Molecular cloning and functional expression of rat leukotriene A4 hydrolase using the polymerase chain reaction.

We isolated a cDNA encoding rat leukotriene A4 (LTA4) hydrolase from mesangial cells by the polymerase chain reaction according to the human amino acid sequence. The deduced amino acid sequence shows that rat LTA4 hydrolase is a 609 amino acid protein with an Mr 69 kDa. Comparison of human LTA4 hydrolase revealed 93% homology, and include zinc-binding motifs of aminopeptidases. COS-7 cells transfected with the cDNA revealed substantial LTA4 hydrolase activity, and their activities were abolished by preincubation with captopril, representing the first reported cDNA expression of recombinant enzyme in mammalian cells. RNA blot analysis indicated that LTA4 hydrolase was expressed in glomerular endothelial, epithelial and mesangial cells.     

Molecular cloning of a cDNA coding for human leukotriene A4 hydrolase. Complete primary structure of an enzyme involved in eicosanoid synthesis

We have isolated a near full-length cDNA encoding human leukotriene A4 hydrolase, which synthesizes a potent chemotactic and spasmogenic compound, leukotriene B4. A human spleen cDNA library was screened with a 48-mer oligonucleotide probe, synthesized according to the partial amino acid sequence of the human leukocyte enzyme. The nucleotide sequence of the cDNA had an open reading frame of 1,833 base pairs, which contained regions coding for the N-terminal amino acid sequence, the amino acid sequence for the probe design, and several other peptide sequences of the enzyme. The complete primary structure of the enzyme composed of 610 amino acid residues (molecular weight, 69,153) was deduced from the cDNA.     

Molecular cloning and expression of a mouse thiamin pyrophosphokinase cDNA

Thiamin pyrophosphokinase (EC 2.7.6.2) catalyzes the pyrophosphorylation of thiamin with adenosine 5'-triphosphate to form thiamin pyrophosphate. A mouse thiamin pyrophosphokinase cDNA clone (mTPK1) was isolated using a combination of mouse expressed sequence tag database analysis, a two-step polymerase chain reaction procedure, and functional complementation screening with a Saccharomyces cerevisiae thiamin pyrophosphokinase-deficient mutant (thi80). The predicted protein contained 243 amino acid residues with a calculated molecular weight of 27,068. When the intact mTPK1 open reading frame was expressed as a glutathione S-transferase fusion protein in Escherichia coli lacking thiamin pyrophosphokinase, marked enzyme activity was detected in the bacterial cells. The corresponding 2.5-kilobase pair mRNA was expressed in a tissue-dependent manner and was found at relatively high levels in the kidney and liver, indicating that the mode of expression of mTPK1 genes differs with cell type. The expression of mTPK1 genes in cultured mouse neuroblastoma and normal liver cells was unaffected by the thiamin concentration in the medium (10 microM versus 3.0 nM). This is the first report on identification of the primary sequence for mammalian thiamin pyrophosphokinase.     

Expression of human leukotriene A4 hydrolase cDNA in Escherichia coli

The cDNA clone encoding human leukotriene A4 hydrolase was inserted into a vector pUC9 and expressed in Escherichia coli as a fusion protein containing the first 10 amino acid residues derived from a vector. The leukotriene A4 hydrolase activity was recovered in the soluble fraction of the transformants. The purified enzyme showed kinetic properties similar to the native enzyme, including inactivation by the substrate and sulfhydryl-modifying reagents. The results demonstrate that a protein with an Mr of 70,000 was expressed in Escherichia coli with a full enzyme activity and structural fidelity. Acquisition of the expression system makes it feasible to elucidate the reaction mechanism of the enzyme.     

Molecular cloning of mouse tumour necrosis factor cDNA and its eukaryotic expression.

Tumour necrosis factor (TNF), released by induced macrophages, causes tumour necrosis in animals and kills preferentially transformed cells in vitro. mRNA induced in the established mouse monocytic PU 5.1.8 cell line by lipopolysaccharide, was converted into double-stranded cDNA and cloned in the pAT153 vector. Recombinant plasmids were screened by plus-minus hybridization and TNF-specific oligonucleotide probes constructed on the basis of partial amino acid sequences of rabbit TNF. A series of TNF specific clones were identified and confirmed by hybrid selection of mouse TNF-specific mRNA. The sequence codes for a 235 amino acids long polypeptide, of which 156 amino acids presumably correspond to the mature product. It can be concluded that mature mouse TNF is a glycosylated dimer. Biologically active TNF was secreted by both Cos-I and CHO-cells transfected with the chimaeric expression vector pSV2d2-mTNF containing the coding region of the mouse TNF cDNA gene.     

Molecular cloning and expression of a mouse muscle cDNA encoding adenylosuccinate synthetase

Adenylosuccinate synthetase (EC 6.3.4.4) catalyzes the first step in formation of AMP from IMP. At least two isozymes exist in vertebrate tissue. An acidic form, present in most tissues, has been suggested to be involved in de novo biosynthesis while a basic isozyme, which predominates in muscle, appears to function in the purine nucleotide cycle. Antibodies specific for the basic isozyme detect a single protein in mouse tissues with highest levels in skeletal muscle, tongue, esophagus, and heart tissue consistent with a role for the enzyme in muscle metabolism. A series of degenerate oligonucleotides were constructed based on peptide sequences from purified rat muscle enzyme and then used to clone a mouse muscle cDNA encoding the basic isozyme. The clone contains a open reading frame of 1356 bases with 452 amino acids. Northern analysis of RNA from mouse tissues showed a tissue distribution similar to that of the protein, indicating a high level of gene expression in muscle. Transfection of COS cells with the mouse muscle cDNA allows expression of a functional protein with a molecular mass of approximately 50 kDa, consistent with the open reading frame and the size of the isolated rat enzyme. The deduced amino acid sequence of the mouse synthetase is 47 and 37% identical to the synthetase sequences from Dictyostelium discoideum and Escherichia coli, respectively. The availability of antibodies and cDNA clones specific for the basic isozyme of adenylosuccinate synthetase from muscle will facilitate future genetic and biochemical analysis of this protein and its role in muscle physiology.     

Molecular evolution and zinc ion binding motif of leukotriene A4 hydrolase

Leukotriene A4 (LTA4) hydrolase belongs to the aminopeptidase N family. In order to investigate the molecular evolution and physiological significance of LTA4 hydrolase, the enzymes belonging to the family were aligned and a phylogenetic tree was constructed. From the alignment, it was found that three residues involved in zinc binding and one residue of the active sites of aminopeptidases N were conserved in LTA4 hydrolase. In agreement with the observation, LTA4 hydrolase is a zinc protein as determined by atomic absorption spectroscopy.     

Molecular cloning and expression of rat betacellulin cDNA

The cDNA encoding an entire open reading frame of rat betacellulin has been cloned from rat kidney. Expression of this cDNA in COS7 cells showed a significant amount of mitogenic activity in the culture media. Western blotting of the cell lysates suggested that the membrane-anchored precursor was cleaved to release its ectodomain very efficiently.     

Molecular cloning and functional expression of rat liver cytosolic acetyl-CoA hydrolase.

A cytosolic acetyl-CoA hydrolase (CACH) was purified from rat liver to homogeneity by a new method using Triton X-100 as a stabilizer. We digested the purified enzyme with an endopeptidase and determined the N-terminal amino-acid sequences of the two proteolytic fragments. From the sequence data, we designed probes for RT-PCR, and amplified CACH cDNA from rat liver mRNA. The CACH cDNA contains a 1668-bp ORF encoding a protein of 556 amino-acid residues (62 017 Da). Recombinant expression of the cDNA in insect cells resulted in overproduction of functional acetyl-CoA hydrolase with comparable acyl-CoA chain-length specificity and Michaelis constant for acetyl-CoA to those of the native CACH. Database searching shows no homology to other known proteins, but reveals high similarities to two mouse expressed sequence tags (91% and 93% homology) and human mRNA for KIAA0707 hypothetical protein (50% homology) of unknown function.     

Molecular cloning and expression of salmon prolactin cDNA

Prolactin was purified from chum salmon pituitaries. It was resolved into two variants by reverse-phase high-performance liquid chromatography. A cDNA library was prepared from Pacific chinook salmon pituitaries. Salmon prolactin gene was screened using a synthetic oligonucleotide based on partial protein sequence. A positive clone (PRL-10) was identified and sequenced. It is a full-size clone containing 1.1 kb and coding for a preprolactin of 211 amino acids. A modified prolactin plasmid (PRL-10A), in which the 5' untranslated sequence and the nucleotide sequence coding for the signal peptide of prolactin were deleted, was reconstructed into an expression vector using the heat-inducible lambda pL promotor. Mature prolactin, a single polypeptide of 22 kDa, was efficiently expressed in the bacteria at an elevated temperature.     

Molecular cloning and expression of mouse brain sialidase.

Sialidase (EC 3.2.1.18) catalyzes the release of sialic acid from sialo-oligosaccharides, gangliosides, or sialo-glycoproteins. In this investigation, we cloned a novel cDNA for mouse brain sialidase and expressed the cDNA in COS-7 cells. This 1,699 bp cDNA codes for a 41.6 kDa protein consisting of 372 deduced amino acid residues. In COS-7 cells transiently transfected with the cDNA, a 250-fold increase was observed in specific activity toward 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid. Similarity searches of the nonredundant GenBank peptide sequence database by the PSI-BLAST program identified rat, hamster, human, and bacterial sialidases homologous to this mouse brain sialidase. Amino acid sequence identities to rat and hamster sialidases (84% and 77%, respectively) suggest that this form of sialidase is conserved in rodents. Sequence identities to human and mouse lysosomal sialidases (30% and 28%, respectively) indicate that the mouse brain sialidase is distinct from the lysosomal enzyme. Mouse brain sialidase has two amino acid sequence motifs common to bacterial sialidases: the 'F/YRIP' motif and the 'Asp-box' motif. The 'F/YRIP' motif is present near the N terminus while two 'Asp-box' motifs are present downstream.     

Azabenzthiazole inhibitors of leukotriene A4 hydrolase

Previously, benzthiazole containing LTA₄H inhibitors were discovered that were potent (1–3), but were associated with the potential for a hERG liability. Utilizing medicinal chemistry first principles (e.g., introducing rigidity, lowering c Log D) a new benzthiazole series was designed, congeners of 1–3, which led to compounds 7a, 7c, 12a–d which exhibited LTA₄H IC₅₀ = 3–6 nM and hERG Dofetilide Binding IC₅₀ = 8.9–> >10 μM.     

Molecular cloning of feline interferon cDNA by direct expression.

A cDNA sequence coding for feline interferon has been cloned for the first time by screening a cDNA library constructed using Okayama-Berg vector and mRNA derived from the feline cells (LSA-I) induced by TPA (12-o-tetradecanoylphorbor 13-acetate) for the ability of transient expression to produce feline interferon in COS1 monkey cells. The amino acid sequence, deduced from the nucleotide sequence by comparing it with the sequences of other mammalian IFNs, consists of 171 amino acids with 6 cysteins and an N-glycosylation site at the amino acid position 79, and has about 60% homology to human IFN alpha 1. The interferon was partially purified through Blue Sepharose, and its molecular weight was estimated to be 2.4 x 10(4). The antiviral activity was acid stable, and glycosylation was suggested.     

Molecular cloning and characterization of cDNA encoding mouse cytokeratin no. 19

We have isolated cDNA clones encoding the mouse cytokeratin No. 19 (Ck 19) from an intestinal cDNA library using synthetic oligodeoxyribonucleotides as probes. We obtained four independent clones, which correspond to about 1.4-kb of ck19 cDNA. Nucleotide sequence analysis revealed that these cDNAs encode a protein of 44,541 Da composed of 403 amino acids (aa). The deduced aa sequence defines an alpha-helical central domain, and suggests that the protein lacks a C-terminal non-alpha-helical tail segment, characteristic of the human and bovine 40-kDa keratins (Ck19). The overall aa identity between mouse Ck19 and human and bovine Ck19 is very high, 82.7% and 82.4%, respectively. The coil-forming central domain of mouse Ck19 has 45-65% similarity to other type-I Ck polypeptides, while it displays only 20-30% similarity to type-II Ck polypeptides. Northern blot analysis showed that mouse ck19 mRNA is strongly expressed in adult intestine, stomach and uterus. Interestingly, it is expressed in a placental cell line and a retinoic acid-treated mouse teratocarcinoma cell line (F9), but not in a parietal yolk sac endoderm-like cell line (PYS-2). This pattern of expression is very similar to that for the mouse gene encoding extra-embryonic endodermal cytoskeletal protein C (EndoC), suggesting they may be the same.     

Molecular cloning and functional expression of human cytosolic acetyl-CoA hydrolase

A cDNA encoding human cytosolic acetyl-CoA hydrolase (CACH) was isolated from a human liver cDNA library, sequenced and functionally expressed in insect cells. The human CACH cDNA encodes a 555-amino-acid sequence that is 81.4%/78.7% identical to those of the mouse/rat homologue, suggesting a conserved role for this enzyme in the human and rodent livers. Bioinformatical study further reveals a high degree of similarity among the human and rodent CACHs as follows: First, the gene is composed of 15 exons ranging in size from 56 to 157 bp. Second, the protein consists of two thioesterase regions and a C-terminal steroidogenic acute regulatory protein-related lipid transfer (START) domain. Third, the promoter region is GC-rich and contains GC boxes, but lacks both TATA and CCAAT boxes, the typical criteria of housekeeping genes. A consensus peroxisome proliferator responsive element (PPRE) present in the rodent CACH promoter regions supports marked CACH induction in rat liver by peroxisome proliferator (PP).     

Molecular cloning and characterization of another leukotriene B4 receptor

Leukotriene B(4) is a potent lipid mediator known to be implicated mainly in inflammatory actions. Previous pharmacological studies indicated the existence of only one class of G protein-coupled receptor for leukotriene B(4), for which a candidate gene, namely BLT, had been identified. Here we report the isolation of another gene encoding a functional G protein-coupled receptor for leukotriene B(4), named JULF2. JULF2 is a novel G protein-coupled receptor of 358 amino acids that shares 36.6% amino acid identity with human BLT. According to genomic information, the JULF2 gene is located on the chromosome 14, about 4 kilobases upstream of the BLT gene. During screening of endogenous ligands for JULF2, we found that leukotriene B(4) induced inhibition of forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells, stably expressing JULF2. Additionally, Chinese hamster ovary cells expressing exogenous JULF2 showed chemotactic responses with leukotriene B(4) in a pertussis toxin-sensitive manner. A large amount of JULF2 mRNA was detected in the human spleen and the peripheral blood leukocytes. Furthermore, JULF2 mRNA was expressed in mononuclear lymphocytes, in which BLT mRNA was barely detected. The discovery of this second leukotriene B(4) receptor will eventually lead to a better understanding of the classification of leukotriene B(4) receptors and reconsideration of the pathophysiological role of leukotriene B(4).     

Molecular cloning and expression of cDNA encoding a galactose/N-acetylgalactosamine-specific lectin on mouse tumoricidal macrophages.

We previously reported that the mouse macrophage galacose and N-acetylgalactosamine-specific lectin (MMGL) may participate in the binding of the macrophages to tumor cells [Oda, S., Sato, M., Toyoshima, S., & Osawa, T. (1989) J. Biochem. 105, 1040-1043]. We now report the cloning and characterization of a cDNA encoding MMGL. The MMGL gene encoded a protein consisting of 304 amino acid residues with a molecular weight of 34,595. The deduced amino acid sequence indicated that MMGL had a single membrane-spanning region, three leucine zipper-like domains, and a carbohydrate recognition domain. Two N-glycosylation sites were found in the extracellular region of MMGL, corresponding to the heavy N-glycosylation in the native MMGL. Comparison of the amino acid sequence of MMGL with those of rat hepatic lectins revealed a high overall sequence homology. The sequence homology was especially high in the putative membrane-spanning region and carbohydrate recognition domain. There was, however, a region of 25 amino acids which did not exist on hepatic lectins. The MMGL cDNA without the region encoding the putative membrane-spanning region and intracellular region was expressed in Escherichia coli. The expressed protein had galactose-binding activity and its sugar-binding specificity was same as that of the native lectin.