Hamster cytochrome P-450 IA gene family, P-450IA1 and P-450IA2 in lung and liver: cDNA cloning and sequence analysis.

Two cDNA clones, 2C19 and 4C1, were isolated from a lung cDNA library of 3-methylcholanthrene (MC)-treated hamster by using rat P-450c cDNA as a probe. The cDNA determined from 2C19 and 4C1 was 2,916 bp long and contained an entire coding region for 524 amino acids with a molecular weight of 59,408. The deduced amino acid sequence showed a 85% identity with that of rat P-450c indicating 2C19 and 4C1 encode the hamster P-450IA1 protein. Another cDNA clone, designated H28, was isolated from a MC-induced hamster liver cDNA library by using the hamster lung 2C19 or 4C1 cDNA clone as a probe. H28 was 1,876 bp long and encoded a polypeptide of 513 amino acids with a molecular weight of 58,079. The N-terminal 20 residues deduced from nucleotide sequence of H28 were identical to those determined by sequence analysis of purified hamster hepatic P-450MCI. The high similarity of the nucleotide and deduced amino acid sequences between H28 and P-450IA2 of other species indicated that H28 encoded a P-450 protein which belongs to the P-450IA2 family. Northern blot analysis revealed that the mRNAs for hamster P-450IA1 and IA2 were about 2.9 and 1.9 kb long, respectively. Hamster P-450IA1 mRNA was induced to the same level in lungs as in livers by MC treatment, whereas hamster P-450IA2 mRNA was induced and expressed only in hamster liver.     

Molecular cloning, cDNA structure and predicted amino acid sequence of bovine 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4 isomerase

We have used our recently characterized human 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) cDNA as probe to isolate cDNAs encoding bovine 3 beta-HSD from a bovine ovary lambda gtll cDNA library. Nucleotide sequence analysis of two overlapping cDNA clones of 1362 bp and 1536 bp in length predicts a protein of 372 amino acids with a calculated molecular mass of 42,093 (excluding the first Met). The deduced amino acid sequence of bovine 3 beta-HSD displays 79% homology with human 3 beta-HSD while the nucleotide sequence of the coding region shares 82% interspecies similarity. Hybridization of cloned cDNAs to bovine ovary poly(A)+ RNA shows the presence of an approximately 1.7 kb mRNA species.     

Correction of the cDNA-derived protein sequence of prostatic spermine binding protein: pivotal role of tandem mass spectrometry in sequence analysis

Spermine binding protein (SBP) is a rat ventral prostate protein that binds various polyamines, and the level of this protein and its mRNA is regulated by androgens. Previously, the cDNA for SBP was cloned and sequenced and an amino acid sequence deduced from the cDNA. Data from cloned and sequenced and an amino acid sequence deduced from the cDNA. Data from partial amino acid sequencing of the purified protein were consistent with the amino acid sequence deduced from the cDNA. However, the amino terminus of the protein was blocked, and therefore, direct protein sequence information confirming the cDNA reading frame of this region could not be obtained by Edman degradation. We have now employed an integrated approach using fast atom bombardment mass spectrometry, tandem mass spectrometry, and conventional sequencing methodologies to establish the amino-terminal sequence of the protein and to identify an amino acid sequence (35 residues) present in the purified protein but missing from the amino acid sequence deduced from cDNA clones for this protein. The missing piece of cDNA corresponds to an exon found in mouse genomic clones for a protein similar to rat SBP. Therefore, the cDNA clones for rat SBP may represent splicing variants that lack the sequence information of one exon. The blocked amino terminus of the protein was identified as 5-oxopyrrolidine-2-carboxylic acid. Mass spectrometry also provided evidence regarding glycosylation of the protein. The first of two potential glycosylation sites clearly carries carbohydrate; the second site is, at most, only partially glycosylated.     

Cloning and sequence analysis of cDNA for rat liver uricase

We have isolated cDNA clones for rat liver uricase using an oligonucleotide corresponding to the N-terminal sequence of 8 amino acids. The nucleotide sequences of the cDNAs have been determined, and the amino acid sequence of the protein deduced. A 867-base open reading frame coding for 289 amino acids, corresponding to a molecular mass of 33,274 daltons, was confirmed by matching eight sequences of a total of 53 amino acids from peptide sequence analyses of the fragments generated by lysyl endopeptidase digestion of purified rat liver uricase. The deduced amino acid sequence of rat liver uricase shares 40% homology with that of soybean nodulin-specific uricase and has an N-terminal extension of 7 amino acids. In contrast, soybean uricase has a C-terminal extension of 12 amino acids, which is presumably the result of local gene duplication. Completely different N- and C-terminal structures of the two uricases suggest that the signals for targeting the proteins to the peroxisome are not located on the terminal continuous stretches of amino acids.     

Molecular characterization of tobacco mitochondrial L-galactono-gamma-lactone dehydrogenase and its expression in Escherichia coli

A cDNA clone encoding L-galactono-gamma-lactone (GAL) dehydrogenase (EC 1.3.2.3) was isolated from tobacco leaves. The cDNA clone contained an open reading frame encoding the protein of 501 amino acids with a calculated molecular mass of 56,926 Da, preceded by a putative mitochondrial targeting signal consisting of 86 amino acid residues. In fact, GAL dehydrogenase was localized in the mitochondria of tobacco cells. The deduced amino acid sequence of the cDNA showed 77 and 82% homology to cauliflower and sweet potato GAL dehydrogenases, respectively. Southern blot analysis showed that tobacco contains one copy of the gene for the enzyme. Northern blot analysis showed that GAL dehydrogenase mRNA (2.0 kb) is expressed in the leaves, stems, and roots in almost equal quantities. We introduced the cDNA clone encoding tobacco GAL dehydrogenase into a pET expression vector to overexpress this protein in Escherichia coli. The partially purified recombinant enzyme was used for comparative studies on the native enzymes from tobacco and other sources; its enzymatic properties were similar to those of other GAL dehydrogenases.     

Isolation, characterization, and expression of cDNA encoding a rat liver endoplasmic reticulum alpha-mannosidase

We have isolated a cDNA encoding an endoplasmic reticulum alpha-mannosidase, an asparagine-linked oligosaccharide processing enzyme, from a rat liver lambda gt11 library. Two degenerate oligonucleotides, based on amino acid sequence data from the purified enzyme, were used as primers in the polymerase chain reaction with liver cDNA as a template to generate an unambiguous cDNA probe. The cDNA fragment (524 base pair) obtained was then used to isolate cDNA clones by hybridization. We isolated two overlapping clones which were used to construct a full-length cDNA of 3392 base pairs. A single open reading frame of 1040 amino acids encodes a protein with a molecular mass of 116 kilodaltons containing the six known peptide sequences. The deduced amino acid sequence revealed no classical signal sequence or membrane-spanning domain. The alpha-mannosidase encoding cDNA can be expressed transiently in COS cells using the mammalian expression vector pXM, causing a 400-fold increase in alpha-mannosidase activity as well as a dramatic increase in immunoreactive polypeptide. The rat liver endoplasmic reticulum alpha-mannosidase bears striking homology to the vacuolar alpha-mannosidase from Saccharomyces cerevisiae.     

Structure and expression of cDNA for an inhibitor of blood coagulation isolated from human placenta: a new lipocortin-like protein

An inhibitor of blood coagulation, a new protein with an apparent molecular weight of 34,000 and an isoelectric point of 4.9, was purified from human placental tissue by EDTA extraction. Five cDNA clones were isolated from the human placental lambda gt11 cDNA library using the mouse monoclonal antibody raised against the coagulation inhibitor as the probe. The longest insert consists of 1,566 nucleotides, and contains 960 nucleotides entirely encoding the 320 amino acids of the inhibitor, and a poly A tail. The deduced amino acid sequence was corroborated by chemical analyses of the protein. The entire amino acid sequence shows homology to those of lipocortin I, lipocortin II, and endonexin-related proteins. The cDNA for the inhibitor was expressed in Escherichia coli under the regulation of the trc promotor of the plasmid pKK233-2. The resulting recombinant protein manifested inhibitory activities against both blood coagulation and phospholipase A2 activity, as did the coagulation inhibitor isolated from human placenta.     

Isolation and characterization of cDNA clones for rat liver 10-formyltetrahydrofolate dehydrogenase.

We have isolated and characterized cDNA clones encoding rat liver cytosol 10-formyltetrahydrofolate dehydrogenase (EC 1.5.1.6). An open reading frame of 2706 base pairs encodes for 902 amino acids of Mr 99,015. The deduced amino acid sequence contains exact matches to the NH2-terminal sequence (28 residues) and the sequences of five peptides derived from cyanogen bromide cleavage of the purified protein. The amino acid sequence of 10-formyltetrahydrofolate dehydrogenase has three putative domains. The NH2-terminal sequence (residues 1-203) is 24-30% identical to phosphoribosylglycinamide formyltransferase (EC 2.1.2.2) from Bacillus subtilis (30%), Escherichia coli (24%), Drosophila melanogaster (24%), and human hepatoma HepG2 (27%). Residues 204-416 show no extensive homology to any known protein sequence. Sequence 417-900 is 46% (mean) identical to the sequences of a series of aldehyde dehydrogenase (NADP+) (EC 1.2.1.3). Intact 10-formyltetrahydrofolate dehydrogenase exhibits NADP-dependent aldehyde dehydrogenase activity. The sequence identity to phosphoribosylglycinamide formyltransferase is discussed, and a binding region for 10-formyltetrahydrofolate is proposed.     

Identification and characterization of cDNA clones encoding hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyltransferase from tobacco.

The sequences of three cDNA clones that include the complete coding region of hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyltransferase (THT) from tobacco are reported. The three cDNAs were isolated by antibody screening of a cDNA expression library produced from poly(A)+RNA purified from tobacco leaves (Nicotiana tabacum cv. Bottom Special), previously infiltrated with an incompatible strain of Ralstonia solanacearum. The identity of these clones was confirmed by the detection of THT activity in extracts of transformed Escherichia coli and by matching the translated polypeptides with tryptic enzyme sequences. cDNA clones tht4 and tht11 differ only by their 5' leader and 3' UTRs and therefore encode the same protein, whereas tht10 and tht11 exhibit 95 and 99% sequence identity at the DNA and deduced amino acid levels, respectively. The three clones encode proteins of 226 amino acids with calculated molecular masses of 26 kDa. The deduced amino acid sequences show no similarity with the sequence of anthranilate hydroxycinnamoyl/benzoyltransferase from Dianthus caryophyllus, the only enzyme exhibiting hydroxycinnamoyltransferase activity to be cloned so far in plants. In contrast, comparison of the THT amino acid sequence with protein sequence databases revealed substantial homology with mammalian diamine acetyltransferases. The THT clones hybridized to a 0.95-kb mRNA from elicited tobacco cell-suspension cultures and also to a mRNA of similar size from wound-healing potato tubers. The messengers for THT were also found to be expressed at relatively high levels in tobacco root tissues. Southern hybridization of tobacco genomic DNA with THT cDNA suggests that several copies of the THT gene occur in the tobacco genome. Inhibition experiments using amino-acid-specific reagents demonstrated that both histidyl and cysteyl residues are required for THT activity. In the course of these experiments THT was also found to be inhibited by (2-hydroxyphenyl) amino sulfinyl acetic acid 1,1-dimethylethyl ester, an irreversible inhibitor of cinnamyl alcohol dehydrogenase.     

Identification and expression of a cDNA from Daucus carota encoding a bifunctional aspartokinase-homoserine dehydrogenase

Aspartokinase (EC 2.7.2.4) and homoserine dehydrogenase (EC 1.1.1.3) catalyze steps in the pathway for the synthesis of lysine, threonine, and methionine from aspartate. Homoserine dehydrogenase was purified from carrot (Daucus carota L.) cell cultures and portions of it were subjected to amino acid sequencing. Oligonucleotides deduced from the amino acid sequences were used as primers in a polymerase chain reaction to amplify a DNA fragment using DNA derived from carrot cell culture mRNA as template. The amplification product was radiolabelled and used as a probe to identify cDNA clones from libraries derived from carrot cell culture and root RNA. Two overlapping clones were isolated. Together the cDNA clones delineate a 3089 bp long sequence encompassing an open reading frame encoding 921 amino acids, including the mature protein and a long chloroplast transit peptide. The deduced amino acid sequence has high homology with the Escherichia coli proteins aspartokinase I-homoserine dehydrogenase I and aspartokinase II-homoserine dehydrogenase II. Like the E. coli genes the isolated carrot cDNA appears to encode a bifunctional aspartokinase-homoserine dehydrogenase enzyme.Abbreviations AK aspartokinase - HSDH homoserine dehydrogenase - PCR polymerase chain reaction - SDS sodium dodecyl sulfate The mention of vendor or product does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over vendors of similar products not mentioned.     

Cytovillin, a microvillar Mr 75,000 protein. cDNA sequence, prokaryotic expression, and chromosomal localization

Cytovillin is a microvillar cytoplasmic peripheral membrane protein, with prominent expression in vivo in placental syncytiotrophoblasts and certain human tumors. Cytovillin cDNA was cloned from a human placental lambda gt11 library using affinity purified antibodies. The identity of cytovillin cDNA clones was confirmed by expression of cytovillin in Escherichia coli and using antibodies raised against the expressed fusion protein in comparison with antibodies against cytovillin purified from cultured human choriocarcinoma cells. In these cells Northern blotting analysis identified a major 3.5-kilobase cytovillin mRNA. The cDNA encodes a protein of 575 amino acids corresponding to a molecular weight of 68,084. According to secondary structure prediction, cytovillin is a hydrophilic protein with an extensive internal alpha-helical region ending in a sequence of 7 consecutive prolines. The predicted alpha-helical region showed limited homology to alpha-helical regions of cytoskeletal proteins and certain other proteins, but no extensive homologies were found in the cytovillin cDNA or the deduced amino acid sequence to other registered DNA or protein sequences. Southern blot analysis of a DNA panel of human mouse somatic cell hybrids localized the cytovillin gene to the end of the long arm of chromosome 6 (6q22-q27). Our results show that cytovillin is representative of a novel class of microvillar proteins.     

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 and nucleotide sequence of cDNA encoding human liver serine-pyruvate aminotransferase

Cloned cDNAs for human liver serine-pyruvate aminotransferase (Ser-PyrAT) were obtained by screening of a human liver cDNA library with a fragment of cDNA for rat mitochondrial Ser-PyrAT as a probe. Two clones were isolated from 50,000 transformants. Both clones contained approximately 1.5 kb cDNA inserts and were shown to almost completely overlap each other on restriction enzyme mapping and DNA sequencing. The nucleotide sequence of the mRNA coding for human liver Ser-PyrAT was determined from those of the cDNA clones. The mRNA comprises at least 1487 nucleotides, and encodes a polypeptide consisting of 392 amino acid residues with a molecular mass of 43,039 Da. The amino acid composition determined on acid hydrolysis of the purified enzyme showed good agreement with that deduced from the nucleotide sequence of the cDNA. In vitro translation of the mRNA derived from one of the isolated clones, pHspt12, as well as that of mRNA extracted from human liver, yielded a product of 43 kDa which reacted with rabbit anti-(rat mitochondrial Ser-PyrAT) serum. Comparison of the deduced amino acid sequences of human Ser-PyrAT and the mature form of rat mitochondrial Ser-PyrAT revealed 79.3% identity. Although human Ser-PyrAT appears to be synthesized as the mature size, the 5'-noncoding region of human Ser-PyrAT mRNA contains a nucleotide sequence which would encode, if translated, an amino acid sequence similar to that of the N-terminal extension peptide of the precursor for rat mitochondrial Ser-PyrAT.     

Two distinct cDNAs for human IMP dehydrogenase

IMP dehydrogenase (EC 1.1.1.205), the rate-limiting enzyme of de novo GTP biosynthesis, is a promising target in antileukemic chemotherapy. We have isolated two distinct cDNA clones (types I and II) encoding IMP dehydrogenase from a human spleen cDNA library. Both clones encode closely related proteins of 514 residues showing 84% sequence identity. Northern hybridization analyses of poly(A)+ RNA from human normal leukocytes and human ovarian tumors demonstrated a striking contrast in mRNA expression in that type I mRNA is the main species in normal leukocytes and type II predominates over type I in the tumor. This is the first report suggesting the existence of two distinct types of human IMP dehydrogenase molecular species which may have different sensitivities to the drugs targeted against IMP dehydrogenase.     

Cloning and expression of cDNA encoding hamster liver 3-hydroxyhexobarbital/17beta(3alpha)-hydroxysteroid dehydrogenase 1

Using RACE techniques we have cloned and sequenced one of the hamster liver 3-hydroxy-hexobarbital dehydrogenases which catalyze not only cyclic alcohols but also 17beta-hydroxy-steroids and 3alpha-hydroxysteroids. The gene specific primers to 3-hydroxyhexobarbital dehydrogenase 1 (G2) were synthesized on the basis of its partial peptide sequences. The sequence of full length cDNA generated by 3'- and 5'-RACE PCR consisted of 1225 nucleotides including an open reading frame of 972 nucleotides encoding a protein of 323 amino acids. The deduced amino acid sequence matched exactly with the partial peptide sequences of hamster liver 3-hydroxyhexobarbital dehydrogenase 1 (G2). The sequence showed 84.5% identity to mouse liver 17beta-dehydrogenase(A-specific), and 74-76% identity to human liver bile acid binding protein/3alpha-hydroxysteroid dehydrogenase (DD2), human liver 3alpha-hydroxysteroid dehydrogenase type I (DD4) and type II (DD3), and rabbit ovary 20alpha-hydroxysteroid dehydrogenase. The protein contains catalytic residues of aldo-keto reductases, Asp50, Tyr55, Lys84, His117. These results suggest that the hamster liver 3-hydroxyhexobarbital/17beta(3alpha)-hydroxysteroid dehydrogenase belongs to aldo-keto reductase superfamily. The insert containing the full-length cDNA of 3-hydroxyhexobarbital dehydrogenase and vector specific overhang produced by PCR was annealed with pET-32 Xa/LIC vector. The plasmid was transformed into BL21 (DE3) cells containing pLysS. The recombinant enzyme was induced 1 mM IPTG. The expressed enzyme was produced as fusion protein and purified by nickel chelating affinity chromatography followed by POROS CM column chromatography and superdex 75 gel filtration. Molecular weight of the recombinant enzyme fused thioredoxin and his*tag was about 55000 and that was 35000 after Factor Xa protease treatment. The recombinant enzyme dehydrogenated 3-hydroxy-hexobarbital, 1-acenaphthenol, 2-cyclohexen-1-ol, testosterone, glycolithocholic acid as well as the native enzyme purified from hamster liver.     

Characterization and expression of the cDNA encoding a new kind of phospholipid transfer protein, the phosphatidylglycerol/phosphatidylinositol transfer protein from Aspergillus oryzae: evidence of a putative membrane targeted phospholipid transfer protein in fungi

The full-length cDNA of a phospholipid transfer protein (PLTP) was isolated from Aspergillus oryzae by a RACE-PCR procedure using degenerated primer pool selected from the N-terminal sequence of the purified phosphatidylinositol/phosphatidylglycerol transfer protein (PG/PI-TP). The cDNA encodes a 173 amino acid protein of 18823 Da. The deduced amino acid sequence from position 38 to 67 is 100% identical to the N-terminal sequence (first 30 amino acids) of the purified PG/PI-TP. This amino acid sequence is preceded by a leader peptide of 37 amino acids which is predicted to be composed of a signal peptide of 21 amino acids followed by an extra-sequence of 16 amino acids, or a membrane anchor protein signal (amino acid 5-29). This strongly suggests that the PG/PI-TP is a targeted protein. The deduced mature protein is 138 amino acids long with a predicted molecular mass of 14933 Da. Comparison of the deduced PG/PI-TP sequence with other polypeptide sequences available in databases revealed a homology with a protein deduced from an open reading frame coding for an unknown protein in Saccharomyces cerevisiae (36% identity and 57% similarity). Apart from this homology, the PG/PI-TP is unique and specific to the filamentous fungi on the basis of comparison of PLTP protein sequences. Northern blot analysis of RNA isolated from A. oryzae cultures grown on glucose or glucose supplemented with phospholipids suggests that the PG/PI-TP is transcribed by only one RNA species and allows us to show that expression of the protein is regulated at the messenger RNA level.     

Nucleotide sequence of cloned cDNA specific for rat ribosomal protein L27

We constructed cDNA libraries from 8-9-S poly(A)-rich RNA from regenerating rat liver, isolated clones specific for ribosomal protein L27 and determined the nucleotide sequences of the cDNA clones. The longest cDNA consists of 15 base pairs from the 5' leading sequence, the entire coding sequence of 411 base pairs, and the 3' trailing sequence of 59 base pairs including the poly(A) tail. The primary structure of protein L27 was deduced from the sequence of nucleotides. Protein L27 contains 135 amino acids and has a molecular mass of 15,666 Da. The amino-terminal sequence agreed well with the published partial amino acid sequence and the calculated amino acid composition is also consistent with the reported composition determined for the hydrolyzate of L27.