and arylsulfatase A. Sequence relationships among these four mRNAs suggest that, assuming equal rates of evolution, the duplication separating the human genes occurred at about the time of separation of the echinoderm and vertebrate lineages. 相似文献
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Hiroi J Maruyama K Kawazu K Kaneko T Ohtani-Kaneko R Yasumasu S 《Development genes and evolution》2004,214(4):176-184
We isolated seven cDNA clones from embryos of the Japanese eel Anguilla japonica. Each deduced amino acid sequence consisted of a signal peptide, a propeptide and a mature enzyme portion belonging to the astacin protease family. A phylogenetic analysis showed that the eel enzymes resembled the high choriolytic enzyme (HCE) of medaka Oryzias latipes, and the hatching enzymes of the zebra fish Danio rerio and masu salmon Oncorhynchus masou. Hatching enzymes of these teleosts belonged to the group of the medaka HCE, and not the medaka low choriolytic enzyme (LCE), another hatching enzyme of medaka. Southern blot analysis showed that the genes of the eel hatching enzymes were multicopy genes like the medaka HCE genes. However, one of the eel hatching enzyme genes comprised eight exons and seven introns, and the exon-intron organization was similar to the medaka LCE gene, which is a single-copy gene. The molecular evolution of the fish hatching enzyme genes is discussed. In addition, whole-mount in situ hybridization and immunocytochemistry showed that the eel hatching enzyme was first expressed in the pillow anterior to the forebrain of early neurula, and finally in the cell mass on the yolk sac of later stage embryos. The early differentiation profile of eel hatching gland cells was similar to that of medaka, masu salmon and zebrafish, whereas the final location of the gland cells was different among fishes.Edited by N. Satoh 相似文献
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A half-smooth tongue-sole, Cynoglossus semilaevis Sox10 (Accession no.: EU070763) was isolated from brain of tongue sole by using homologous cloning and RACE method. The complete cDNA of the tongue sole Sox10 contains a 35 bp 5′UTR, a 1338 bp open reading frame (ORF) encoding 445 amino acids and a 1155 bp 3′UTR. A condensed phylogenetic tree was constructed based on the amino acid sequences of tongue sole Sox10 and other well-defined vertebrate Sox. The overall topology of the tree showed the tongue sole Sox10 clusters with all Sox10. Alignment of amino acid residues of the tongue sole Sox10 gene with those from other vertebrate indicated high level conservation of amino acid sequence. The RT-PCR analysis demonstrated that the tongue sole Sox10 was highly expressed in brain, gills, skin and eyes, intermediately in spleen, heart, head–kidney and muscles, weakly expressed in kidneys and intestine and no expression in liver and gonad. The Sox10 was also expressed weakly in germ cell and zygote. We cannot detect the expression of the Sox10 in 8-cells stage. However it resumed expression weakly from blastula stage to middle of gastrula. And it expressed highly from neurula stage to 25 dah (day after hatching). It suggested that the Sox10 was involved in the development of embryos and larvae in tongue sole. 相似文献
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以UVS.2为探针从第25期非洲爪蟾胚胎头背部的cDNA文库中筛选出了一个1.8kb的孵化酶基因(xhe),其转录产物最早出现于第17期胚胎的头背部,在第30期转录量达到高峰,随后便逐渐减少。该基因含有编码514个氨基酸的一个开框阅读框架,含有信号肽和原酶序列。所推测出的成熟蛋白有425个氨基酸,包括位于N一端的含有200个氨基酸的金属蛋白酶序列和位于C端的两个各110个氨基酸的CUB重复区。而UVS.2只代表该基因C端大约3/4的部分。同时还发现该酶分子量为60kDa,是一种胰蛋白酶类型的金属蛋白酶。它很不稳定,在纯化过程中极易降解为40kDa分子。60kDa分子具有很强的卵黄膜溶解活性和蛋白酶活性。其中CUB重复区很可能在介导卵黄膜和40kDa分子中起着重要作用,而40kDa分子很可能是在纯化操作过程中,由60kDa分子发生降解或自身降解丢失了两个CUB重复区而形成的,它只是60kDa分子中的一个金属蛋白酶主功能区,所以它没有卵黄膜溶解活性,尽管仍具有很强的蛋白酶活性。 相似文献
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用RACE-PCR方法从原肠期SMART文库中扩增到银鲫pou2基因的全长cDNA,其全长为2421bp,开放阅读框为1416bp,编码471个氨基酸,与斑马鱼pou2基因的氨基酸序列一致性高达91.0%。我们用RT-PCR和整体原位杂交的方法研究了银鲫pou2基因在胚胎发育过程中的时空表达图式。RT-PCR结果显示,银鲫pou2基因有母源转录本,其合子基因在高囊胚期强烈表达,在50%下包期和90%下包期也有高量的转录本,但在100%下包期表达量急剧降低,至体节期时已经完全检测不到其转录本。胚胎整体原位杂交结果显示其母源转录本在所有的胚盘细胞中。在高囊胚期和50%下包期,高度表达的合子转录本仍在所有的胚盘细胞中,但至90%下包期时,pou2的表达向胚胎背部的正中线汇聚,集中在神经板的两侧区域和脑部的两条横向条带。在100%下包期时,pou2的表达集中在神经板的中间区域以及预期形成的中后脑区域。至体节期时,转录本消失,这与RT-PCR结果高度一致。银鲫pou2基因的表达图式提示该基因在胚胎发育的早期具有重要作用,它可能参与调控神经板的形成和中后脑细胞的发育命运。 相似文献
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陆地棉叶绿体铜锌超氧化物歧化酶基因的克隆与表达 总被引:1,自引:0,他引:1
以陆地棉‘CRI36'的叶片为材料,使用RACE技术克隆到了棉花叶绿体Cu/Zn-SOD酶基因。基因序列全长共1 043 bp,含有完整的开放阅读框。推导的氨基酸序列分析显示含有叶绿体信号肽,和已知植物的叶绿体Cu/Zn-SOD酶蛋白的氨基酸残基的同源性在66%~74%之间。基因的表达谱分析显示:棉花叶绿体Cu/Zn-SOD酶基因主要在叶片、茎中表达,根、花和下胚轴中没有检测到信号,即基因的表达主要在棉花的绿色组织。不同生育期的表达谱结果证实:该基因主要在苗期表达,以后表达逐渐减少。用pET-21a(+)构建了原核表达载体,在大肠杆菌BL21(DE3)的表达结果显示:表达后得到一个29.0 kD的新蛋白,其分子量与预期目标一致。对SOD酶活性的分析证实,重组菌的酶活性显著增加,证明克隆的基因具有活性。 相似文献
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Shih LJ Chen CA Chen CP Hwang SP 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2002,131(2):143-151
A bone morphogenetic protein 2/4 (BMP2/4) gene has been cloned from the starfish, Archaster typicus, for the purpose of investigating the expression pattern of the BMP4 gene in echinoderm embryos which do not produce micromeres. The isolated gene (named AtBMP2/4) contained two exons that encoded the entire coding region. The deduced AtBMP2/4 protein sequence contained 509 amino acids. Sequence comparison showed that it shared high amino acid similarity with sea urchin BMP2/4 and Xenopus BMP2 and BMP4. Northern blot analyses indicated that AtBMP2/4 mRNA initially appears at the blastula stage and has a maximal expression level at the gastrula stage. Whole-mount in situ hybridization revealed that AtBMP2/4 mRNA is expressed in the archenteron, coelomic vesicles, and ectodermal cells of gastrula stage embryos. The observed spatial distribution pattern vastly differs from that of sea urchin SpBMP2/4, which is expressed mainly in the oral ectoderm region of the mesenchyme blastula and early gastrula embryos. 相似文献
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Rat liver beta-glucuronidase. cDNA cloning, sequence comparisons and expression of a chimeric protein in COS cells. 总被引:3,自引:1,他引:2
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P P Powell J W Kyle R D Miller J Pantano J H Grubb W S Sly 《The Biochemical journal》1988,250(2):547-555
A cDNA for rat liver beta-glucuronidase was isolated, its sequence determined and its expression after transfection into COS cells studied. The deduced amino acid sequence of the rat liver clone showed 77% homology with that from the cDNA for human placental beta-glucuronidase and 47% homology with that deduced from the cDNA for Escherichia coli beta-glucuronidase. Several differences were found between the cDNA from rat liver and that previously reported from rat preputial gland. Only one change leads to an amino acid difference in the mature enzyme. A chimeric clone was constructed by using a fragment encoding the first 18 amino acid residues of the signal sequence from the human placental cDNA clone and a fragment from the rat clone encoding four amino acid residues of the signal sequence, all 626 amino acid residues of the mature rat enzyme, and all of the 3' untranslated region. After transfection into COS cells the chimeric clone expressed beta-glucuronidase activity that was specifically immunoprecipitated by antibody to rat beta-glucuronidase. The Mr value of 76,000 of the expressed gene product was characteristic of the glycosylated rat enzyme. It was proteolytically processed in COS cells to Mr 75,000 6 h after metabolic labelling. At least 50% of the expressed enzyme was secreted at 60 h post-transfection, but the secreted enzyme did not undergo proteolytic processing. These results provide evidence that the partial cDNA isolated from a rat liver library contains the complete coding sequence for the mature rat liver enzyme and that the chimeric signal sequence allows normal biosynthesis and processing of the transfected rat liver enzyme in COS cells. 相似文献
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Primary structure and gene localization of human prolidase 总被引:15,自引:0,他引:15
F Endo A Tanoue H Nakai A Hata Y Indo K Titani I Matsuda 《The Journal of biological chemistry》1989,264(8):4476-4481
Complementary DNA clones of prolidase (imidodipeptidase, EC 3.4.13.9) were isolated from human liver and placental cDNA libraries. Two clones named lambda PL21 and lambda PP6 from the liver and placental cDNA libraries, respectively, were analyzed in detail. The first clone, lambda PL21, carried a cDNA insert of 1.7 kilobase pairs and covered all the coding region of human prolidase mRNA. The second clone, lambda PP6, contained a 1.8-kilobase insert with a full-length 3'-untranslated region. Comparison of the amino acid sequence predicted from the nucleotide sequence of the cDNA insert of the two clones with the partial amino acid sequence determined by Edman degradation of peptides derived from human erythrocyte prolidase established that both clones code for human prolidase. The amino terminus of the human mature enzyme is blocked and seems to begin with the sequence X-Ala-Ala-Ala. Presumably no processing occurs at the carboxyl terminus. The mature enzyme is composed of 492 residues, corresponding to Mr 54,305. The sequence of prolidase is unique and not similar to any known protein, except for a significant similarity to regions of F1-ATPase alpha and beta subunits from various sources. The gene has been mapped to the short arm of chromosome 19 (19p13.2). Elucidation of the complete amino acid sequence and the gene location of prolidase should provide the basis for understanding structure-function relationships and also inherited disorders caused by deficiency of this metabolically important enzyme. 相似文献
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In plants the enzyme coproporphyrinogen oxidase catalyzes the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX in the heme and chlorophyll biosynthesis pathway(s).We have isolated a soybean coproporphyrinogen oxidase cDNA from a cDNA library and determined the primary structure of the corresponding gene. The coproporphyrinogen oxidase gene encodes a polypeptide with a predicted molecular mass of 43 kDa. The derived amino acid sequence shows 50% similarity to the corresponding yeast amino acid sequence. The main difference is an extension of 67 amino acids at the N-terminus of the soybean polypeptide which may function as a transit peptide.A full-length coproporphyrinogen oxidase cDNA clone complements a yeast mutant deleted of the coproporphyrinogen oxidase gene, thus demonstrating the function of the soybean protein.The soybean coproporphyrinogen oxidase gene is highly expressed in nodules at the stage where several late nodulins including leghemoglobin appear. The coproporphyrinogen oxidase mRNA is also detectable in leaves but at a lower level than in nodules while no mRNA is detectable in roots.The high level of coproporphyrinogen oxidase mRNA in soybean nodules implies that the plant increases heme production in the nodules to meet the demand for additional heme required for hemoprotein formation. 相似文献
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Geeta B. Vohra G. Brian Golding Nora Tsao Ronald E. Pearlman 《Journal of molecular evolution》1992,34(5):383-395
Summary We have determined the nucleotide sequence of both genomic and complementary DNA (cDNA) for the gene encoding the glycolytic enzyme phosphoglycerate kinase from the ciliated protozoan Tetrahymena thermophila. The amino acid sequence for the enzyme has also been derived from the cDNA sequence. The gene contains an open reading frame of 1260 nucleotides encoding 420 amino acids. Coding sequence in genomic DNA is interrupted by two introns at positions corresponding to introns 3 and 4 in mammalian phosphoglycerate kinase genes. The derived amino acid sequence was used to prepare a phylogeny by aligning the Tetrahymena sequence with 25 other phosphoglycerate kinase amino acid sequences. The Tetrahymena sequence is a typical eukaryotic sequence. There is recognizable and clear homology across species that cover nearly the complete range of life forms. The phylogenetic reconstruction of these sequences generally supports the conclusions that have been reached using rRNA sequences.Offprint requests to: R.E. Pearlman 相似文献