Molecular cloning of a cDNA encoding 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from liver of Sparus aurata: nutritional regulation of enzyme expression

A cDNA clone encoding full-length 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/Fru-2, 6-P2ase) was isolated and sequenced from a Sparus aurata liver cDNA library. The 2527 bp nucleotide sequence of the cDNA contains a 73 bp 5'-untranslated region (5'-UTR), an open reading frame that encodes a 469 amino acid protein and 1041 bp at the 3'-UTR. The deduced amino acid sequence is the first inferred 6PF-2-K/Fru-2, 6-P2ase in fish. The kinase and bisphosphatase domains, where the residues described as crucial for the mechanism of reaction of the bifunctional enzyme are located, present a high degree of homology with other liver isoenzymes. However, within the first 30 amino acids at the N-terminal regulatory domain of the fish enzyme a low homology is found. Nutritional regulation of the 6-phosphofructo-2-kinase activity, together with immunodetectable protein and mRNA levels of 6PF-2-K/Fru-2,6-P2ase, was observed after starvation and refeeding. In contrast to results previously described for rat liver, the decrease in immunodetectable protein and kinase activity caused by starvation was associated in the teleostean fish to a decrease in mRNA levels.     

Cloning and expression of the turtle (<Emphasis Type="Italic">Trachemys scripta</Emphasis>) immunoglobulin joining (J)-chain cDNA

The J-chain protein is a M(r) 15000 polypeptide associated with polymeric IgA and IgM. The complete cDNA sequences of human, mouse, cow, brushtail possum, chicken and frog J chains have been previously reported, but nothing is known about the cDNA and amino acid sequences of reptilian J chain. Here, we determined a turtle J-chain cDNA sequence by RT-PCR and RACE, and examined J-chain mRNA and protein expression by Northern blotting and immunohistochemistry. This turtle J-chain cDNA was 1934 bp and had an open reading frame of 477 nucleotides, encoding 159 amino acids. The mature J-chain protein is composed of 137 amino acids, M(r) approximately 15000. The deduced amino acid sequence of the turtle J chain was highly homologous to that of human (60%), mouse (61%), cow (60%), rabbit (60%), chicken (69%), brushtail possum (65%), Rana catesbeiana (47%) and Xenopus laevis (58%). Eight cysteine residues were located at the same positions as in these other species, with the exception of X. laevis. PROSITE database analysis indicated the presence of two N-glycosylation sites in turtle, one of which was novel. Northern blot analysis revealed that turtle J-chain mRNA was expressed in lung, stomach, spleen and intestine. In addition, immunohistochemistry showed J-chain-positive plasma cells in the intestine and spleen. These results suggest the presence of a mucosal immune system mainly composed of J-chain-containing Ig in the turtle.     

Sequence of a cDNA encoding turtle high mobility group 1 protein

In order to understand sequence information about turtle HMG1 gene, a cDNA encoding HMG1 protein of the Chinese soft-shell turtle (Pelodiscus sinensis) was amplified by RT-PCR from kidney total RNA, and was cloned, sequenced and analyzed. The results revealed that the open reading frame (ORF) of turtle HMG1 cDNA is 606 bp long. The ORF codifies 202 amino acid residues, from which two DNA-binding domains and one polyacidic region are derived. The DNA-binding domains share higher amino acid identity with homologues sequences of chicken (96.5%) and mammalian (74%) than homologues sequence of rainbow trout (67%). The polyacidic region shows 84.6% amino acid homology with the equivalent region of chicken HMG1 cDNA. Turtle HMG1 protein contains 3 Cys residues located at completely conserved positions. Conservation in sequence and structure suggests that the functions of turtle HMG1 cDNA may be highly conserved during evolution. To our knowledge, this is the first report of HMG1 cDNA sequence in any reptilian.     

鳜胰岛素样生长因子-ⅠcDNA全长克隆及组织表达分析

采用RT-PCR、cDNA末端快速扩增法(RACE)等技术克隆了鳜(Siniperca chuatsi)肝组织胰岛素样生长因子-I(IGF-I)cDNA全长序列.结果表明,鳜IGF-I cDNA全长1 784 bp,包括5'端非翻译区233bp,3'端非翻译区990 bp和开放阅读框561 bp,共编码186个氨基酸;...     

Primary structure of sorghum malate dehydrogenase (NADP) deduced from cDNA sequence. Homology with malate dehydrogenase (NAD)

Malate dehydrogenase (NADP) (NADP-MDH) is an important enzyme of the photosynthetic CO2 fixation pathway of C4 plants. We have isolated two clones from a sorghum lambda gt11 cDNA library (CM3, 932 bp, and CM7, 1441 bp). Nucleotide sequence analysis of the cDNAs CM3 and CM7 showed the existence of two NADP-MDH mRNA species encoding different enzyme subunits. Microsequencing of the N-terminus of the mature protein indicated that a specific cleavage of 13 amino acids occurred during the purification steps of the enzyme. The full-length cDNA CM7 contains a large open reading frame encoding an NH2-terminal transit peptide of 40 amino acids and a mature protein of 389 amino acids (42.207 kDa). Alignment of the NADP-MDH sequence with those of several malate dehydrogenases revealed some similarities with NAD-MDHs.     

鳜两种生长抑素受体cDNA全长克隆与组织表达

利用cDNA末端快速扩增(RACE)技术克隆了鳜(Siniperca chuatsi)脑中2种生长抑素受体(somatostatin receptor,SSTR2和SSTR3)cDNA全长序列。结果显示,鳜SSTR2 cDNA全长1 820 bp,含开放阅读框1 146 bp,编码382个氨基酸;SSTR3 cDNA全长1 874 bp,含开放阅读框1 458 bp,编码486个氨基酸。SSTR均由5个结构区域组成:N端、7个转膜区(TMD)、3个细胞外袢(ECLs)、4个细胞内袢(ICLs)和C末端。NJ系统进化树分析显示,鳜SSTR2和SSTR3分别形成相对独立的分支,两者间的氨基酸序列相似度为51.2%,表明它们是由不同基因编码而成。利用实时荧光定量RT-PCR技术检测了鳜SSTR2和SSTR3 mRNA的组织表达特征,它们均在多种组织中广泛表达,SSTR2 mRNA在肝中表达量最高,SSTR3 mRNA在胃中表达量最高。SSTR2、SSTR3表达差异反映它们可能参与不同生理调控作用。     

Rat apolipoprotein E mRNA. Cloning and sequencing of double-stranded cDNA

A 900-base pair clone corresponding to rat liver apolipoprotein E (apo-E) mRNA, and containing a 3'-terminal poly(A) segment, was identified from a library of rat liver cDNA clones in the plasmid pBR322 by specific hybrid selection and translation of mRNA. A restriction endonuclease DNA fragment from this recombinant plasmid was used to clone the 5'-terminal region of the apo-E mRNA by primed synthesis of cDNA. A portion of the double-stranded cDNA corresponding to the 3'-terminal region of apo-E mRNA was subcloned into the bacteriophage M13mp7 and employed as a template for the synthesis of a radioactively labeled, cDNA hybridization probe. This cDNA probe was used in a RNA-blot hybridization assay that showed the length of the apo-E mRNA to be about 1200 nucleotides. The hybridization assay also demonstrated that apo-E mRNA is present in rat intestine, but at about a 100-fold lower level than that of the rat liver. The nucleotide sequence of rat liver apo-E mRNA was determined from the cloned, double-stranded cDNAs. The amino acid sequence of rat liver apo-E was inferred from the nucleotide sequence, which showed that the mRNA codes for a precursor protein of 311 amino acids. A comparison to the NH2-terminal amino acid sequence of rat plasma apo-E indicated that the first 18 amino acids of the primary translation product are not present in the mature protein and are probably removed during co-translational processing. The coding region was flanked by a 3'-untranslated region of 109 nucleotides, which contained a characteristic AAUAAA sequence that ended 13 nucleotides from a 3'-terminal poly(A) segment. At the 5'-terminal region of the mRNA, 23 nucleotides of an untranslated region were also determined. The inferred amino acid sequence of mature rat apo-E, which contains 293 amino acids, was compared to the amino acid sequence of human apo-E, which contains 299 amino acids. Using an alignment that permitted a maximum homology of amino acids, it was found that overall, 69% of the amino acid positions are identical in both proteins. The amino acid identities are clustered in two broad domains separated by a short region of nonhomology, an NH2-terminal domain of 173 residues where 80% are identical, and a COOH-terminal domain of 84 residues where 70% are identical. These two domains may be associated with specific functional roles in the protein.     

中华鳖转铁蛋白基因序列特征及表达研究

转铁蛋白是呼吸链的关键因子, 参与体液和免疫系统的调节, 具有抗菌抗病毒等功能。研究从嗜水气单胞菌诱导的中华鳖肝脏SMART cDNA文库中, 分离克隆了中华鳖转铁蛋白基因cDNA和gDNA全序列, 对其序列特征、组织表达及原核表达进行了分析研究。结果显示, 中华鳖转铁蛋白对应全长cDNA的基因组DNA全长为19100 bp, 由17个外显子和16个内含子组成; cDNA全长为2359 bp, 开放阅读框为 2094 bp, 编码697个氨基酸; 转铁蛋白的分子量为76.6 kD, 包含2个糖基化位点, 存在4个二硫键形成区域的缺失, 两个同源结构域的相似性为37%, 信号肽位于第119位点。荧光定量PCR结果显示, 正常组中华鳖转铁蛋白基因在肝脏中的表达量最大; 以嗜水气单胞菌刺激后, 其在心、肝、脾和肾脏组织中的表达, 均有一个上调后减少的趋势。此外, 实验还进行了重组转铁蛋白原核表达和Western blot 检测鉴定, 为深入研究中华鳖转铁蛋白功能的提供了基础数据。       

Isolation and characterization of a cDNA for rat liver cysteine dioxygenase

Cysteine dioxygenase is a key enzyme of cysteine metabolism in mammals. The cDNA clones for rat liver cysteine dioxygenase were isolated by immunological screening and plaque hybridization from a rat liver cDNA library. The longest clone contained an insert of 1458 bp and encoded a polypeptide of 200 amino acids. The clone included the corresponding nucleotide sequence to amino acid sequences obtained from four lysyl endopeptidase-digested fragments of purified rat liver cysteine dioxygenase. The calculated molecular weight of rat liver cysteine dioxygenase was 23,025. Northern blot analysis revealed a single cysteine dioxygenase mRNA species of about 1.7 kb. A computer homology search indicated that this protein showed no homology with any known protein.     

Characterization of the cDNA encoding human nucleophosmin and studies of its role in normal and abnormal growth

A cDNA encoding human nucleophosmin (protein B23) was obtained by screening a human placental cDNA library in lambda gtll first with monoclonal antibody to rat nucleophosmin and then with confirmed partial cDNA of human nucleophosmin as probes. The cDNA had 1311 bp with a coding sequence encoding a protein of 294 amino acids. The identity of the cDNA was confirmed by the presence of encoded amino acid sequences identical with those determined by sequencing pure rat nucleophosmin (a total of 138 amino acids). The most striking feature of the sequence is an acidic cluster located in the middle of the molecule. The cluster consists of 26 Asp/Glu and 1 Phe and Ala. Comparison of human nucleophosmin and Xenopus nucleolar protein NO38 shows 64.3% sequence identity. The N-terminal 130 amino acids of human nucleophosmin also bear 50% identity with that of Xenopus nucleoplasmin. Northern blot analysis of rat liver total RNA with a partial nucleophosmin cDNA as probe demonstrated a homogeneous mRNA band of about 1.6 kb. Similar observations were made in hypertrophic rat liver and Novikoff hepatoma. However, the quantity of nucleophosmin mRNA is 50- and 5-fold higher in Novikoff hepatoma and hypertrophic rat liver, respectively, when compared with normal rat liver. Dot blot analysis also showed a nucleophosmin mRNA ratio of 64:5:1 in the three types of rat liver. When the protein levels were compared with Western blot immunoassays, Novikoff hepatoma showed 20 times more nucleophosmin, while only about 5 times more nucleophosmin was observed in hypertrophic rat liver than in unstimulated normal liver.     

Purification of juvenile hormone esterase and molecular cloning of the cDNA from Manduca sexta.

Juvenile hormone esterase (JHE) is a highly specific enzyme important for regulating the onset of metamorphosis in lepidopteran insects. After affinity chromatography of the hemolymph proteins of Manduca sexta, the pure JHE protein was digested with Lys-C and the resultant peptides were purified by microbore HPLC. Two peptides were selected for sequencing. Based upon these amino acid sequences, degenerate RT-PCR was performed in order to amplify a partial cDNA sequence from mRNA from the fat body of M. sexta. A 1512bp partial cDNA was generated and found to be highly homologous to the JHE from Heliothis virescens. 5' and 3' RACE were performed to obtain the full length cDNA sequence. The cDNA has a total length of 2220bp, with a 1749bp coding region. The deduced protein sequence contains 573 amino acids.     

特异种质烟草HZNH的Fe-SOD基因的克隆与表达

超氧化物歧化酶(superoxidedismutase,SOD)是一种广泛存在于动物、植物、微生物体内的金属酶,按其结合的金属性离子可分为Fe SOD、Mn SOD和CuZn SOD三种,它们通过催化超氧阴离子自由基O·-2发生歧化反应,达到清除O·-2的效果,具有防御氧毒性、增强机体抗辐射损伤能力、防衰老,治疗某些肿瘤、炎症、自身免疫疾病等功效,在农业、医药、食品、化工等产业中的应用前景广阔,因此广受国内外科研工作者的关注和重视[1].而试图通过转SOD基因技术来培育高抗逆农作物新品种和基因克隆与表达技术来实现SOD的大规模发酵生产,已成为国内外SOD…     

Molecular Cloning of cDNA to mRNA for a Cerebellar Spot 35 Protein

The nucleotide sequence of mRNA for rat cerebellar spot 35 protein, a Ca-binding protein, was determined from recombinant complementary DNA (cDNA) clones. The sequence was composed of 1,714 base pairs (bp) which included the 783 bp of the complete coding region, the 130 bp of the 5'-noncoding region, and the 801 bp of the 3'-noncoding region containing a polyadenylation signal. In addition, a polyadenylic acid [poly(A)] tail was also found. Because the size of spot 35 mRNA was estimated to be about 1,900 bases by Northern blot analysis, the longest insert was verified to contain a nearly full-length cDNA sequence including the poly(A) tail. The amino acid sequence of the protein deduced from the nucleotide sequence contains 261 amino acids and at least five Ca-binding domains. There was a high homology in the amino acid sequences (79%) and the nucleotide sequences (77%) between spot 35 protein and chick intestinal Ca-binding protein (28K).     

Isolation and characterization of a cDNA clone for a novel serine-rich neutrophil protein.

A cDNA expression library from pig blood neutrophils was immunoscreened with a rabbit antiserum raised against a 32 kDa neutrophil membrane phosphoprotein. Previous work indicated this protein as a component of the superoxide-forming NADPH oxidase enzyme complex (1,2). Only one cDNA clone (B+) was highly positive. The B+ clone contained a 1109 bp insert, with an open reading frame encoding for 284 amino acids. The deduced B+ amino acid sequence contained a 72 amino acid domain with proline and glutamine repeats and two domains extremely enriched with serine residues. The isolated cDNA hybridizes with a 3.1 kb mRNA expressed in pig and human leukocytes.     

Cloning and nucleotide sequence of a full-length cDNA for human liver gamma-glutamylcysteine synthetase.

We have cloned and sequenced a full-length cDNA for human liver gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in glutathione biosynthesis. The cDNA consists of 2634 bp containing an open reading frame encoding a protein of 367 amino acids and having a calculated M(r) = 72,773. The nucleotide sequence of the cDNA for human liver GCS shares an 84% overall similarity with the composite rat GCS sequence deduced from three overlapping partial cDNAs (Yan and Meister, JBC 265: 1588-1593, 1990). The deduced amino acid sequences are 94% similar. Comparison of Northern blots of total RNA isolated from rat kidney or liver with that from human kidney revealed the GCS mRNA to be larger in the human tissue (approximately 4.0 kb vs. approximately 3.7 kb). (The sequence for the human liver GCS cDNA has been assigned accession number M90656 in GenBank/EMBL databases.     

Molecular cloning and characterization of the cDNA encoding the rat liver gamma-butyrobetaine hydroxylase

Carnitine biosynthesis from lysine and methionine involves five enzymatic reactions. γ-butyrobetaine hydroxylase (BBH; EC 1.14.11.1) is the last enzyme of this pathway. It catalyzes the reaction of hydroxylation of γ-butyrobetaine to carnitine. The cDNA encoding this enzyme has been isolated and characterized. The cDNA contained an open reading frame of 1161 bp encoding a protein of 387 amino acids with a deduced molecular weight of 44.5 kDa. The sequence of the cDNA showed an important homology with the human cDNA recently isolated. Northern analysis showed γ-butyrobetaine hydroxylase expression in the liver and in some extend in the testis and the epididymis. During this study, it also appeared that BBH mRNA expression was undetectable by Northern analysis during the perinatal period. During the development of the rat, the amount of BBH mRNA appeared after the weaning of the young rat and reached a maximal expression at the adult stage.