Molecular cloning, complementary deoxyribonucleic acid structure and predicted full-length amino acid sequence of the hormone-inducible regulatory subunit of 3'-5'-cyclic adenosine monophosphate-dependent protein kinase from human testis

In this study, we report the isolation and characterization of a full-length cDNA clone for the hormone-inducible regulatory subunit RII beta (formerly called RII51) of type II cAMP-dependent protein kinase from a human testis cDNA library. The cloned cDNA demonstrated tissue-specific expression of RII beta mRNA in human tissues, with the highest mRNA levels in testis and ovary. The isolated human cDNA clone was 3.3 kilobases (kb) in length and contained 166 base pairs (bp) of G/C-rich 5'-noncoding sequence, an open reading frame of 1254 bp and an A/T-rich 3'-nontranslated region containing 1836 bp followed by an 89 nucleotide long poly(A)-tail. The predicted protein contains 418 amino acids including the start methionine, and the estimated mol wt of human RII beta is 53,856. The nucleotide sequence within the open reading frame and the predicted amino acid sequence of human RII beta are highly conserved compared with partial rat RII beta sequences, displaying 91% and 97% similarity, respectively. Codon preference analysis of the cloned cDNA sequence indicated that the two cAMP-binding domains and the hinge region are highly conserved through evolution, whereas the dimerization domain displayed a codon preference pattern indicative of appearance at a later stage of evolution. The isolated human cDNA detected an FSH- and cAMP-inducible mRNA of 3.2 kb in rat Sertoli cells, thus confirming that the cloned cDNA represents the hormone-inducible regulatory subunit of cAMP-dependent protein kinase. This is the first report documenting the isolation of a full-length cDNA clone for the RII beta of cAMP-dependent protein kinase.     

Complete primary structure of the transacylase (E2b) subunit of the human branched chain alpha-keto acid dehydrogenase complex

We isolated from a placental cDNA library by immunoscreening a cDNA clone encoding the transacylase (E2b) precursor of the human branched chain alpha-keto acid dehydrogenase (BCKDH) complex. The cDNA insert consists of 2,649 base pairs with an open reading frame of 1,431 base pairs which can be translated into 477 amino acids and a 3'-untranslated region of 1,205 base pairs. The deduced amino acid sequence includes a leader peptide of 56 amino acid residues, a lipoyl-bearing domain, a E3-binding domain and an inner core domain. A mature human E2b subunit is likely to contain 421 amino acid residues with a calculated Mr 46,322. The nucleotide sequence of the open reading frame and the deduced amino acid sequence of the human E2b shows 91.6% and 92.0% homology with those of the bovine E2b subunit, respectively.     

南方鲇Vasa基因两种亚型cDNA的克隆及其表达

采用RT-PCR和RACE相结合的方法,从南方鲇分离到Vasa基因的两个亚型scVasa和scVaga-s。它们是同一基因在5′端经选择性剪接的产物,其cDNA全长分别为2525bp和2438bp,编码662和641个氨基酸。两者均具有DEAD-box家族成员特有的8个保守基序和Vasa的典型特征。南方鲇Vasa与银鲫相似性最高（73．3％）。两个亚型均特异地表达于雌雄性腺中。原位杂交结果表明：scVasa主要在卵巢Ⅰ、Ⅱ时相的卵母细胞和精巢的精原细胞和初级精母细胞中表达。半定量PCR结果显示,在生殖周期中,两种亚型在以Ⅱ时相卵母细胞为主体的卵巢恢复期表达均高于以Ⅲ-Ⅳ时相卵母细胞为主体的卵黄生成期[动物学报54（6）：1051—1060,2008]。     

Nucleotide sequence of a full-length complementary DNA clone and amino acid sequence of human phenylalanine hydroxylase

A full-length human phenylalanine hydroxylase complementary DNA (cDNA) clone was isolated from a human liver cDNA library, and the nucleotide sequence encoding the entire enzyme was determined. The cDNA clone contains an inserted DNA fragment of 2448 base pairs, including 19 base pairs of poly(A) at the 3' end. The first methionine codon occurs at nucleotide position 223, followed by an open reading frame of 1353 base pairs, encoding 451 amino acids. Translation of the nucleotide sequence in the open reading frame predicts the amino acid sequence of human phenylalanine hydroxylase. The human protein shows a 96% amino acid sequence homology with the corresponding rat enzyme. The determination of the complete primary structure for phenylalanine hydroxylase represents the first among mixed-function oxidases.     

菠菜甜菜碱醛脱氢酶基因的克隆和序列分析

以耐盐的菠菜mRNA为模板,经反转录合成甜菜碱醛脱氢酶(BADH)基因第一链cDNA。在人工合成的两端引物引导下,通过多聚酶链式反应(PCR),扩增获得双链cDNA。把重组有BADH基因的pUC19转化至E.coli DH5α菌株,亚克隆后测定了基因的全序列。所得到的BADH基因全长序列为1491bp,编码497个氨基酸。与文献报道的相比较,核苷酸序列同源性99.8％,氨基酸序列同源性达99.6％。在此基础上,构建了BADH基因的高等植物表达载体。     

The nucleotide sequence of boar transition protein 2 (TNP2) cDNA and haploid expression of the gene during spermatogenesis

A cDNA clone coding for boar transition protein 2 (TNP 2) was isolated from a randomly primed cDNA library of boar testis. Sequence analysis revealed an open reading frame of 414 bp (corresponding to 138 amino acids), 33 bp of the 5' untranslated and about 300 bp of the 3' untranslated region. As compared to TNP 2 of mouse and rat, similarity with TNP 2 of the boar is approximately 70% at the nucleotide level and only about 40% on the basis of amino acid sequence. The similarity between boar and bull TNP2 is 77% and 64%, respectively. Northern blot experiments with RNA of different boar tissues and in situ hybridization on mature boar testis sections revealed testis-specific expression of the TNP 2 gene which is restricted to haploid germ cells. Hybridization experiments of boar TNP2 cDNA with testicular RNA of boar, bull, rat and mouse revealed decreasing intensities of the hybridization signals. With human testicular RNA no hybridization could be obtained.     

Molecular cloning of an alanine aminotransferase from NAD-malic enzyme type C4 plant Panicum miliaceum

We have determined the nucleotide sequence of a cDNA encoding AlaAT-2, which is believed to function in the C4-pathway of Panicum miliaceum. An open reading frame (1446 bp) encodes a protein of 482 amino acid residues. The deduced amino acid sequence of AlaAT-2 shows 44.2 and 44.8% homology with the amino acid sequences of AlaATs from rat and human livers, respectively. Northern blot analysis showed that the gene encoding AlaAT-2 in mesophyll and bundle sheath cells was the same and transcribed similarly in the cells. The level of translatable mRNA for AlaAT-2 increased dramatically during greening.     

Differential mRNA expression levels and gene sequences of a putative carboxylesterase-like enzyme from two strains of the parasitoid Anisopteromalus calandrae (Hymenoptera: Pteromalidae).

Carboxylesterase-like enzyme cDNAs have been cloned and sequenced from malathion-resistant and susceptible strains of the parasitoid Anisopteromalus calandrae (Howard) (Hymenoptera: Pteromalidae). The cDNAs consist of 1963 nucleotides including a 35 bp untranslated 5'-end, a 1596 bp open reading frame, and a 332 bp untranslated 3'-end. The open reading frame encodes 532 amino acid residues. The predicted protein sequence from these cDNAs includes 2 potential N-glycosylation sites, a carboxylesterase type-B serine active site FGGDSENVTIFGESAG, and conserved residues Ser187, Glu317, and His432 to function as the catalytic triad. The predicted carboxylesterase-like enzyme sequence is most similar to that of the carboxylesterase from the peach-potato aphid, Myzus persicae with 45% sequence identity. Alignment of the parasitoid carboxylesterase-like enzyme cDNAs revealed that there are two nucleotide differences in the open reading frame between the parasitoid strains, including a silent mutation and a point mutation that presumably causes a gene product difference. A nucleotide thymine at position 658 in the susceptible strain cDNA is replaced by a guanine in the resistant strain cDNA. This substitution leads to an amino acid change from tryptophan (Trp220) in the susceptible strain to glycine (Gly220) in the resistant strain. This substitution is genetically linked to resistance but it is not known how or if this amino acid substitution affects detoxification of malathion. Northern blot analyses demonstrated that expression level of the carboxylesterase-like enzyme mRNA in adult A. calandrae is approximately 30-fold higher in the resistant strain relative to that in the susceptible strain. Southern analysis indicated that Pst I or Eco RI restriction sites are different in the two strains. Both a modified gene structure and an increase in expression of carboxylesterase may be responsible for the high level of resistance found in this beneficial wasp.     

汉坦病毒中国疫苗株Z37M片段的克隆及序列分析

汉坦病毒Z37株是从褐家鼠体内分离到的,用于生产双价肾综合征出血热疫苗的病毒毒株之一,血清分型为SEO型。利用RT-PCR方法扩增Z37株M基因片段cDNA,克隆入质粒载体,进行核苷酸序列测定及分析。Z37株M基因片段由3651个核苷酸组成,只有一个开放读码框架,共编码1133个氨基酸。与HTN型病毒(76-118、A9、HV-114)的核苷酸和氨基酸同源性分别为71.8%～72.1%、76.2%～76.7%,与SEO型(R22、L99、80-39)的核苷酸和氨基酸同源性分别为95.3%～96.1%、95.3%～98.9%。这一结果的获得进一步从分子水平确定了Z37株的型别,并为研制M基因片段重组疫苗打下基础。     

北京鸭卵磷脂胆固醇酰基转移酶的cDNA和蛋白质的结构分析

为获得不易感动脉粥样硬化动物北京鸭卵磷脂胆固醇酰基转移酶 (LCAT)的cDNA和蛋白质序列 ,分析其结构特点 .以从北京鸭肝脏mRNA反转录获得的cDNA一链为模板 ,应用SMART RACE技术 ,获得了北京鸭LCAT的cDNA序列 ,推导出其蛋白质氨基酸序列 ,应用分子生物学软件对该蛋白的一级、二级结构进行分析和比较 .北京鸭LCATcDNA (在GenBank中的注册号为AF32 4 887)全长 195 3bp ,其中开放阅读框架 135 6bp ,编码 4 5 1个氨基酸 ,包括一个由 2 3个氨基酸构成的疏水性信号肽和一个由 4 2 8个氨基酸组成的成熟蛋白 .该成熟蛋白比人LCAT在C端多 12个氨基酸 ,其与鸡、人、家兔的同源性依次为 98%、83%和 82 % .与其它种属LCAT蛋白序列的比较结果表明 ,北京鸭LCAT蛋白质序列虽然在长度上和结构上与其它种属有一定的差异 ,但序列中与酶催化活性相关的序列均非常保守     

Molecular cloning of the alcohol/hydroxysteroid form (hSTa) of sulfotransferase from human liver.

A cDNA encoding the human alcohol/hydroxysteroid sulfotransferase (h-ST-a), which catalyzes the sulfo-conjugation of many drugs and hormones, was isolated from a human liver cDNA library using a rat STa (rSTa) cDNA probe. The cDNA, designated as hSTa, consists of 1069 base pairs (bp) and contains an 855-nucleotide open reading frame beginning at nucleotide 65, which encodes a 285 amino acid polypeptide of 33.76 kDa. A second cDNA clone (1563 bp) was truncated 5' at nucleotide 231 (lacking the first 15 amino acids) with identical coding region, however, it had a much longer 3' untranslated region (UTR). Both clones contained a short segment of poly(A)+ tail. Northern blot analysis of an adult human liver showed that there are at least 2 mature mRNA with sizes ranging from approximately 1.1 kb to 1.7 kb, verifying the authenticity of the obtained cDNA clones. From the sequence alignment, the hSTa shares 62%/74%, 39%/59%, 35%/48%, 36%/54% identity with rSTa, rSTp (phenol), rSTe (estrogen), and bovine STe (bSTe) at the deduced amino acid and DNA levels, respectively, indicating that there are at least three subfamilies (alcohol, phenol and estrogen) of genes that encode for sulfotransferases in mammals.     

Complete nucleotide sequence of a CDNA encoding porcine tumor necrosis factor‐alpha

Abstract

Tumor necrosis factor‐alpha (TNF‐α), produced by immune cells, is a cytokine with a central role in the mediation of inflammatory responses to infection and injury. We report the nucleotide and corresponding amino acid sequence of a full length porcine TNF‐α cDNA. The complete cDNA nucleotide sequence is 1650 bp in length (not including the poly A tail) and has an open reading frame of 696 bp encoding a 232 amino acid protein. The porcine TNF cDNA sequence shows homologies of 86, 77, and 82% to human, murine, and lapine TNF cDNA sequences in the coding regions, respectively. The 5’ untranslated region of the cDNAs shows little sequence similarity. However, the 3´ untranslated region contains highly conserved sequences among all species, especially the TTATTTAT motif characteristic of cytokine messages.     

Nucleotide sequence of the yeast glutathione S-transferase cDNA

The nucleotide sequence (658 bp) of the cDNA coding for glutathione S-transferase Y-2 of yeast Issatchenkia orientalis was obtained. The cDNA clone contains an open reading frame of 570 nucleotides encoding a polypeptide comprising 190 amino acids with a molecular weight of 21,520. The primary amino acid sequence of the enzyme exhibits only 25.0% and 21.1% identity with 177 and 151 amino acid residues of maize glutathione S-transferase I and rat glutathione S-transferase Yb2, respectively.     

Molecular cloning and sequence analysis of human dipeptidyl peptidase IV, a serine proteinase on the cell surface.

The cDNA coding for the human dipeptidyl peptidase IV (DPPIV) has been isolated and sequenced. The nucleotide sequence (3465 bp) of the cDNA contains an open reading frame encoding a polypeptide comprising 766 amino acids, one residue less than those of rat DPPIV. The predicted amino acid sequence exhibits 84.9% identity to that of the rat enzyme, and contains nine potential N-linked glycosylation sites, one site more than those in the rat enzyme. A putative catalytic triad for serine proteinases, serine, aspartic acid and histidine, are found in a completely conserved COOH-terminal region (positions 625-752).