Molecular cloning and sequence analysis of cDNA encoding delta 4-3-ketosteroid 5 beta-reductase of rat liver.

A cDNA clone encoding delta 4-3-ketosteroid 5 beta-reductase was isolated from rat liver cDNA libraries using antibodies specific for the enzyme and oligonucleotides as probes. The cDNA contained 981-base pair open reading frame encoding 327 amino acid residues (Mr 37,376) and an unusually long 3'-untranslated region rich in AT sequence in the total length of 3189 base pairs. The predicted amino acid sequence contains the sequences similar to the putative NADPH- and steroid-binding regions.     

Isolation and cDNA sequence of human postheparin plasma hepatic triglyceride lipase

Hepatic triglyceride lipase (H-TGL) was isolated from human postheparin plasma by column chromatography on heparin-Sepharose and phenyl-Sepharose and immunoaffinity chromatography with monoclonal antibodies. The purified enzyme had an apparent molecular weight of 65,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an amino-terminal sequence of Leu-Gly-Gln-Ser-Leu-Lys-Pro-Glu. Partial amino acid sequences of seven cyanogen bromide peptides were obtained. A human hepatoma cDNA library was screened with synthetic oligonucleotides derived from the partial protein sequence. The cloned H-TGL cDNA of 1569 nucleotides predicts a mature protein of 477 amino acids plus a leader sequence of 22 amino acids. Blot hybridization analysis of poly(A)+ mRNA with a putative H-TGL cDNA clone gave a single hybridizing band of 1.7 kilobases. The protein contains four consensus N-glycosylation sequences based on the cDNA sequence. Comparison of the enzyme sequence with that of other lipases reveals highly conserved sequences in regions of putative lipid and heparin binding. The carboxyl terminus of H-TGL contains a highly basic sequence which is not reported to be present in rat H-TGL or other members of the lipase gene family.     

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.     

Purification and cDNA cloning of rat 6-pyruvoyl-tetrahydropterin synthase

6-Pyruvoyl-tetrahydropterin synthase, which catalyzes the second step in the biosynthesis of tetrahydrobiopterin, was purified approximately 18,000-fold to apparent homogeneity from rat liver. The molecular mass of the native enzyme was estimated to be 83 kDa by gel filtration. The enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis corresponding to a molecular mass of 17 kDa. Up to 24 residues of the NH2-terminal sequence were determined by Edman degradation, which released a single amino acid at each step. These results indicate that the enzyme consists of identical subunits. The purified enzyme was digested with lysyl endopeptidase or V8 protease, and 11 peptide fragments were isolated. On the basis of the sequences of these peptides, oligonucleotides were synthesized and used to screen a rat liver cDNA library, and one cDNA clone was isolated. The complete nucleotide sequence of the 1176-base pair cDNA was then determined. The deduced amino acid sequence contained 144 amino acid residues, but a NH2-terminal four-amino acid sequence was not found in the purified protein. Therefore, the mature protein consists of 140 amino acids. A single mRNA band of 1.3 kilobases was obtained by RNA blot analysis of rat liver. The predicted amino acid sequence of 6-pyruvoyl-tetrahydropterin synthase was compared with the Protein Sequence Database of the National Biomedical Research Foundation, revealing significant local similarity to large T antigens from the polyomavirus family.     

Molecular cloning and nucleotide sequence of a cDNA clone coding for the cell attachment domain in human fibronectin

A cDNA clone coding for the cell attachment domain in human fibronectin has been isolated using synthetic oligonucleotides. Three sets of mixed tetradecamer oligonucleotides were synthesized based on amino acid sequences in the 108-amino acid cell attachment domain (Pierschbacher, M. D., Ruoslahti, E., Sundelin, J., Lind, P., and Peterson, P. A. (1982) J. Biol. Chem. 257, 9593-9597). One of these sets was made complementary to amino acids located near the COOH terminus of the cell attachment domain and synthesized as a mixture of 24 sequences. This oligonucleotide mixture was used to prime cDNA synthesis with mRNA prepared from a human fibrosarcoma as a template. A cDNA library was constructed with the oligonucleotide-primed sequences in the vector pBR322. Colonies that hybridized with the primer were isolated from the library and further identified by hybridization with oligonucleotides deduced from an amino acid sequence located 45 amino acid residues NH2-terminal of the primer sequence. One clone which hybridized to both probes was characterized in detail. The insert was 380 base pairs long and its nucleotide sequence agreed completely with the corresponding amino acid sequence of human plasma fibronectin, showing that the sequences for this region are identical in plasma fibronectin and fibronectin from a cell line. This clone should be useful for studies on the expression of fibronectins and may also allow for the production of the biologically active cell attachment domain of fibronectin in bacteria.     

Phenol hydroxylase from Trichosporon cutaneum: gene cloning, sequence analysis, and functional expression in Escherichia coli.

A cDNA clone encoding phenol hydroxylase from the soil yeast Trichosporon cutaneum was isolated and characterized. The clone was identified by hybridization screening of a bacteriophage lambda ZAP-based cDNA library with an oligonucleotide probe which corresponded to the N-terminal amino acid sequence of the purified enzyme. The cDNA encodes a protein consisting of 664 amino acids. Amino acid sequences of a number of peptides obtained by Edman degradation of various cleavage products of the purified enzyme were identified in the cDNA-derived sequence. The phenol hydroxylase cDNA was expressed in Escherichia coli to yield high levels of active enzyme. The E. coli-derived phenol hydroxylase is very similar to the T. cutaneum enzyme with respect to the range of substrates acted upon, inhibition by excess phenol, and the order of magnitude of kinetic parameters in the overall reaction. Southern blot analysis revealed the presence of phenol hydroxylase gene-related sequences in a number of T. cutaneum and Trichosporon beigelii strains and in Cryptococcus elinovii but not in Trichosporon pullulans, Trichosporon penicillatum, or Candida tropicalis.     

cDNA and deduced amino acid sequence of a mouse DNA repair enzyme (APEX nuclease) with significant homology to Escherichia coli exonuclease III

We purified a mouse DNA repair enzyme having apurinic/apyrimidinic endonuclease, DNA 3'-phosphatase, 3'-5'-exonuclease and DNA 3' repair diesterase activities, and designated the enzyme as APEX nuclease. A cDNA clone for the enzyme was isolated from a mouse spleen cDNA library using probes of degenerate oligonucleotides deduced from the N-terminal amino acid sequence of the enzyme. The complete nucleotide sequence of the cDNA (1.3 kilobases) was determined. Northern hybridization using this cDNA showed that the size of its mRNA is about 1.5 kilobases. The complete amino acid sequence for the enzyme predicted from the nucleotide sequence of the cDNA (APEX nuclease cDNA) indicates that the enzyme consists of 316 amino acids with a calculated molecular weight of 35,400. The predicted sequence contains the partial amino acid sequences determined by a protein sequencer from the purified enzyme. The coding sequence of APEX nuclease was cloned into pUC18 SmaI and HindIII sites in the control frame of the lacZ promoter. The construct was introduced into BW2001 (xth-11, nfo-2) strain cells of Escherichia coli. The transformed cells expressed a 36.4-kDa polypeptide (the 316 amino acid sequence of APEX nuclease headed by the N-terminal decapeptide of beta-galactosidase) and were less sensitive to methyl methanesulfonate than the parent cells. The fusion product showed priming activity for DNA polymerase on bleomycin-damaged DNA and acid-depurinated DNA. The deduced amino acid sequence of mouse APEX nuclease exhibits a significant homology to those of exonuclease III of E. coli and ExoA protein of Streptococcus pneumoniae and an intensive homology with that of bovine AP endonuclease 1.     

Isolation of partial complementary DNA encoding human thromboxane synthase

Thromboxane synthase catalyzes the biosynthesis of thromboxane A2 which plays a key role in the proaggregatory and vasoconstrictive processes. In this communication, we reported the successful cloning of thromboxane synthase cDNA from a human lung cDNA library. Oligonucleotides were synthesized according to the direct amino acid sequence of 2 peptides derived from purified human thromboxane synthase. Polymerase chain reaction was carried out using these oligonucleotides as primers to isolate a complementary DNA from human lung cDNA library. The longest cDNA thus obtained was 687 base pairs in length. Amino acid sequences deduced from the cDNA contained all three peptide sequences reported, confirming the authenticity of the cDNA clone.     

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.     

cDNA cloning and expression of bovine aspartyl (asparaginyl) beta-hydroxylase.

Aspartyl (asparaginyl) beta-hydroxylase which specifically hydroxylates 1 Asp or Asn residue in certain epidermal growth factor-like domains of a number of proteins, has been previously purified to apparent homogeneity from detergent-solubilized bovine liver microsomes (Wang, Q., VanDusen, W. J., Petroski, C. J., Garsky, V. M., Stern, A. M., and Friedman, P. A. (1991) J. Biol. Chem. 266, 14004-14010). Three oligonucleotides, corresponding to three amino acid sequences of the purified hydroxylase, were used to screen bovine cDNA libraries. Several overlapping positive cDNA clones containing a full length open reading frame of 754 amino acids encoding a 85-kDa protein were isolated, and a cDNA, containing the full length open reading frame, was constructed from two of these clones. The resulting clone was then transcribed and translated in vitro to produce recombinant protein which possessed Asp beta-hydroxylase activity. These results constitute proof that the protein purified from bovine liver is an Asp beta-hydroxylase. Comparisons of deduced amino acid sequences of two other alpha-ketoglutarate-dependent dioxygenases, prolyl-4-hydroxylase and lysyl hydroxylase, with that of Asp beta-hydroxylase showed no significant homologies. Indeed, Asp beta-hydroxylase appears to be unique as no striking homology was found with known protein sequences. Furthermore, structural predictions derived from the deduced amino acid sequence are in accord with earlier Stokes' radius and sedimentation coefficient determinations of the enzyme, suggesting that the enzyme contains a relatively compact carboxyl-terminal catalytic domain and an extended amino terminus. This amino-terminal region has a potential transmembrane type II signal-anchor domain that could direct the catalytic domain into the lumen of the endoplasmic reticulum.     

Cloning of NAD-dependent sorbitol dehydrogenase from apple fruit and gene expression

Partial amino acid sequences of NAD-dependent sorbitol dehydrogenase (NAD-SDH) were used to identify a full-length cDNA from apple fruit. This clone consisted of 1,433 bp containing an open reading frame of 1,137 bp that could code for a polypeptide with 379 amino acids. To our knowledge, this is the first report about cloning of NAD-SDH cDNA from a plant source. The deduced amino acids from cDNA revealed 43.7% identity to human NAD-SDH. The activity of this enzyme to convert sorbitol to fructose with the reduction of NAD was certified by the fusion protein of this clone expressed in Escherichia coli. Northern blot analysis showed that the mRNA was expressed in matured apple fruit.     

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.     

cDNA cloning of porcine brain prolyl endopeptidase and identification of the active-site seryl residue

Prolyl endopeptidase is a cytoplasmic serine protease. The enzyme was purified from porcine kidney, and oligonucleotides based on peptide sequences from this protein were used to isolate a cDNA clone from a porcine brain library. This clone contained the complete coding sequence of prolyl endopeptidase and encoded a polypeptide with a molecular mass of 80,751 Da. The deduced amino acid sequence of prolyl endopeptidase showed no sequence homology with other known serine proteases. [3H]Diisopropyl fluorophosphate was used to identify the active-site serine of prolyl endopeptidase. One labeled peptide was isolated and sequenced. The sequence surrounding the active-site serine was Asn-Gly-Gly-Ser-Asn-Gly-Gly. This sequence is different from the active-site sequences of other known serine proteases. This difference and the lack of overall homology with the known families of serine proteases suggest that prolyl endopeptidase represents a new type of serine protease.     

Glutathione S-transferase from the spruce budworm, Choristoneura fumiferana: identification, characterization, localization, cDNA cloning, and expression.

A 23-kDa protein that was present at higher levels in diapausing 2nd instar larvae than in feeding 2nd instar larvae of Choristoneura fumiferana was purified, and polyclonal antibodies were raised against this protein. The antibodies were subsequently used to screen a cDNA library that was constructed using RNA from 2nd instar larvae. Eight identical cDNA clones were isolated. The cDNA clone had a 665-bp insert and the longest open reading frame coded for a 203-amino acid protein with a predicted molecular mass of 23.37 kDa. The deduced amino acid sequence showed high similarity to glutathione S-transferases and therefore, the cDNA clone was named C. fumiferana glutathione S-transferase (CfGST). Identity of CfGST was confirmed by using affinity-purification as well as enzyme activity assay. CfGST was closer in similarity to insect GST2 members than GST1 members. The apparent Vmax of the purified CfGST towards the substrates glutathione and 1-chloro-2,4-dinitrobenezene (CDNB) were similar. However, the enzyme had a three-fold higher affinity towards CDNB than glutathione. Analyses using Northern blot, immunoblot and immunocytochemistry demonstrated that the fat body was the major tissue where the enzyme was synthesized and stored. Higher levels of CfGST protein were present in diapausing 2nd instar larvae compared to feeding 2nd and 6th instar larvae, suggesting that besides detoxification CfGST may have other roles during insect development that are not readily apparent at present. The CfGST cDNA was expressed in a recombinant baculovirus expression system and an active enzyme was produced.     

Molecular characterization of a novel quinolizidine alkaloid O-tigloyltransferase: cDNA cloning, catalytic activity of recombinant protein and expression analysis in Lupinus plants

A novel acyltransferase committed to the final step of quinolizidine alkaloid biosynthesis, tigloyl-CoA:(-)-13alpha-hydroxymultiflorine/(+)-13alpha-hydroxylupanine O-tigloyltransferase, has been purified from Lupinus albus. The internal amino acid sequences were determined with protease-digested fragments of 25 and 30 kDa bands, allowing design of primers for amplification of cDNA fragments by polymerase chain reaction. Using an amplified fragment as the probe, a full-length cDNA clone was isolated. Sequence analysis revealed that the cDNA encodes a protein of 453 amino acids with a molecular mass of 51.2 kDa. Phylogenetic analysis of the deduced amino acid sequences indicated that this alkaloid acyltransferase belongs to a unique subfamily of a plant acyl-CoA-dependent acyltransferase gene family. The cDNA was expressed in bacterial cells as a recombinant protein fused to glutathione S-transferase. The fusion protein was affinity purified and cleaved to yield the recombinant enzyme for the study of catalytic properties. The recombinant enzyme catalyzed the acyltransfer reaction from tigloyl-CoA to (-)-13alpha-hydroxymultiflorine and (+)-13alpha-hydroxylupanine. Benzoyl-CoA could also serve efficiently as an acyl donor for these hydroxylated alkaloids. RNA blot analysis suggested that the gene was expressed in roots and hypocotyls but not in cotyledons and leaves. These results indicated that this specialized acyltransferase, isolated for the first time as tigloyltransferase from nature, is committed to control the quinolizidine alkaloid patterns in a tissue-specific manner.     

Stachyose synthesis in seeds of adzuki bean (Vigna angularis): molecular cloning and functional expression of stachyose synthase

Stachyose is the major soluble carbohydrate in seeds of a number of important crop species. It is synthesized from raffinose and galactinol by the action of stachyose synthase (EC 2.4.1.67). We report here on the identification of a cDNA encoding stachyose synthase from seeds of adzuki bean (Vigna angularis Ohwi et Ohashi). Based on internal amino acid sequences of the enzyme purified from adzuki bean, oligonucleotides were designed and used to amplify corresponding sequences from adzuki bean cDNA by RT-PCR, followed by rapid amplification of cDNA ends (RACE-PCR). The complete cDNA sequence comprised 3046 nucleotides and included an open reading frame which encoded a polypeptide of 857 amino acid residues. The entire coding region was amplified by PCR, engineered into the baculovirus expression vector pVL1393 and introduced into Spodoptera frugiperda (Sf21) insect cells for heterologous expression. The recombinant protein was immunologically reactive with polyclonal antibodies raised against stachyose synthase purified from adzuki bean and was shown to be a functional stachyose synthase with the same catalytic properties as its native counterpart. High levels of stachyose synthase mRNA were transiently accumulated midway through seed development, and the enzyme was also present in mature seeds and during germination.     

Isolation of several cDNAs encoding yeast peroxisomal enzymes

Several candidate clones carrying partial cDNAs for yeast peroxisomal enzymes, such as catalase, carnitine acetyltransferase, isocitrate lyase, malate synthase and acyl-CoA oxidase, were efficiently isolated at a single plating from a phage lambda gt11 recombinant cDNA library prepared with poly(A)-rich RNA from an n-alkane-grown yeast, Candida tropicalis, with a mixture of antibodies against the respective purified enzymes. Among them, one candidate clone carrying partial cDNA for catalase was subcloned and subjected to nucleotide sequence analysis. We succeeded in determining that the amino acid sequence deduced from the nucleotide analysis included the sequences derived from the two peptide fragments obtained from the purified enzyme.     

Catalytic component of calmodulin-independent adenylate cyclase from bovine brain. Isolation and determination of partial amino acid sequence

The catalytic component of calmodulin-independent adenylate cyclase of cattle cerebral cortex was solubilized and purified to the homogeneous state. The conditions for preparative obtaining of the enzyme on the column with immobilized antibodies to adenylate cyclase were found. These antibodies were proved to interact with the calmodulin-independent rather than the calmodulin-dependent form of the enzyme. Molecular mass of the calmodulin-independent adenylate cyclase determined electrophoretically is 140 +/- 10 kDa. Amino acid composition of the enzyme and sequences of its fragments (in total 300 amino acid residues) obtained upon treatment with lysyl-specific proteinase from Achromobacter liticus were determined. Clone containing a cDNA 605 bp insertion coding for the 183 amino acid residue fragment of adenylate cyclase was isolated from the bovine brain cDNA library. Homology of this fragment to the known sequences of Escherichia coli and Bordetella pertussis adenylate cyclases was revealed.