Molecular cloning and sequencing of a cDNA coding for mature human kidney cathepsin H

A cDNA library was established from human kidney RNA and screened with an extended oligonucleotide probe derived from the amino-acid sequence of human cathepsin H. A recombinant clone, pRF15, was isolated and characterized. DNA sequence analysis of its 1106-nucleotide-long insert revealed that pRF15 encodes the complete protein sequence of mature cathepsin H plus 28 amino acids of a propeptide, thus confirming that cathepsin H is synthesized as a larger precursor molecule and posttranslationally processed. Northern blot analysis indicated that cathepsin H is predominantly synthesized in kidney. A high degree of sequence homology was observed with rat cathepsin H, especially within the propeptide. The part of the prosequence coding for the "minichain" is conserved in the prosequence of aleurain, a plant thiol protease.     

Molecular cloning of human apolipoprotein B cDNA.

In this paper we describe the isolation of cDNA clones which code for parts of apolipoprotein B (apoB). The clones were obtained by immunoscreening of an expression library (lambda gt 11) derived from a human hepatoma cell line (Hep G2). The relationship between positive clones and apoB was established with immunochemical techniques using polyclonal as well as monoclonal antibodies. Recombinants, expressing nonoverlapping regions of apoB are described, all hybridizing with a very large mRNA (approximately 20,000 bases long). The nucleotide sequence obtained predicts a primary protein structure with a composition suitable for the formation of stretches of an amphipatic alpha-helix.     

Purification of a human urinary carboxypeptidase (kininase) distinct from carboxypeptidases A, B, or N

A carboxypeptidase which cleaves basic C-terminal amino acids from peptides was purified from concentrated human urine by a three-step procedure: chromatography on Affi-Gel Blue, arginine-Sepharose affinity chromatography, and gel filtration by HPLC on a TSK-G3000SW column. Urinary carboxypeptidase was purified 406-fold with an 11% yield and a specific activity of 49 mumol/min/mg with benzoylglycylargininic acid as substrate. It migrated as a single band of Mr 75,700 in polyacrylamide gel electrophoresis with sodium dodecyl sulfate. It cleaved benzoylglycylarginine, benzoylglycyllysine, benzoylglycylargininic acid, benzoylalanyllysine, and benzoylphenylalanyllysine at different relative rates than human plasma carboxypeptidase N, the Mr 48,000 active subunit of carboxypeptidase N or human pancreatic carboxypeptidase B. Urinary carboxypeptidase did not hydrolyze benzoylglycylphenylalanine, a substrate of carboxypeptidase A, but readily cleaved bradykinin with a Km of 46 microM and a Kcat of 32 min-1. Its activity was enhanced by CoCl2 and inhibited by cadmium acetate, o-phenanthroline, or DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid. The enzyme had a pH optimum of 7.0 and its activity dropped at pH 6.0 by 60%. It was stable for at least 2 h at 37 degrees C (pH 8.0) but was unstable at room temperature below pH 4.5. The molecular weight, electrophoretic mobility, and activity of urinary carboxypeptidase was not affected by trypsin. The effect of pH and stability further distinguished the urinary carboxypeptidase from other human carboxypeptidases. Urinary carboxypeptidase was immunologically distinct from carboxypeptidase N when analyzed by the "Western blot" technique. Thus, human urine contains a basic carboxypeptidase, different from known carboxypeptidases, which may be released into the urine by the kidney. Here it could inactivate kinins and other peptides containing a basic C-terminal amino acid.     

Molecular cloning and cDNA sequence analysis of carboxypeptidases A1, A2 and B from the Japanese flounder Paralichthys olivaceus

Although pancreatic serine proteases have been cloned in teleosts, no sequence data are currently available on members of the carboxypeptidase (CP) family. Here, we cloned cDNAs coding for two preproCPAs, corresponding to mammalian preproCPA1 and preproCPA2, and one preproCPB from a pancreatic cDNA library of the Japanese flounder, Paralichthys olivaceus. The activation peptides of flounder proCPs completely retained the sequences for inhibition of enzymatic activity of proCPs just like mammalian proCPs. Of 306–309 amino acids in total, 95 amino acids are completely conserved between bovine CPA1 and CPB and flounder CPs. Notably, amino acid residues for Zn²⁺ ligands, catalysis and substrate anchoring are completely conserved between flounder and bovine CPs. Three species of flounder preproCPs are all expressed in the pancreas of first feeding larvae.     

Molecular cloning and nucleotide sequencing of human immunoglobulin epsilon chain cDNA.

DNA complementary to mRNA of human immunoglobulin E heavy chain (epsilon chain) isolated and purified from U266 cells has been synthesized and inserted into the PstI site of pBR322 by G-C tailing. This recombinant plasmid was used to transform E. coli chi 1776 to screen 1445 tetracycline resistant colonies. Nine clones (pGETI - 9) containing cDNA coding for the human epsilon chain were recognized by colony hybridization and Southern blotting analysis with a nick-translated human IgE genome fragment. The nucleotide sequence of the longest cDNA contained in pGET2 was determined. The results indicate that the sequence of 1657 nucleotides codes for 494 amino acids covering a part of the variable region and all of the constant region of the human epsilon chain. Most of the amino acid sequence deduced from the nucleotide sequence is in substantial agreement with that reported. Furthermore a termination codon after the -COOH terminal amino acid marks the beginning of a 3' untranslated region of 125 nucleotides with a poly A tail. Taking this into account, the structure of the human epsilon chain mRNA, except a part of the 5' end, is conserved fairly well in the cDNA insert in pGET2.     

Molecular cloning and partial sequencing of hepatitis A viral cDNA.

Hepatitis A virus was purified from infected monkey kidney cell cultures, and the viral RNA was used to synthesize double-stranded cDNA. This cDNA was cloned either after insertion into a plasmid-primed synthesis system or after insertion into the PstI site of pBR322. The resulting clones were mapped by restriction endonuclease analysis and by cross hybridization of the viral inserts to generate a composite map which represented at least 97% of the viral genome, lacking ca. 220 bases from the 5' end of the genome. The clones were verified to be hepatitis A virus specific based on their positive hybridization to viral RNA and to total hepatitis A virus-infected cellular RNA from a heterologous marmoset host system. The nucleotide sequence of 3,054 base pairs of cDNA homologous to the 5' half of the viral genome was determined, and an open reading frame of 854 consecutive coding triplets was identified. In addition, sequences which encode the VP-1 and VP-3 viral structural proteins were located in the nucleotide sequence.     

Molecular cloning, sequencing and expression of human interferon-gamma-inducible indoleamine 2,3-dioxygenase cDNA

The antiproliferative action of human interferon (HuIFN)-gamma on human cells and the inhibition of intracellular pathogens, e.g. Toxoplasma gondii and Chlamydia psittaci, is at least in part due to an induction of indoleamine 2,3-dioxygenase (IDO) enzyme which degrades tryptophan, an essential amino acid. A cDNA clone (called C42) was isolated from a cDNA library made from poly(A)+ RNA obtained from HuIFN-gamma-treated human fibroblasts. Its nucleotide sequence revealed an open reading frame coding for a polypeptide of 403 amino acids, but no homology with any known gene in GenBank database was found. Evidence was obtained indicating that this cDNA codes for IDO: (i) Hybrid selected C42 specific poly(A)+ RNA from IFN-gamma-treated human cells coded for a polypeptide in vitro of approximately 42 kD (reported size of IDO, approximately 40 kD) which was immunoprecipitated by monoclonal anti-IDO antibody but not by a control antibody; and (ii) transfection of human fibroblasts with an expression plasmid containing C42 cDNA transcribed from chicken beta-actin promoter led to constitutive expression of C42 specific RNA as well as IDO activity. This cDNA clone will be useful in studying the role of IDO in the biological effects of IFN-gamma, and the regulation of IDO gene by IFN-gamma.     

Molecular cloning and sequencing of human cDNA for phosphoribosyl pyrophosphate synthetase subunit II

cDNA clones for human phosphoribosyl pyrophosphate synthetase subunit II (PRS II) were isolated. The five overlapping clones contained 2457 base pairs (bp) covering a 954-bp complete coding region for 318 amino acid residues. Homologies between human and rat PRS II were 99% of the amino acids and 88% of the nucleotides in the coding region. This amino acid homology seems to be the highest so far reported for enzymes involved in nucleotide metabolism and glycolysis. The highly conserved structure may be required for unique catalysis and rigid regulation of this enzyme.     

Molecular cloning and sequencing of a cDNA encoding a human alpha 1A adrenergic receptor.

DNA from a rat hippocampus cDNA library and sets of highly degenerate oligonucleotide primers directed toward conserved regions of previously cloned G-protein receptors were used in the polymerase chain reaction to selectively amplify and clone new members of this gene family. A human hippocampus cDNA library was screened with a 610 base pair fragment generated by PCR and a cDNA clone, H318/3, was isolated. The deduced amino acid sequence of this clone encoded a protein of 501 amino acids that showed strong sequence homology to previously cloned G-protein receptors. Nucleotide sequence analysis revealed clone H318/3 was 78% homologous to a rat alpha 1A adrenergic receptor with homology being 95% when comparisons were made in the region that lies between the first to the seventh transmembrane domains. Based on this high degree of sequence homology, we conclude that clone H318/3 represents a cDNA for a human alpha 1A adrenergic receptor.     

Molecular cloning and sequencing of a cDNA encoding N alpha-acetyltransferase from Saccharomyces cerevisiae

Acetylation is the most frequently occurring chemical modification of the alpha-NH2 group of eukaryotic proteins and is catalyzed by an N alpha-acetyltransferase. Recently, a eukaryotic N alpha-acetyltransferase was purified to homogeneity from Saccharomyces cerevisiae, and its substrate specificity was partially characterized (Lee, F.-J. S., Lin L.-W., and Smith, J. A. (1988) J. Biol. Chem. 263, 14948-14955). This article describes the cloning from a yeast lambda gt11 cDNA library and sequencing of a full length cDNA encoding yeast N alpha-acetyltransferase. DNA blot hybridizations of genomic and chromosomal DNA reveal that the gene (so-called AAA1, amino-terminal, alpha-amino, acetyltransferase) is present as a single copy located on chromosome IV. The use of this cDNA will allow the molecular details of the role of N alpha-acetylation in the sorting and degradation of eukaryotic proteins to be determined.     

Molecular cloning and sequencing of the cDNA for vacuolar H+-pyrophosphatase from Chara corallina1

We have cloned a cDNA for vacuolar proton-translocating pyrophosphatase of Chara corallina that is one of the closest green algae to the land plants. The deduced protein consists of 793 amino acid residues. Its sequence is 71% identical to the H+-pyrophosphatases of land plants, and is less than 46% identical to those of marine alga and phototrophic bacterium.     

Molecular cloning, sequencing, and expression of cDNA for rat liver microsomal aldehyde dehydrogenase.

The cDNA clone for rat liver microsomal aldehyde dehydrogenase (msALDH) was isolated and sequenced. The deduced amino acid sequence consisting of 484 amino acid residues revealed that the carboxyl-terminal region of msALDH has a hydrophobic segment, which is probably important for the insertion of this enzyme into the endoplasmic reticulum membrane. COS-1 cells transfected with the expression vector pcD containing the full-length cDNA showed that the active enzyme was expressed and localized mainly on the cytoplasmic surface of the endoplasmic reticulum membranes. It has been proposed that ALDH isozymes form a superfamily consisting of class 1, 2, and 3 ALDHs (Hempel, J., Harper, K., and Lindahl, R., (1989) Biochemistry 28, 1160-1167). Comparison of the amino acid sequence of rat liver msALDH with those of rat other class ALDHs showed that msALDH was 24.2, 24.0, and 65.5% identical to phenobarbital-inducible ALDH (variant class 1), mitochondrial ALDH (class 2), and tumor-associated ALDH (class 3), respectively. Several amino acid residues common to the other known ALDHs, however, were found to be conserved in msALDH. Based on these results, we proposed to classify msALDH as a new type, class 4 ALDH.     

Molecular cloning of cDNA for the import precursor of human subunit B of H(+)-ATP synthase in mitochondria.

The nucleotide sequence of the import precursor of subunit b of human H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a human kidney cDNA library with a cDNA for rat subunit b as a probe. The sequence was composed of 1,134 nucleotides including a coding region for the import precursor of subunit b and noncoding regions on the 5'- and 3'-sides. The import precursor of subunit b and its mature polypeptide deduced from the open reading frame were found to consist of 256 and 214 amino acid residues with molecular weights of 28,893 and 24,610, respectively. The presequence of 42 amino acids could be the import signal peptide for directing the protein into the mitochondrial matrix.     

Molecular cloning, sequencing, and expression of human myocardial fatty acid ethyl ester synthase-III cDNA

Fatty acid ethyl ester synthase-III (FAEES-III), previously purified to homogeneity from human heart, metabolizes ethanol nonoxidatively. Using a derived partial amino acid sequence and corresponding oligonucleotide probes, the cDNA for this enzyme has been cloned from a human heart lambda gtll library. Of the five positive clones obtained, one contained a complete coding region (630 base pairs) and the entire 3'-noncoding region (41 base pairs). From this nucleotide sequence the complete 210 amino acid sequence of FAEES-III (Mr 23,307) is reported. Comparison of its amino acid sequence with that of glutathione S-transferase pi-1 suggests that they belong to the same gene family since they differ in only six nucleotides and four amino acids. The sequence of FAEES-III was also compared with those of placental glutathione S-transferase and the basic glutathione S-transferase. FAEES-III was 84% homologous with placental glutathione S-transferase but only less than 10% homologous with the basic glutathione S-transferase. Northern blots demonstrate expression of FAEES-III mRNA in normal human liver, placenta, and heart. In all cases, the mRNA for the enzyme is 0.7 kilobase in size. MCF-7 cells transfected with FAEES-III cDNA have a 14-fold increase in synthase activity and a 12-fold increase in glutathione S-transferase (GST) activity compared with control cells. MCF-7 cells transfected with GST pi-1 cDNA have a 13-fold increase in GST activity compared with control cells but no increase in synthase activity. When the supernatant of COS-7 cells transfected with FAEES-III cDNA were immunoblotted with rabbit FAEES-III antibody, a band at 24 kilodaltons was demonstrated. Thus, we have obtained the first cDNA and amino acid sequence for a human FAEES-III which also has significant GST activity, and we have identified 4 residues potentially responsible for conferring ethanol recognition to GSTs.     

cDNA cloning and complete primary structure of the small, active subunit of human carboxypeptidase N (kininase 1)

The human plasma metallo-protease carboxypeptidase N of Mr 280,000 consists of two small, enzymatically active subunits of Mr 50,000 and two large subunits. Only the large subunits are glycosylated. They may have a function in stabilizing the complex in plasma. The N-terminal sequence of the small subunit was determined from the isolated protein and used to specify a unique 59-mer oligonucleotide probe. A cDNA clone of 1.7 kbp containing the entire coding sequence of the small subunit of carboxypeptidase N was isolated from a human-liver cDNA library. The cDNA clone encodes a signal sequence of 20 amino acids and the 438 amino acids of the mature subunit. There is a remarkable primary structure similarity of 49% to bovine carboxypeptidase E (enkephalin convertase). A more distant relationship to the bovine pancreatic, digestive carboxypeptidases A and B or even to the metallo-endopeptidases is based mainly on the occurrence of conserved, mechanistically important residues.