Human microsomal xenobiotic epoxide hydrolase. Complementary DNA sequence, complementary DNA-directed expression in COS-1 cells, and chromosomal localization

A lambda gt11 expression library constructed from human liver mRNA was screened with an antibody against human microsomal xenobiotic epoxide hydrolase. The clone pheh32 contains an insert of 1742 base pairs with an open reading frame coding for a protein of 455 amino acids with a calculated Mr of 52,956. The nucleotide sequence is 77% similar to the previously reported rat xenobiotic epoxide hydrolase cDNA sequence. The deduced amino acid sequence of the human epoxide hydrolase is 80% similar to the previously reported rabbit and 84% similar to the deduced rat protein sequence. The NH2-terminal amino acids deduced from the human xenobiotic epoxide hydrolase cDNA are identical to the published 19 NH2-terminal amino acids of the purified human xenobiotic epoxide hydrolase protein. Northern blot analysis revealed a single mRNA band of 1.8 kilobases. Southern blot analysis indicated that there is only one gene copy/haploid genome. The human xenobiotic epoxide hydrolase gene was assigned to the long arm of human chromosome 1. Several restriction fragment length polymorphisms were observed with the human epoxide hydrolase cDNA. pheh32 was expressed as enzymatically active protein in cultured monkey kidney cells (COS-1).     

Chicken apolipoprotein A-I: cDNA sequence,tissue expression and evolution

Using an antibody against chicken apolipoprotein (apo) A-I, we identified multiple cDNA clones for the protein in two intestinal cDNA libraries in λgtll. The complete nucleotide sequence of chicken apoA-I cDNA was determined. The sequence predicts a mature protein of 240 amino acids, a 6-amino acid propeptide and an 18-amino acid signal peptide. Using a ³²P-cDNA probe, we detected the presence of apoA-I mRNA in 21 day old chicken intestine, liver, kidney, spleen, breast muscle and brain. The primary sequence of apoA-I contains numerous tandem repeats of 11 and 22 residues in a manner similar to the mammalian proteins. Our analysis of apoA-I sequences from human, rabbit, dog, rat, and chicken indicates that the rate of amino acid substitution is considerably faster in the rat lineage than in other mammalian lineages.     

Nucleotide sequence of the cDNA and the derived amino acid sequence for the major antigenic protein of foot and mouth disease virus, type Asia 1 63/72.

A 0.9 kb cDNA for the foot and mouth disease virus (FMDV) type Asia 1 63/72, cloned in the plasmid pUR222 by dC/dG tailing method, was expressed into a protein which was immunogenic in guinea pigs and cattle. The protein purified to homogeneity was found to be basic and of 38 kDa. A sequence of 879 nucleotides of the inserted cDNA was obtained. The nucleotide sequence was 65% GC-rich and was homologous to the gene for VPI of FMDV types A5, OIK and C3 to the extent of 35-40%. From the nucleotide sequence, a sequence of 293 amino acids was derived which contained 43 arginine, 4 lysine, 7 glutamic acid and 18 aspartic acid residues making the protein highly basic. The molecular weight was calculated to be 31.6 kDa. The 38 kDa protein produced by the cloned cDNA is a fused protein composed of the 293 amino acids; 5 and 55 amino acids of the alpha-complementation protein of the beta-galactosidase at the N and C terminal, respectively, and 5 amino acid coded by the dG/dC tails used for cloning the cDNA.     

Bacillus megaterium spore protein C-3: nucleotide sequence of its gene and the amino acid sequence at its spore protease cleavage site

The nucleotide sequence of the Bacillus megaterium gene coding for spore-specific protein C-3 has been determined. The gene codes for 65 amino acids and the coding sequence is preceded by an efficient ribosome-binding site. The predicted protein C-3 sequence agrees with both the amino acid composition and the amino terminal sequence of protein C-3, and shows homology (approx. 65 % of all residues are identical) with the sequences of the analogous proteins A and C of B. megaterium. Protein C-3 is cleaved by the sequence-specific B. megaterium spore protease, and the amino acid sequence at the new amino-terminus generated is identical to that predicted from the gene sequence, and homologous to the spore protease cleavage sites in the A and C proteins. The protein C-3 gene also shares a number of features with the previously sequenced protein C gene in both upstream and downstream flanking sequence.     

Amino acid sequence of the b subunit of human factor XIII, a protein composed of ten repetitive segments

Factor XIII is a plasma protein that participates in the final stages of blood coagulation. The complete amino acid sequence of the b subunit of human factor XIII was determined by a combination of cDNA cloning and amino acid sequence analysis. A lambda gt11 cDNA library prepared from human liver mRNA was screened with an affinity-purified antibody against the b subunit of human factor XIII. Nine positive clones were isolated from 2 X 10(6) phage and plaque-purified. The largest cDNA insert was sequenced and shown to contain 2180 base pairs coding for a portion of the leader sequence (19 amino acids), the mature protein (641 amino acids), a stop codon (TGA), a 3' noncoding region (187 nucleotides), and a poly(A) tail. When the b subunit of human factor XIII was digested with cyanogen bromide, nine peptides were isolated by gel filtration and reverse-phase high-performance liquid chromatography. Amino acid sequence analyses of these peptides were performed with an automated sequenator, and 299 amino acid residues were identified. These amino acid sequences were in complete agreement with the amino acid sequence predicted from the cDNA. The b subunit of factor XIII contained 10 repetitive homologous segments, each composed of about 60 amino acids and 4 half-cystine residues. Each of these repeated segments is a member of a family of repeats present in human beta 2-glycoprotein I, complement factor B, and haptoglobin alpha 1 chain. Three potential Asn-linked carbohydrate attachment sites were also identified in the b subunit of factor XIII.     

Complete cDNA sequence of chicken vigilin, a novel protein with amplified and evolutionary conserved domains.

The complete cDNA (4375 bp), coding for a new protein called vigilin, was isolated from chicken chondrocytes. The cDNA shows an open reading frame of 1270 amino acids which are organized in 14 tandemly repeated homologous domains. Each domain consists of two subdomains, one with a conserved sequence motif of 35 amino acids (subdomain A) and another one with a presumptive alpha-helical structure of 21-33 amino acids (subdomain B). 149 amino acids at the N-terminus and 71 amino acids at the C-terminus of vigilin do not show the characteristic domain structure. No sequence characteristic of a signal peptide has been found, which argues for an intracellular localisation of vigilin. Vigilin is highly expressed in freshly isolated chicken chondrocytes but little in chondrocytes after prolonged time in culture. Vigilin mRNA exists in two size species, 4.4 kb and 6.5 kb in length due to the usage of different polyadenylation sites. Comparison of the vigilin sequence with data bases showed a remarkable similarity to protein HX from Saccharomyces cerevisiae [Delahodde, A., Becam, A. M., Perea, J. & Jacq, C. (1986) Nucleic Acids Res. 14, 9213-9214]. The yeast protein consists of eight homologous domains with 11 conserved amino acid residues within a set of 35 amino acids. The N-terminal and C-terminal regions of vigilin and protein HX do not reveal any sequence similarity. These results, together with the demonstration of the characteristic vigilin sequence motif in a human cDNA clone, suggest that the repeats represent evolutionary conserved autonomous domains within a family of proteins found in yeast, chicken and man.     

Isolation and sequence analysis of a complementary DNA encoding rat liver L-gulono-gamma-lactone oxidase, a key enzyme for L-ascorbic acid biosynthesis

L-Gulono-gamma-lactone oxidase, one of the microsomal flavin enzymes, catalyzes the last step of L-ascorbic acid biosynthesis in many animals; however, it is missing in scurvy-prone animals such as humans, primates, and guinea pigs. A cDNA clone for this enzyme was isolated by screening a rat liver cDNA expression library in lambda gt11 using antibody directed against the enzyme. The cDNA clone contained 2120 nucleotides and an open reading frame of 1320 nucleotides encoding 440 amino acids of the protein with a molecular weight of 50,605. The amino-terminal sequence (residues 1-33) of the enzyme isolated from rat liver completely coincided with the corresponding part of the deduced amino acid sequence. The identity of the cDNA clone was further confirmed by the agreement of the composition of the deduced amino acids with that determined by amino acid analysis of the enzyme. Hydropathy analysis of the deduced amino acid sequence revealed several hydrophobic regions, suggesting that they anchor the protein into the microsomal membrane. The deduced amino acid sequence showed no obvious homology with the flavin-binding regions of other eight flavoenzymes.     

Human apolipoprotein A-II: complete nucleic acid sequence of preproapoA-II

The complete cDNA nucleic acid sequence of preproapolipoprotein (apo) A-II, a major protein constituent of high density lipoproteins, has been determined on clones from a human liver ds-cDNA library. Clones containing ds-cDNA for apoA-II were identified in the human liver ds-cDNA library using synthetic oligonucleotides as probes. Of 3200 clones screened, 4 reacted with the oligonucleotide probes. The DNA sequence coding for amino acids ?17 to +17 of apoA-II were determined by Maxam-Gilbert sequence analysis of restriction fragments isolated from one of these clones, pMDB2049. The remainder of the cDNA sequence was established by sequence analysis of a primer extension product synthesized utilizing a restriction fragment near the 5'-end of clone pMDB2049 as primer with total liver mRNA. The apoA-II mRNA encodes for a 100 amino acid protein, preproapoA-II that has an 18 amino acid prepeptide and a 5 amino acid propeptide terminating with a basic dipeptide (Arg-Arg) at the cleavage site to mature apoA-II.     

Nuclear-encoded chloroplast ribosomal protein L27 of Nicotiana tabacum: cDNA sequence and analysis of mRNA and genes.

A tobacco (Nicotiana tabacum cv. Petite Havana) leaf cDNA library was constructed in the expression vector lambda gt11. Immunological and nucleic acid hybridization screening yielded several cDNAs encoding an M(r) 19,641 precursor to an M(r) 14,420 mature protein which is homologous to Escherichia coli ribosomal protein L27. One cDNA (L27-1; 882 nucleotides long) contains 104 bp of 5'-noncoding sequence, 51 codons for a transit peptide, 128 codons for the predicted mature L27 polypeptide, and 241 bp of 3'-noncoding sequence, including the poly(A)29 tail. A beta-galactosidase-L27 fusion protein was bound to nitrocellulose filters, expressed, and used as an affinity matrix to purify monospecific antibody to L27 protein from an antiserum of rabbits immunized with 50S chloroplast ribosomal proteins. Using this monospecific antibody, protein L27 was identified among HPLC-purified tobacco chloroplast ribosome 50S subunit proteins. The predicted amino terminus of the mature L27 protein was confirmed by partial sequencing of the HPLC-purified L27 protein. The mature L27 protein has 66%, 61%, 56%, and 48% amino acid sequence identity with the L27-type ribosomal proteins of Bacillus subtilis, E. coli, Bacillus stearo-thermophilus, and yeast mitochondria (MRP7), respectively, in the homologous overlapping regions. The transit peptide of tobacco chloroplast ribosomal protein L27 has 41% amino acid sequence similarity with the MRP7 mitochondrial targeting sequence. Tobacco chloroplast L27 protein also has a 40 amino acid long carboxyl-terminal extension (compared to its bacterial counterparts) which is similar to the corresponding portion of yeast MRP7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and sequence of rat testis cDNA for a calcium binding polypeptide similar to the regulatory subunit of calcineurin.

We have cloned and sequenced rat testis cDNAs coding for a calcium binding polypeptide similar to calcineurin beta subunit, the Ca(2+)-binding subunit of the Ca2+/calmodulin stimulated protein phosphatase. Rat testis cDNA library was screened with a monoclonal antibody Va1 raised against bovine brain calcineurin beta subunit. The deduced amino acid sequence is similar to that of human brain calcineurin beta subunit with respect to containing four putative calcium binding sites. However, distinct differences were found: 1) The cloned cDNA had six amino acids polypeptide tail at carboxy-terminal which is absent in human brain calcineurin beta subunit. This amino acids tail makes the carboxy-terminal highly hydrophilic in contrast to the human brain beta subunit which is hydrophobic at carboxy-terminal; 2) eleven amino acids at the N terminal of the cloned cDNA were completely different from the corresponding region of the brain calcineurin beta subunit.     

The amino acid sequence of the human RNA polymerase II 33-kDa subunit hRPB 33 is highly conserved among eukaryotes

We have cloned and sequenced a cDNA of 1766 base pairs in length encoding the 275 amino acids of hRPB 33, the third largest subunit of human RNA polymerase II. The DNA was isolated by screening of a human lambda gt11 cDNA library with oligonucleotides designed on the basis of the amino acid residue analysis of the bovine material. The hRPB 33 amino acid sequence is highly conserved between Saccharomyces cerevisiae and human. Overall, 45% of the amino acid residues are identical with the yeast homologue RPB 3, and 65% of the amino acids are identical in the two major conserved regions at residues 0-103 and 151-197. hRPB 33 is also homologous to yeast RPC 5. The amino acid sequence of hRPB 33 showed no obvious homology with bacterial RNA polymerase or with any of its sigma factors.     

Characterization of cDNA encoding mouse DNA repair protein O6-methylguanine-DNA methyltransferase and high-level expression of the wild-type and mutant proteins in Escherichia coli.

A mouse cDNA clone encoding O6-methylguanine-DNA methyltransferase (MGMT), responsible for repair of mutagenic O6-alkylguanine in DNA, was cloned from a lambda gt11 library. On the basis of an open reading frame in cDNA, the mouse protein contains 211 amino acids with a molecular mass of 22 kDa. The size and the predicted N-terminal sequence of the mouse protein were confirmed experimentally. The deduced amino acid sequence of the mouse MGMT is 70% homologous to that of the human MGMT. Cysteine-149 was shown to be the only alkyl acceptor residue in the mouse protein, in confirmation of the prediction based on conserved sequences of different MGMTs. Mouse MGMT protein is recognized by some monoclonal antibodies specific for human MGMT. Site-directed mutagenesis was utilized to reclone the mouse cDNA in a T7 promoter-based vector for overexpression of the native repair protein in Escherichia coli. The mouse protein has a tetrapeptide sequence, Pro-Glu-Gly-Val at positions 56-59, absent in the human protein. Neither deletion of this tetrapeptide nor substitution of valine-169 with alanine affected the activity of the mutant proteins.     

Amino acid sequence of human histidine-rich glycoprotein derived from the nucleotide sequence of its cDNA

A lambda gt 11 library containing cDNA inserts prepared from human liver mRNA has been screened with an affinity-purified antibody to human histidine-rich glycoprotein (HRG) and then with a restriction fragment isolated from the 5' end of the largest cDNA insert obtained by antibody screening. A number of positive clones were identified and shown to code for HRG by DNA sequence analysis. A total of 2067 nucleotides were determined by sequencing 3 overlapping cDNA clones, which included 121 nucleotides of 5'-noncoding sequence, 54 nucleotides coding for a leader sequence of 18 amino acids, 1521 nucleotides coding for the mature protein of 507 amino acids, a stop codon of TAA, and 352 nucleotides of 3'-noncoding sequence followed by a poly(A) tail of 16 nucleotides. The length of the noncoding sequence of the 3' end differed in several clones, but each contained a polyadenylylation or processing sequence of AATAAA followed by a poly(A) tail. More than half of the amino acid sequence of HRG consisted of five different types of internal repeats. Within the last 3 internal repeats (type V), there were 12 tandem repetitions of a 5 amino acid segment with a consensus sequence of Gly-His-His-Pro-His. This repeated portion, referred to as a "histidine-rich region", contained 53% histidine and showed a high degree of similarity to a histidine-rich region of high molecular weight kininogen.     

Cloning and sequence analysis of the cDNA for human placental NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase.

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase catalyzes the oxidation of many prostaglandins at C-15, resulting in a subsequent reduction in their biological activity. We report the isolation of the cDNA for this enzyme. A human placental lambda gt11 cDNA library was screened using polyclonal antibodies prepared against the human placental enzyme. A 2.5-kilobase cDNA containing the entire coding region for the enzyme was isolated. The cDNA encodes for a protein of 266 amino acids with a calculated Mr of 28,975. Identification of the cDNA as that coding for 15-hydroxyprostaglandin dehydrogenase was based on the comparison of the deduced amino acid sequence with the amino acid sequence of two peptides, one from the rabbit lung enzyme and the other from the human placental enzyme. This cDNA hybridizes with two species of poly(A+) RNA isolated from human placenta: one of 3.4 kilobases and the other of 2.0 kilobases. Isolation of the cDNA for 15-hydroxyprostaglandin dehydrogenase should facilitate studies on the structure, function, and regulation of this enzyme.     

Cloning and sequence analysis of cnaA gene encoding the catalytic subunit of calcineurin from Aspergillus oryzae

Calcineurin has been implicated in ion-homeostasis, stress adaptation in yeast and for hyphal growth in filamentous fungi. Genomic DNA and cDNA encoding the catalytic subunit of calcineurin (cnaA) were isolated from Aspergillus oryzae. The cnaA open reading frame extended to 1727 bp and encoded a putative protein of 514 amino acids. Comparative analysis of the nucleotide sequence of cnaA genomic DNA and cDNA confirmed the presence of three introns and a highly conserved calmodulin binding domain. The deduced amino acid sequence was homologous to calcineurin A from Aspergillus nidulans (92%), Neurospora crassa (84%), human (67%), Saccharomyces cerevisiae (58%) and Schizosaccharomyces pombe (54%). Further, A. oryzae cnaA cDNA complemented S. cerevisiae calcineurin disruptant strain (Deltacmp1 Deltacmp2), which was not viable in the presence of high concentrations of NaCl (1.2 M) and at alkaline pH 8.5.     

The complete amino acid sequence of human complement factor H.

The complete amino acid sequence of the human complement system regulatory protein, factor H, has been derived from sequencing three overlapping cDNA clones. The sequence consists of 1213 amino acids arranged in 20 homologous units, each about 60 amino acids long, and an 18-residue leader sequence. The 60-amino-acid-long repetitive units are homologous with those found in a large number of other complement and non-complement proteins. Two basic C-terminal residues, deduced from the cDNA sequence, are absent from factor H isolated from outdated plasma. A tyrosine/histidine polymorphism was observed within the seventh homologous repeat unit of factor H. This is likely to represent a difference between the two major allelic variants of factor H. The nature of the cDNA clones indicates that there is likely to be an alternative splicing mechanism, resulting in the formation of at least two species of factor H mRNA.     

Cloning and sequence analysis of beta-4 cDNA: an integrin subunit that contains a unique 118 kd cytoplasmic domain

The alpha 6 beta 4 complex is a member of the integrin superfamily of adhesion receptors. A human keratinocyte lambda gt11 cDNA library was screened using a monoclonal antibody directed against the beta 4 subunit. Two cDNAs were selected and subsequently used to isolate a complete set of overlapping cDNA clones. The beta 4 subunit consists of 1778 amino acids with a 683 amino acid extracellular domain, a 23 amino acid transmembrane domain and an exceptionally long cytoplasmic domain of 1072 residues. The deduced amino-terminal sequence is in good agreement with the published amino-terminal sequence of purified beta 4. The extracellular domain contains five potential N-linked glycosylation sites and four cysteine-rich homologous repeat sequences. The extracellular part of the beta 4 subunit sequence shows 35% identify with other integrin beta subunits, but is the most different among this class of molecules. The transmembrane region is poorly conserved, whereas the cytoplasmic domain shows no substantial identity in any region to the cytoplasmic tails of the known beta sequences or to other protein sequences. The exceptionally long cytoplasmic domain suggests distinct interactions of the beta 4 subunit with cytoplasmic proteins.