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.     

Cloning and expression of a cDNA coding for a rat liver plasma membrane ecto-ATPase. The primary structure of the ecto-ATPase is similar to that of the human biliary glycoprotein I

The amino acid sequence of the ecto-ATPase from rat liver was deduced from analysis of cDNA clones and a genomic clone. Immunoblots with antibodies raised against a peptide sequence deduced from the cDNA sequence indicated that the determined amino acid sequence is that of the ecto-ATPase. The deduced sequence predicts a 519-amino acid protein with a calculated molecular mass of 57,388 daltons. There are 16 potential asparagine-linked glycosylation sites in the protein. Hydropathy analysis of the deduced amino acid sequence indicates that the protein has two hydrophobic stretches. One is located at the N-terminal and the other is near the C-terminal end. A full-length clone encoding the ecto-ATPase was expressed transiently in mouse L cells and human HeLa cells. The cell lysate from the transfected cells contained immunoreactive ecto-ATPase and Ca2+-stimulated ATPase activities. The expressed protein is glycosylated and has an apparent molecular weight (100,000) similar to that of the rat liver plasma membrane ecto-ATPase.     

Molecular characterization of the interferon-induced 15-kDa protein. Molecular cloning and nucleotide and amino acid sequence

We have isolated a cDNA clone for an interferon-induced 15-kDa protein. The cDNA clone was prepared from mRNA isolated from interferon-beta-treated human Daudi cells. The clone of 635 base pairs contains an open reading frame coding for a protein of 145 amino acids, and suggests for the mRNA a 75-base pair 5' untranslated and a 125-base pair 3' untranslated region. Approximately 85% of the amino acid sequence of the 15-kDa protein has been independently obtained from 2 nmol of material using microsequencing technology on the N terminus of the intact protein and on tryptic and chymotryptic peptides. The amino acid sequence of the isolated protein is identical to the amino acid sequence deduced from the cDNA. Northern blot analysis confirmed that the mRNA for the 15-kDa protein is undetectable in untreated cells, but is greatly induced following interferon treatment.     

Bovine hydroxyindole-O-methyltransferase. Significant sequence revision.

Hydroxyindole-O-methyltransferase (HIOMT) catalyzes the final step in melatonin synthesis. The nucleotide and deduced amino acid sequences of bovine HIOMT have been reported. Our laboratory recently isolated a cDNA clone encoding human HIOMT. Comparison of the human and bovine nucleotide sequences revealed several discrepancies which prevented perfect alignment and produced defined regions of virtually no homology in the deduced amino acid sequence. Consequently, we repeated sequence analysis of the original bovine HIOMT cDNA clone, the results of which are reported here. The revised nucleotide sequence includes 23 differences from the published sequence. This completely changes the deduced amino acid sequence in two regions, encompassing a total of 96 residues, or 28% of the protein. The revised deduced amino acid sequence predicts different post-translational modifications as compared to that of the original deduced sequence. This information will make it possible in future investigations of HIOMT to design improved polymerase chain reaction primers, peptides for the generation of antisera, and probes for various types of analysis and screening of libraries.     

Characterization of a cDNA clone encoding a Brassica napus 12 S protein (cruciferin) subunit. Relationship between precursors and mature chains

Cruciferin (12 S globulin) is a large, neutral, oligometric protein synthesized in rapeseed (Brassica napus) during seed development. It is the major seed protein and is composed of six subunit pairs. Each of these pairs is synthesized as a precursor containing one heavy alpha-chain and one light beta-chain. Electrophoretic analysis of cruciferin showed that four different alpha- and four different beta-chains exist. A cruciferin clone was selected from an embryo cDNA library. This clone, pCRU1, contains a 1518-base pair open reading frame corresponding to a truncated NH2-terminal signal sequence followed by an alpha-chain of 296 and a beta-chain of 190 amino acid residues. Individual cruciferin chains as well as peptides thereof were subjected to NH2-terminal amino acid sequence analysis. The sequences obtained from a specific alpha- and beta-chain pair (alpha 1 and beta 1) showed total identity with the deduced amino acid sequence from pCRU1. Further comparisons revealed that a previously characterized cruciferin cDNA clone encodes one of the precursors for the closely related alpha 2/ alpha 3-beta 2/beta 3 subunits. The deduced amino acid sequences of the two cDNA clones display 64% similarity.     

3-Ketoacyl-acyl carrier protein synthase III from spinach (Spinacia oleracea) is not similar to other condensing enzymes of fatty acid synthase.

A cDNA clone encoding spinach (Spinacia oleracea) 3-ketoacyl-acyl carrier protein synthase III (KAS III), which catalyzes the initial condensing reaction in fatty acid biosynthesis, was isolated. Based on the amino acid sequence of tryptic digests of purified spinach KAS III, degenerate polymerase chain reaction (PCR) primers were designed and used to amplify a 612-bp fragment from first-strand cDNA of spinach leaf RNA. A root cDNA library was probed with the PCR fragment, and a 1920-bp clone was isolated. Its deduced amino acid sequence matched the sequences of the tryptic digests obtained from the purified KAS III. Northern analysis confirmed that it was expressed in both leaf and root. The clone contained a 1218-bp open reading frame coding for 405 amino acids. The identity of the clone was confirmed by expression in Escherichia coli BL 21 as a glutathione S-transferase fusion protein. The deduced amino acid sequence was 48 and 45% identical with the putative KAS III of Porphyra umbilicalis and KAS III of E. coli, respectively. It also had a strong local homology to the plant chalcone synthases but had little homology with other KAS isoforms from plants, bacteria, or animals.     

Cloning of rat brain protein kinase C complementary DNA

Four peptides derived from rat brain protein kinase C were partially sequenced. Using synthetic oligonucleotides deduced from the amino acid sequences as probes, a clone of complementary DNA (cDNA) was isolated from a cDNA library prepared from the same tissue. The nucleotide sequence of this cDNA clone revealed the primary structure of the carboxyl-terminal region as having 224 amino acids, with significant sequence homology with cyclic AMP-dependent and cyclic GMP-dependent protein kinases.     

Molecular cloning and nucleotide sequence of full-length cDNA for ascorbate oxidase from cultured pumpkin cells

A lambda gt11 cDNA expression library was constructed from size-fractionated poly(A)-rich RNA of cultured pumpkin cells. A full-length cDNA clone for ascorbate oxidase mRNA was selected from the library by screening with synthetic oligonucleotides designed from the amino-terminal sequence of ascorbate oxidase protein. The identity of the clone was confirmed by comparing the amino acid sequence deduced by nucleotide sequence analysis with that determined for the amino-terminal sequence of pumpkin ascorbate oxidase. The nucleotide sequence of the cDNA insert was found to contain an open reading frame of 579 codons corresponding to a signal peptide of 30 amino acids and the mature 549-residue ascorbate oxidase. Furthermore, it was found that the amino acid sequence deduced from the nucleotide sequence of the cDNA insert contained four potential N-glycosylation sites and copper-binding amino acid residues located in four regions where the sequence was identical or nearly identical to those of the other known blue multicopper oxidases Neurospora crassa laccase and human ceruloplasmin.     

Complete nucleotide sequence of hepatic 5-aminolaevulinate synthase precursor

Chick embryo liver mitochondrial matrix protein, 5-aminolaevulinate synthase, is synthesised initially as a larger cytosolic precursor. In this report we present the complete nucleotide sequence of a cDNA clone coding for the precursor together with corresponding confirmatory amino acid sequence of peptides derived from purified mature mitochondrial enzyme. The deduced amino acid sequence shows that the precursor consists of mature enzyme of 579 amino acids and an N-terminal extension of 56 amino acids. The latter presequence is highly basic in character as found with other mitochondrial preproteins.     

cDNA molecular cloning of Geotrichum candidum lipase

The cDNA clone of Geotrichum candidum (Geo.) lipase was isolated from the Geo. cDNA library by colony hybridization using 32P-labeled oligonucleotides corresponding to a partial amino acid sequence of this enzyme. The nucleotide sequence of the cDNA determined by the dideoxy chain terminating method included some partial amino acid sequences determined by Edman degradation, and the overall amino acid composition deduced from the cDNA coincided with that from amino acid analysis of this protein. The cloned cDNA coded a protein of 554 amino acids and a hydrophobic signal sequence of 19 amino acids. Geo. lipase contained the -Gly-X-Ser-X-Gly- sequence which is believed to form part of the interfacial lipid recognition site.     

Bacillus licheniformis APase I gene promoter: a strong well-regulated promoter in B. subtilis

The 5' regulatory region and the portion of the structural gene coding for the amino-terminal sequence of alkaline phosphatase I (APase I) were isolated from Bacillus licheniformis MC14 using a synthetic oligodeoxynucleotide deduced from the amino acid sequence of the enzyme. The DNA sequence analysis of this region revealed an open reading frame of 129 amino acids containing the amino-terminal sequence of the mature APase protein. The protein sequence was preceded by a putative signal sequence of 32 amino acid residues. The predicted amino acid sequence of the partial APase clone as well as the experimentally determined amino acid sequence of the enzyme indicated that B. licheniformis APase retains the important features conserved among other APases of Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae, and various human tissues. Heterologous expression studies of the promoter using a fusion with the lacZ gene indicated that it functions as a very strong inducible promoter in B. subtilis that is tightly regulated by phosphate concentration.     

Molecular cloning of the complementary DNA for a human folate binding protein

The complementary DNA for a human folate binding protein has been cloned from a lambda gt11-cDNA library prepared from cultured KB cells. A number of clones were selected by immunoscreening with a monospecific antiserum and by oligonucleotide probes corresponding to the NH2-terminal sequence of the folate binding protein. A partial nucleotide sequence of the cDNA was determined directly from the lambda gt11 phage and after subcloning into M13. The 18 amino acids deduced from the initial 19 codons were exactly the same as the amino acid sequence obtained by peptide analysis of the purified protein providing proof that this clone is the folate binding protein cDNA.     

Cloning of the human cDNA for the U1 RNA-associated 70K protein.

Anti-RNP sera were used to isolate a cDNA clone for the largest polypeptide of the U1 snRNP, a protein of mol. wt 70 kd designated 70K, from a human liver cDNA library constructed in the expression vector pEX1. The cro-beta-galactosidase-70K fusion protein reacted with various anti-RNP patient sera, a rabbit anti-70K antiserum, as well as with a monoclonal antibody specific for this protein. The sequences of four 70K peptides were determined and they match parts of the deduced amino acid sequence of the 1.3 kb insert of p70.1 indicating that it is a genuine 70K cDNA. Screening of a new cDNA library constructed from polysomal mRNA of HeLa cells with the p70.1 clone yielded an overlapping clone, FL70K, which was 2.7 kb long and covered the complete coding and 3'-untranslated sequence of the 70K protein in addition to 680 nucleotides upstream of the putative initiation codon, The predicted mol. wt of the encoded protein is approximately 70 kd. Amino acid analysis of the purified HeLa 70K protein yielded values close or identical to those deduced from the nucleotide sequence of the full-length cDNA. The 70K protein is rich in arginine (20%) and acidic amino acids (18%). Extremely hydrophilic regions containing mixed-charge amino acid clusters have been identified at the carboxyl-terminal half of the protein, which may function in RNA binding. A sequence comparison with two recently cloned RNA binding proteins revealed homology with one region in the U1 RNP 70K protein. This domain may also be responsible for RNA binding.     

Nucleotide sequence of cloned cDNA specific for rat ribosomal protein S11

A cDNA clone specific for rat ribosomal protein S11 was isolated by hybrid-selected translation from the cDNA library made for 8-9 S poly(A) RNA from regenerating rat liver. Since this cDNA had not enough length, another clone was selected by colony hybridization using a fragment of isolated cDNA as a probe. The nucleotide sequence of the cDNA was determined. The sequence contains 2 base pairs from the 5' noncoding region, the entire coding region of 477 base pairs, and the 3' noncoding region of 55 base pairs besides the poly(A) tail. The primary structure of the protein S11 was deduced from the nucleotide sequence. It consists of 157 amino acids. Its molecular weight is 18,299. The calculated amino acid composition is consistent with the reported composition of S11 determined on the protein hydrolysate. The amino acid sequence showed a marked homology with that of S16 of Halobacterium cutirubrum and an appreciable homology with that of S17 of Escherichia coli.     

The primary structure of a plant storage protein: zein.

The protein sequence of a representative of the zeins, the major storage proteins of maize, has been derived from the nucleotide sequence of a zein cDNA clone. This cDNA was sequence both by the Maxam and Gilbert and the M13-dideoxy techniques. The nucleotide sequence encompasses the non-translated 3' terminus of the mRNA, the entire coding sequence specifying both the mature zein protein and a small signal peptide, and a portion of the non-translated 5' region. The deduced amino acid composition and the amino-terminal amino acid sequence closely resemble those derived from chemical analysis of the zein protein fraction. The data presented represent the first complete amino acid sequence of a plant storage protein.     

cDNA cloning and sequencing of tobacco chloroplast ribosomal protein L12

Tobacco chloroplast ribosomal protein L12 was isolated as a ssDNA-cellulose-binding protein from a chloroplast soluble protein fraction. Based on the N-terminal amino acid sequence of chloroplast L12, a cDNA clone was isolated and characterized. The precursor protein deduced from the DNA sequence consists of a transit peptide of 53 amino acid residues and a mature L12 protein of 133 amino acid residues. The chloroplast L12 protein was synthesized with a reticulocyte lysate and subjected to nucleic acid-binding assays. L12 synthesized in vitro does not bind to ssDNA, dsDNA nor ribonucleotide homopolymers, but it binds to cellulose matrix.