Cloning and sequencing of chloroperoxidase cDNA.

An oligod-d(T) 12-18 primed cDNA library has been prepared from Caldariomyces fumago mRNA. A clone containing a full-length insert was sequenced on the supercoiled plasmid, pBR322. The complete primary sequence of chloroperoxidase has been derived. We have also determined about 73% of the peptide sequence by amino acid sequencing. The DNA sequence data matches all of the available known peptide sequences. The mature polypeptide contains 300 amino acids having a combined molecular weight of 32,974 daltons. A putative signal peptide of 21 amino acids is proposed from DNA sequence data. The chloroperoxidase gene encodes three potential glycosylation sites recognized as Asn-X-Thr/Ser sequences. Three cysteine residues are found in the protein sequence. A small region around Cys87 bears a minimal homology to the active site of cytochrome P450cam. No other heme protein homologues can be detected. We propose that Cys87 serves as a thiolate ligand to the iron of heme prosthetic group. A rare arginine codon, AGG, is used three times out of twelve in contrast to the very infrequent use of this codon in E. coli or yeast.     

Molecular cloning and nucleotide sequence of cDNA coding for rat brain cholecystokinin precursor

A mixture of 14-mer oligodeoxynucleotides was used for the screening of a cDNA clone coding for a cholecystokinin (CCK) precursor from a cDNA library for rat brain microsomal poly(A)RNA. The longest insert is 718 bp long which was verified to contain a nearly full-length cDNA sequence coding for rat CCK precursor, because the size of CCK mRNA was estimated to be about 850 bases long by Northern blotting analysis. Sequence analysis revealed 110 bp in the 5'-untranslated region, 345 bp in the amino acid coding region corresponding to the CCK precursor and 263 bp in the 3'-noncoding region which contains polyadenylation signal AUUAAA and the poly(A) sequence. The precursor may contain a 28 amino acid signal peptide and 12 additional amino acids at the carboxyl terminus.     

Isolation of a cDNA clone for transition protein 1 (TP1), a major chromosomal protein of mammalian spermatids

We have isolated a cDNA clone for rat transition protein 1 (TP1), a major chromosomal protein of mammalian spermatids. The clone was identified initially by hybrid selection of TP1 mRNA. The sequence of the 251-nucleotide cDNA includes the entire coding region for the protein, thereby confirming the identity of the clone as well as predicting two changes in the published amino acid sequence.     

Rat Brain Glutamic Acid Decarboxylase Sequence Deduced from a Cloned cDNA

A cDNA clone complementary to the rat brain glutamic acid decarboxylase mRNA was isolated from a rat brain cDNA expression library using an antibody specific to the enzyme. The cDNA insert has been shown to direct the synthesis of an active protein in Escherichia coli. In this study, the nucleotide sequence of this clone, which includes the complete coding region, is presented. The predicted protein is 593 amino acids in length. The first 557 residues display a 95% identity when compared with the corresponding cat sequence. However, the deduced amino acid sequence of the carboxy-terminal end of the rat protein, downstream of residue 557, is totally different from the cat, whereas it agrees with a published partial peptidic sequence of the rat protein.     

Molecular cloning and sequence analysis of cDNA for the catalytic subunit 1 alpha of rat kidney type 1 protein phosphatase, and detection of the gene expression at high levels in hepatoma cells and regenerating livers as compared to rat livers.

A cDNA clone containing the full coding sequence of a type 1 protein phosphatase catalytic subunit 1 alpha has been isolated from a rat kidney lambda gt 10 library. The protein sequence deduced from the cDNA contains 330 amino acid residues with a molecular mass of 38 kDa. The cDNA clone from rat kidney was 89% identical at the nucleotide level in the coding region to type 1 protein phosphatase 1 alpha from rabbit skeletal muscle. However, the two protein sequences were completely identical. The type 1 alpha protein phosphatase from rat kidney shows 49% homology of amino acid sequence to the rat type 2A alpha protein phosphatase. Thus, the protein sequence of type 1 alpha protein phosphatase was completely conserved between rat and rabbit. The mRNA levels of type 1 protein phosphatase were determined in rat liver, AH13, a strain of rat hepatoma, and regenerating rat liver by Northern blot analysis using the cDNA fragment as a probe, under which conditions a single mRNA of 1.5 kb was detected. The mRNA levels of AH13 were remarkably increased when compared to those of normal ivers, whereas the mRNA levels of regenerating livers were slightly but significantly increased. These results demonstrate a marked increase in gene expression of type 1 protein phosphatase in hepatoma cells, suggesting an important role of the type 1 protein phosphatase in hepatocarcinogenesis.     

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.     

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.     

Cloning, expression, and catalytic mechanism of the low molecular weight phosphotyrosyl protein phosphatase from bovine heart.

The first representative of a group of mammalian, low molecular weight phosphotyrosyl protein phosphatases was cloned, sequenced and expressed in Escherichia coli. Using a 61-mer oligonucleotide probe based on the amino acid sequence of the purified enzyme, several overlapping cDNA clones were isolated from a bovine heart cDNA library. A full-length clone was obtained consisting of a 27-bp 5' noncoding region, an open reading frame encoding the expected 157 amino acid protein, and an extensive 3' nontranslated sequence. The identification of the clone as full length was consistent with results obtained in mRNA blotting experiments using poly(A)+ mRNA from bovine heart. The coding sequence was placed downstream of a bacteriophage T7 promoter, and protein was expressed in E. coli. The expressed enzyme was soluble, and catalytically active and was readily isolated and purified. The recombinant protein had the expected Mr of 18,000 (estimated by SDS-PAGE), and it showed cross-reactivity with antisera that had been raised against both the bovine heart and the human placenta enzymes. The amino acid sequence of the N-terminal region of the expressed protein showed that methionine had been removed, resulting in a sequence identical to that of the enzyme isolated from the bovine tissue, with the exception that the N-terminal alanine of the protein from tissue is acetylated. A kinetically competent phosphoenzyme intermediate was trapped from a phosphatase-catalyzed reaction. Using 31P NMR, the covalent intermediate was identified as a cysteinyl phosphate. By analogy with the nomenclature used for serine esterases, these enzymes may be called cysteine phosphatases.     

Cloning of the C alpha catalytic subunit of the bovine cAMP-dependent protein kinase.

The bovine C alpha type catalytic subunit of the cAMP-dependent protein kinase was cloned. A partial cDNA was isolated from a bovine heart cDNA library. This clone contained 120 bp of the coding sequence and the entire 3' untranslated region of 1431 bp. The complete coding region was cloned by PCR amplification from total bovine heart and skeletal muscle RNA. The sequence of the 3' oligonucleotide was taken from the partial cDNA clone whereas the 5' oligonucleotide was chosen by comparison of sequences of published C alpha subunits from other species. In the deduced amino acid sequence there is one deviation from the published bovine C alpha protein sequence, aspartic acid 286 is exchanged by an asparagine. The C alpha mRNA was found to be expressed differentially in various bovine tissues.     

Transient expression of human adenosine deaminase cDNAs: identification of a nonfunctional clone resulting from a single amino acid substitution.

Human adenosine deaminase (ADA) is an important purine catabolic enzyme which irreversibly deaminates adenosine and deoxyadenosine. Severe genetic deficiency of ADA leads to an immunological deficiency state in which T-lymphoid cells are selectively destroyed by the accumulation of toxic levels of deoxyadenosine and deoxy-ATP. In preparation for transfer of ADA sequences into a variety of cell types, we explored expression of ADA cDNAs transfected into cultured cells within a simian virus 40-based expression vector. After transfection into monkey kidney (COS) cells, ADA cDNA encompassing the entire coding region of the protein generated human ADA activity. An unexpected finding, however, was the identification of a cDNA clone that failed to produce either human enzyme activity or immunoreactive ADA protein. As this pattern is typical of many naturally occurring mutant ADA alleles, we characterized the molecular defect in this clone. DNA sequence analysis revealed a single nucleotide substitution in amino acid position 50 (glycine-valine). Northern blotting with a unique 17-mer oligonucleotide demonstrated the absence of the mutant sequence in the mRNA from which the cDNA library giving rise to the mutant cDNA was constructed. Therefore, the substitution in the variant cDNA was created during cloning. These data define one critical region of the human ADA protein molecule and suggest a convenient strategy for characterization of the phenotypes associated with naturally occurring mutant alleles.     

Major acute-phase alpha(1)protein in the rat: structure, molecular cloning, and regulation of mRNA levels

Rat major acute-phase alpha(1)protein (MAP) was characterized by determining its secondary structure, ligand binding and partial amino acid sequence. A cDNA clone expressing MAP and coding for the entire mature protein was isolated from a cDNA library in E. coli prepared from rat liver mRNA. By hybridization to nick translated cDNA, mRNA for MAP was found only in liver, where it increased 17-fold during acute inflammation. Constant proportions of rates of leucine incorporation into MAP over mRNA levels in liver indicated that the regulation of the synthesis of MAP is due to a change in the rate of synthesis and/or the stability of mRNA for MAP, but not the rate of its translation.     

A single WAP domain-containing protein from Litopenaeus vannamei hemocytes

A cDNA clone coding for a single WAP domain (SWD) protein was isolated from a hemocyte cDNA library of Litopenaeus vannamei. The full-length cDNA sequence is 0.4kb long and encodes a 93-amino acid protein. Using this sequence as a probe a similar clone coding for a 92-amino acids protein was found in a cDNA library from Penaeus monodon hemocytes. The mRNA size was confirmed by Northern blot as well as that gene is expressed in hemocytes, but not in hepatopancreas. mRNA levels of the shrimp SWD protein were modified after injection of Vibrio alginolyticus, indicating the probable role of this protein in the immune response. Although amino acid sequence seems to be similar to those of other WAP domain-containing proteins, shrimp SWD protein does not have any other functional domain, similar to a mouse single WAP motif (SWAM) protein reported in mouse; however, the phylogenetic analysis shows that shrimp SWD is more related to other WAP proteins than to mouse SWAM.     

Structure of soybean Kunitz trypsin inhibitor mRNA determined from cDNA by using oligodeoxynucleotide primers

Oligodeoxynucleotides complementary to the deduced mRNA sequence of soybean Kunitz trypsin inhibitor (KTI) were used to prime the synthesis of cDNA from soybean cotyledon total poly(A) RNA. The primed cDNA was used to select clones from a Glycine max cotyledon cDNA library. Two out of twelve hybridizing clones were shown to contain KTI cDNA. The nucleotide sequence of one clone, pSTI 9-2, was determined and it was found to encompass the complete protein coding region of KTI excet for three C-terminal residues. Trypsin inhibitor is synthesized with a 25 amino acid hydrophobic N-terminal sequence presumed to be a signal peptide. The mature polypeptide encoded by pSTI 9-2 agrees with the published amino acid composition of KTI, but contains two discrepancies at the peptide sequence level.     

Isolation and characterization of a cDNA clone encoding rat nucleoside diphosphate kinase

A cDNA clone for cytosolic nucleoside diphosphate (NDP) kinase was isolated from a cDNA library of rat skeletal muscle using synthetic oligonucleotides as probes. The clone constitutes a 621-base pair cDNA sequence including the 456-base pair coding region and 137-base pair 3'-untranslated one with polyadenylation site. The complete primary structure of NDP kinase was deduced from the coding sequence. An NH2-terminal amino acid sequence analysis suggested that the translated enzyme protein suffered proteolytic cleavage followed by modification at the alpha-NH2 group of the newly produced NH2-terminal amino acid residue. Taking this into account, it was tentatively concluded that the mature NDP kinase consists of 147 amino acid residues with a molecular weight of 16,724. Northern blot hybridization analysis showed that NDP kinase mRNA could be detected in total RNA fractions of brain, spleen, heart, lung, liver, kidney, testis as well as skeletal muscle, and that there was no difference in the size of mRNAs from these tissues. Tissue distribution of the mRNA nearly paralleled those of protein moiety and activity of the enzyme.     

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.     

A chimaeric tryptophan decarboxylase gene as a novel selectable marker in plant cells

A cDNA clone, pMA1949, detects two mRNA species in wheat seedling tissue that are late embryogenesis-abundant (LEA) and dehydration stress-inducible. Sequence analysis of the pMA1949 clone shows it to be a 991 bp partial cDNA encoding a polypeptide of 317 amino acids with homology to two group 3 LEA proteins, carrot (DC8) and a soybean protein encoded by pGmPM2 cDNA. Molecular analysis of the deduced protein reveals a 33 kDa acidic and extremely hydrophilic protein with potential amphiphilic -helical regions. In addition, the protein contains eleven similar, contiguous repeats of 11 amino acids, which are separated by 118 amino acids from two additional and unique repeats of 36 residues each at the carboxyl end of the protein. Comparisons of sequences of reported group 3 LEA proteins revealed that there are two types, separable by sequence similarity of the 11 amino acid repeating motifs and by the presence or absence of a certain amino acid stretch at the carboxyl terminus. Based on resuls from these comparisons, we propose a second type of group 3 LEA proteins, called group 3 LEA (II).     

Isolation and structural organization of the Neurospora crassa copper metallothionein gene.

The Neurospora crassa copper metallothionein gene was cloned and its complete nucleotide sequence is reported. Enriched metallothionein mRNA was used as a template for cDNA synthesis, primed by a metallothionein-specific, synthetic undecanucleotide. The sequence of the cDNA obtained allowed the synthesis of a unique 21-mer which was used to screen a genomic DNA library of N. crassa. In agreement with the published amino acid sequence, the gene codes for a polypeptide 26 amino acid residues in length. The coding region is interrupted by a small intron (94 nucleotides). The gene structure is compared with those of mammalian metallothioneins. In both cases, the coding regions are split by introns, the intron-exon boundaries, however, are in different positions. The neurospora copper metallothionein gene is, to our knowledge, the smallest gene interrupted by an intron isolated so far.     

Identification of the fifth axial heme ligand of chloroperoxidase

Chloroperoxidase from Caldariomyces fumago catalyzes the peroxidative chlorination of organic acceptor molecules. From a variety of spectroscopic data, it had long been thought that chloroperoxidase possessed an active site structure similar to that of cytochrome P-450cam. Resonance Raman studies conducted with isotopically substituted enzyme proved conclusively that the fifth axial ligand to the ferriprotoporphyrin IX moiety of chloroperoxidase is indeed a cysteine thiolate (Bangcharoenpaurpong, O., Champion, P. M., Hall, K. S., and Hager, L. P. (1986) Biochemistry 25, 2374-2378). In this study, Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid), was used to ascertain which of the 3 cysteine residues in the primary structure of chloroperoxidase serves as the fifth axial heme ligand; two of the cysteine residues were earlier shown to be involved in a disulfide linkage. Apoprotein was labeled under denaturing conditions with 5,5'-dithiobis(2-nitrobenzoic acid). A unique peptide, containing the thionitrobenzoate adduct, was isolated via reverse phase HPLC following digestion with endoproteinase Glu-C. Amino acid and Edman sequence analysis revealed the fifth axial ligand in chloroperoxidase to be cysteine 29. Under reducing and denaturing conditions, incubation of apochloroperoxidase with Ellman's reagent resulted in 3 labeled residues. Proteolysis and isolation of the labeled peptides using reverse phase HPLC and subsequent Edman sequence analysis detected and identified the thionitrobenzoate adducts of each of the three cysteinyl peptides of chloroperoxidase.