Molecular cloning, sequencing, and expression of mouse ferrochelatase

The cDNA encoding mouse ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) was isolated from a mouse erythroleukemia (MEL) cell cDNA library in lambda gt11 expression vector, by immunoscreening with a polyclonal antibody. Two full-length clones containing cDNA inserts of 2.2 and 2.90 kilobases were obtained. These clones have the same entire enzyme coding region, but alternative putative polyadenylation sites in the 3'-noncoding regions. From the deduced primary structure, a putative leader sequence of 53 amino acid residues resulted in a precursor protein of 420 amino acid residues (Mr 47,130) and a mature protein of 367 residues (Mr 41,692). The cDNA allows for the expression of active ferrochelatase by transfected culture cells. RNA blot analysis showed two species of ferrochelatase mRNA consistent with findings of two polyadenylation sites. Both the mRNAs increased by treatment of the MEL cells with dimethyl sulfoxide. The band pattern of the RNA of the mouse liver was the same as that of the MEL cells. Based on these results, we deduce that ferrochelatase in erythroid and hepatic cells can be only of one type.     

Molecular cloning of cDNA for trehalase from the European honeybee, Apis mellifera L., and its heterologous expression in Pichia pastoris

cDNA encoding the bound type trehalase of the European honeybee was cloned. The cDNA (3,001 bp) contained the long 5' untranslated region (UTR) of 869 bp, and the 3' UTR of 251 bp including a poly(A) tail, and the open reading frame of 1,881 bp consisting of 626 amino acid residues. The Mr of the mature enzyme comprised of 591 amino acids, excluded a signal sequence of 35 amino acid residues, was 69,177. Six peptide sequences analyzed were all found in the deduced amino acid sequence. The amino acid sequence exhibited high identity with trehalases belonging to glycoside hydrolase family 37. A putative transmembrane region similar to trehalase-2 of the silkworm was found in the C-terminal amino acid sequence. Recombinant enzyme of the trehalase was expressed in the methylotrophic yeast Pichia pastoris as host, and displayed properties identical to those of the native enzyme except for higher sugar chain contents. This is the first report of heterologous expression of insect trehalase.     

Characterization of a cDNA clone encoding the complete amino acid sequence of cotton isocitrate lyase

A cDNA clone encoding the glyoxysomal enzyme isocitrate lyase (ICL) (EC 4.1.3.1) was isolated from a library prepared from cotton (Gossypium hirsutum L.) cotyledon poly(A)+ RNA. The clone is 1893 basepairs (bp) in length and contains a 1728 bp open reading frame encoding a polypeptide of 576 residues (Mr = 64,741). The deduced amino acid sequence of cotton ICL is 85.2%, 90.3% and 41.1% identical to ICL from rapeseed, castor bean and E. coli, respectively. Cotton ICL has a C-terminal tripeptide of A-R-M which is a putative trafficking signal for peroxisome (glyoxysome) proteins.     

Amino acid sequence of rat argininosuccinate lyase deduced from cDNA

Argininosuccinate lyase [EC 4.3.2.1] is an enzyme of the urea cycle in the liver of ureotelic animals. The enzymes of the urea cycle, including argininosuccinate lyase, are regulated developmentally and in response to dietary and hormonal changes, in a coordinated manner. The nucleotide sequence of rat argininosuccinate lyase cDNA, which was isolated previously (Amaya, Y., Kawamoto, S., Oda, T., Kuzumi, T., Saheki, T., Kimula, S., & Mori, M. (1986) Biochem. Int. 13, 433-438), was determined. The cDNA clone contained an open reading frame encoding a polypeptide of 461 amino acid residues (predicted Mr = 51,549), a 5'-untranslated sequence of 150 bp, and a 3'-untranslated sequence of 41 bp. The amino acid composition of rat liver argininosuccinate lyase predicted from the cDNA sequence is in close agreement with that determined on the purified enzyme. The predicted amino acid sequences of the human and yeast enzymes along the entire sequences (94 and 39%, respectively), except for a region of 66 residues of the human enzyme near the COOH terminus. However, the sequence of this region of the human enzyme predicted from another reading frame of the human enzyme cDNA is homologous with the corresponding sequences of the rat and yeast enzymes. Therefore, the human sequence should be re-examined. Lysine-51, the putative binding site for argininosuccinate, and the flanking sequences are highly conserved among the rat, steer, human, and yeast enzymes.     

Molecular cloning, primary structure, and expression of the human growth factor-activatable Na+/H+ antiporter

We present the complete sequence of a cDNA encoding the human amiloride-sensitive Na+/H+ antiporter. After functional complementation of a mouse fibroblast mutant by gene transfer, we isolated a 0.8 kb genomic probe from a third-cycle mouse transformant. The probe detects gene amplification in Na+/H+ antiporter "overexpressers" and a single class of mRNA of ca. 5.6 kb in human, mouse, and hamster cells. With this probe we isolated a 4 kb cDNA from a library constructed from a mouse transformant in which the transfected human gene was amplified. This cDNA includes a noncoding leader of 407 bp, a 2682 bp open reading frame, and a 3' noncoding sequence containing a mouse B1 repeated element. The amino acid sequence predicts a protein of Mr = 99,354 with an N-terminal amphipathic domain that contains 10 putative transmembrane-spanning segments and two potential glycosylation sites, followed by a hydrophilic stretch of 395 residues, presumably cytoplasmic. Stable expression of the transfected cDNA in Na+/H+ antiporter-deficient cells restored the key functional features of this transporter: H+i-activated Na+ influx, amiloride sensitivity, and pHi regulation.     

Molecular cloning and characterization of a putative protein kinase gene from the thermophilic fungus Thermomyces lanuginosus.

Based on the conserved amino acid sequence (DLKPEN) of serine-threonine protein kinase from several fungi, a degenerate primer was designed and synthesized. Total RNA was isolated from the thermophilic fungus Thermomyces lanuginosus. Using RACE-PCR, full-length cDNA of a putative serine-threonine protein kinase gene was cloned from T. lanuginosus. The full-length cDNA of T. lanuginosus protein kinase was 2551 bp and contained an 1806 bp open reading frame encoding a putative protein kinase precursor of 601 amino acid residues. Sequencing analysis showed that the cloned cDNA of T. lanuginosus had consensus protein kinase sequences. Conservative amino acid subdomains which most serine-threonine kinases contain can be found in the deduced amino acid sequence of T. lanuginosus putative protein kinase. Comparison results showed that the deduced amino acid sequence of T. lanuginosus putative protein kinase was highly homologous to that of Neurospora crassa dis1-suppressing protein kinase Dsk1. The putative protein kinase contained three arginine/serine-rich (SR) regions and two transmembrane domains. These showed that it might be a novel putative serine-threonine protein kinase.     

Isolation and sequence analysis of a cDNA encoding rat liver alpha-L-fucosidase.

cDNA clones for alpha-L-fucosidase were isolated from a rat liver lambda gt11 expression library by using both monospecific polyclonal antibodies against the affinity-purified enzyme and biotinylated rat liver fucosidase cDNA sequences as probes. The largest clone, lambda FC9, contained a 1522 bp full-length cDNA insert (FC9) that encoded the 434-amino acid-residue subunit (Mr 50439) of rat liver alpha-L-fucosidase. A putative signal peptide 28 amino acid residues in length preceded the sequence for the mature protein. In addition, FC9 specified for 11 nucleotide residues of 5' untranslated sequence, 78 nucleotide residues of 3' untranslated sequence and a poly(A) tail. The deduced amino acid sequence from FC9 in conjunction with the experimentally determined N-terminus of the mature enzyme suggested that rat liver fucosidase did not contain a pro-segment. However, there was the possibility of limited N-terminal processing (one to five amino acid residues) having occurred after removal of the predicted signal peptide. Amino acid sequences deduced from FC9 were co-linear with amino acid sequences measured at the N-terminus of purified fucosidase and on two of its CNBr-cleavage peptides. An unusual aspect of rat liver alpha-L-fucosidase protein structure obtained from the FC9 data was its high content of tryptophan (6%). The coding sequence from FC9 showed 82% sequence identity with that from a previously reported incomplete human fucosidase sequence [O'Brien, Willems, Fukushima, de Wet, Darby, DiCioccio, Fowler & Shows, (1987) Enzyme 38, 45-53].     

Molecular cloning and sequence analysis of cDNA encoding human cholesterol 7 alpha-hydroxylase

A complete cDNA clone encoding human cholesterol 7 alpha-hydroxylase has been isolated using a rat P-450ch7 alpha cDNA insert [(1989) FEBS Lett. 257, 97-100] as a probe and totally sequenced. The cDNA contained 1512-base pair open reading frame encoding 504 amino acid residues (Mr 57,630), 39-base pair 5'-untranslated region 1322-base pair 3'-ultranslated region including 20 nucleotides of poly A tail in the total length of 2873 base pairs. The deduced amino acid sequence showed 82% similarity to rat P-450ch7 alpha. Unique amino acid residues were observed in putative binding domains for heme and steroid which are highly conserved in most steroidogenic P-450s.     

Rat liver beta-glucuronidase. cDNA cloning, sequence comparisons and expression of a chimeric protein in COS cells.

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.     

Molecular cloning and sequence analysis of cDNA encoding delta 4-3-ketosteroid 5 beta-reductase of rat liver.

A cDNA clone encoding delta 4-3-ketosteroid 5 beta-reductase was isolated from rat liver cDNA libraries using antibodies specific for the enzyme and oligonucleotides as probes. The cDNA contained 981-base pair open reading frame encoding 327 amino acid residues (Mr 37,376) and an unusually long 3'-untranslated region rich in AT sequence in the total length of 3189 base pairs. The predicted amino acid sequence contains the sequences similar to the putative NADPH- and steroid-binding regions.     

褐飞虱乙酰胆碱酯酶基因全长cDNA的克隆及序列分析

利用反转录聚合酶链式反应(RT_PCR)结合快速扩增cDNA末端(RACE)技术克隆了褐飞虱Nilaparvata lugens 乙酰胆碱酯酶基因cDNA。该cDNA全长2 467 bp,包含一个1 938 bp的开放阅读框(GenBank登录号AJ852420); 在推导出的646个氨基酸残基的前体蛋白中, N端的前30个氨基酸残基为信号肽,随后的616个氨基酸残基是成熟的乙酰胆碱酯酶序列,其预测的分子量为69 418 D。在一级结构中,形成催化活性中心的3个氨基酸残基(Ser242,Glu371和His485),以及在亚基内形成二硫键的6个半胱氨酸完全保守; 位于催化功能域的14个芳香族氨基酸中有10 个完全保守。该酶的氨基酸序列与黑尾叶蝉的同源性最高,达83%。对来自23种昆虫（包括褐飞虱）的30个乙酰胆碱酯酶的聚类分析显示,褐飞虱的乙酰胆碱酯酶与其中6个Ⅱ型乙酰胆碱酯酶（AChE2）同属一个支系; 此外,只存在于昆虫AChE2中的超变区及特异的氨基酸残基,也存在于褐飞虱的乙酰胆碱酯酶中。以上结果表明,所克隆的褐飞虱的乙酰胆碱酯酶基因是一个与黑腹果蝇的orthologous型基因同源的AChE2基因。     

The cDNA sequence of mouse Pgp-1 and homology to human CD44 cell surface antigen and proteoglycan core/link proteins

We describe the isolation and sequencing of a cDNA encoding mouse Pgp-1. An oligonucleotide probe corresponding to the NH2-terminal sequence of the purified protein was synthesized by the polymerase chain reaction and used to screen a mouse macrophage lambda gt11 library. A cDNA clone with an insert of 1.2 kilobases was selected and sequenced. In Northern blot analysis, only cells expressing Pgp-1 contained mRNA species that hybridized with this Pgp-1 cDNA. The nucleotide sequence of the cDNA has a single open reading frame that yields a protein-coding sequence of 1076 base pairs followed by a 132-base pair 3'-untranslated sequence that includes a putative polyadenylation signal but no poly(A) tail. The translated sequence comprises a 13-amino acid signal peptide followed by a polypeptide core of 345 residues corresponding to an Mr of 37,800. Portions of the deduced amino acid sequence were identical to those obtained by amino acid sequence analysis from the purified glycoprotein, confirming that the cDNA encodes Pgp-1. The predicted structure of Pgp-1 includes an NH2-terminal extracellular domain (residues 14-265), a transmembrane domain (residues 266-286), and a cytoplasmic tail (residues 287-358). Portions of the mouse Pgp-1 sequence are highly similar to that of the human CD44 cell surface glycoprotein implicated in cell adhesion. The protein also shows sequence similarity to the proteoglycan tandem repeat sequences found in cartilage link protein and cartilage proteoglycan core protein which are thought to be involved in binding to hyaluronic acid.     

The primary structure of human Rieske iron-sulfur protein of mitochondrial cytochrome bc1 complex deduced from cDNA analysis

We isolated a cDNA encoding human Rieske Fe-S protein of mitochondrial cytochrome bc1 complex from a fibroblast cDNA library by colony hybridization. The cDNA contains the nucleotide sequence encoding all of the amino acids (274 residues) comprising the putative precursor to the protein. Based on the known amino acid sequence of bovine Rieske Fe-S protein, the N-terminal extension sequence is presumed to be composed of 78 amino acids with a molecular weight of 8053. The mature protein consists of the same number of amino acid residues as that of its rat and bovine counterparts, having a homology of about 92% with the latter.     

Cloning and sequence analysis of a cDNA encoding human non-selective type of endothelin receptor.

A cDNA encoding non-selective type (ETB) of endothelin receptor was isolated from a human liver cDNA library. The cDNA had an open reading frame encoding a protein of 442 amino acid residues with a relative Mr of 49,643. The deduced amino acid sequence of human ETB receptor was 88% and 64% identical to those of rat lung ETB receptor and bovine lung ET-1-specific (ETA) receptor, respectively, and contained a relatively long and proline-rich extracellular N-terminal region in addition to a significant similarity with the G protein-coupled receptor super-family with seven transmembrane segments.     

Human lysosomal acid phosphatase: cloning, expression and chromosomal assignment.

A 2112-bp cDNA clone (lambda CT29) encoding the entire sequence of the human lysosomal acid phosphatase (EC 3.1.3.2) was isolated from a lambda gt11 human placenta cDNA library. The cDNA hybridized with a 2.3-kb mRNA from human liver and HL-60 promyelocytes. The gene for lysosomal acid phosphatase was localized to human chromosome 11. The cDNA includes a 12-bp 5' non-coding region, an open reading frame of 1269 bp and an 831-bp 3' non-coding region with a putative polyadenylation signal 25 bp upstream of a 3' poly(A) tract. The deduced amino acid sequence reveals a putative signal sequence of 30 amino acids followed by a sequence of 393 amino acids that contains eight potential glycosylation sites and a hydrophobic region, which could function as a transmembrane domain. A 60% homology between the known 23 N-terminal amino acid residues of human prostatic acid phosphatase and the N-terminal sequence of lysosomal acid phosphatase suggests an evolutionary link between these two phosphatases. Insertion of the cDNA into the expression vector pSVL yielded a construct that encoded enzymatically active acid phosphatase in transfected monkey COS cells.     

Cloning and characterization of a novel human leptin receptor overlapping transcript-like 1 gene (LEPROTL1)

A new full-length cDNA encoding a novel protein was isolated from our human fetal brain cDNA library. The cDNA consists of 2701 bp and has a putative open reading frame encoding 131 amino acids which possesses a JAK binding site (Pro(46)-Ile-Pro(48) which is preceded by a cluster of hydrophobic residues) and is highly homologous to the leptin receptor gene-related protein (OB-RGRP). Northern blot analysis showed that this new gene is widely expressed in human tissues and radiation hybrid mapping placed the gene to human chromosome 8p21.1-8p21.2.     

Cloning and sequence of a cDNA encoding human carbamyl phosphate synthetase I: molecular analysis of hyperammonemia.

Carbamyl phosphate synthetase I (CPSI) is the first enzyme involved in urea synthesis. CPSI deficiency is an autosomal recessive disorder characterized by hyperammonemic coma in the neonatal period. To analyze the enzyme and gene structures, and to elucidate the nature of mutations in CPSI deficiency, we isolated cDNA clones encoding human liver CPSI. Oligo(dT)-primed and random primer human liver cDNA libraries in lambda gt11 were screened using 5', middle, and 3' fragments of the rat CPSI cDNA as probes. Seven positive clones covered the full-length cDNA sequence with an open reading frame encoding a precursor polypeptide of 1500 amino acids (aa) (deduced Mr, 164,828) with a putative N-terminal presequence of 38 or 39 aa, a 5'-untranslated sequence of 118 bp and a 3'-untranslated sequence of 597 bp. Comparison with the rat CPSI cDNA showed that the deduced aa sequence of the human liver CPSI precursor is 94.4% identical to the rat enzyme precursor. A molecular analysis was made of the genomic DNA from three patients with CPSI deficiency. Heterogeneity of hybridized fragments that may or may not be the cause of the deficiency was apparent on the DNA blots from tissues from one patient.