Human tissue factor: cDNA sequence and chromosome localization of the gene

A human placenta cDNA library in lambda gt11 was screened for the expression of tissue factor antigens with rabbit polyclonal anti-human tissue factor immunoglobulin G. Among 4 million recombinant clones screened, one positive, lambda HTF8, expressed a protein that shared epitopes with authentic human brain tissue factor. The 1.1-kilobase cDNA insert of lambda HTF8 encoded a peptide that contained the amino-terminal protein sequence of human brain tissue factor. Northern blotting identified a major mRNA species of 2.2 kilobases and a minor species of approximately 3.2 kilobases in poly(A)+ RNA of placenta. Only 2.2-kilobase mRNA was detected in human brain and in the human monocytic U937 cell line. In U937 cells, the quantity of tissue factor mRNA was increased severalfold by exposure of the cells to phorbol 12-myristate 13-acetate. Additional cDNA clones were selected by hybridization with the cDNA insert of lambda HTF8. These overlapping isolates span 2177 base pairs of the tissue factor cDNA sequence that includes a 5'-noncoding region of 75 base pairs, an open reading frame of 885 base pairs, a stop codon, a 3'-noncoding region of 1141 base pairs, and a poly(A) tail. The open reading frame encodes a 33-kilodalton protein of 295 amino acids. The predicted sequence includes a signal peptide of 32 or 34 amino acids, a probable extracellular factor VII binding domain of 217 or 219 amino acids, a transmembrane segment of 23 amino acids, and a cytoplasmic tail of 21 amino acids. There are three potential glycosylation sites with the sequence Asn-X-Thr/Ser.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and characterization of bovine insulin-like growth factor binding protein-3 (bIGFBP-3).

We report for the first time the isolation of a cDNA encoding the complete amino acid sequence for bovine growth hormone-dependent insulin-like growth factor binding protein-3 (bIGFBP-3). The deduced amino acid sequence from the cDNA revealed a mature polypeptide consisting of 264 amino acids and a 27 amino acid putative signal peptide. The amino acid sequence is over 80% homologous with human IGFBP-3 with complete conservation of the 18 cysteine residues and the 3 Asn-linked glycosylation sites. Between the two species there are 44 amino acid substitutions. Northern analysis of the bIGFBP-3 mRNA in bovine tissue revealed a single mRNA species of 1.65 kilobases.     

Molecular cloning and characterization of onchocystatin, a cysteine proteinase inhibitor of Onchocerca volvulus.

A cDNA clone designated OV7 encodes a polypeptide that corresponds to a highly antigenic Onchocerca volvulus protein. OV7 has significant amino acid sequence homology to the cystatin superfamily of cysteine proteinase inhibitors. In this report we establish that the OV7 recombinant protein is active as a cysteine proteinase inhibitor, and we have named it onchocystatin. It contains a cystatin-like domain that inhibits the activity of cysteine proteinases at physiological concentrations. Recombinant glutathione S-transferase-OV7 (GST-OV7, 1 microM) and maltose-binding protein-OV7 (MBP-OV7, 4 microM) fusion polypeptides inhibit 50% of the enzymatic activity of the bovine cysteine proteinase cathepsin B. Neither fusion polypeptide inhibits serine or metalloproteinases activity. The Ki for GST-OV7 fusion polypeptide is 170 nM for cathepsin B and 70 pM or 25 nM for cysteine proteinases purified from a protozoan parasite Entamoeba histolytica or the free living nematode Caenorhabditis elegans, respectively. The 5' end of the OV7 clone was isolated by polymerase chain reaction and sequenced, thus extending the previous cDNA clone to 736 base pairs. This represents the complete coding sequence of the mature onchocystatin (130 amino acids). A hydrophobic leader sequence of 32 amino acids was found, indicating a possible extracellular function of the onchocerca cysteine proteinase inhibitor.     

Complete primary structure of the transacylase (E2b) subunit of the human branched chain alpha-keto acid dehydrogenase complex

We isolated from a placental cDNA library by immunoscreening a cDNA clone encoding the transacylase (E2b) precursor of the human branched chain alpha-keto acid dehydrogenase (BCKDH) complex. The cDNA insert consists of 2,649 base pairs with an open reading frame of 1,431 base pairs which can be translated into 477 amino acids and a 3'-untranslated region of 1,205 base pairs. The deduced amino acid sequence includes a leader peptide of 56 amino acid residues, a lipoyl-bearing domain, a E3-binding domain and an inner core domain. A mature human E2b subunit is likely to contain 421 amino acid residues with a calculated Mr 46,322. The nucleotide sequence of the open reading frame and the deduced amino acid sequence of the human E2b shows 91.6% and 92.0% homology with those of the bovine E2b subunit, respectively.     

Energy-linked anion transport. Cloning, sequencing, and characterization of a full length cDNA encoding the rat liver mitochondrial proton/phosphate symporter

A full length cDNA clone encoding the precursor of the rat liver mitochondrial phosphate transporter (H+/Pi symporter) has been isolated from a cDNA library using a bovine heart partial length phosphate transporter clone as a hybridization probe. The entire clone is 1263 base pairs in length with 5'- and 3'-untranslated regions of 16 and 168 base pairs, respectively. The open reading frame encodes for the mature protein (312 amino acids) preceded by a presequence of 44 amino acids enriched in basic residues. The polypeptide sequence predicted from the DNA sequence was confirmed by analyzing the first 17 amino-terminal amino acids of the pure phosphate transporter protein. The rat liver phosphate transporter differs from the bovine heart transporter in 32 amino acids (i.e. approximately 10%). It contains a region from amino acid 139 to 159 which is 37% identical with the beta-subunit of the liver mitochondrial ATP synthase. Amino acid sequence comparisons of the Pi transporter with Pi binding proteins, other H+-linked symporters, and the human glucose transporter did not reveal significant sequence homology. Analysis of genomic DNA from both rat and S. cerevisiae by Southern blots using the rat liver mitochondrial Pi carrier cDNA as a probe revealed remarkably similar restriction patterns, a finding consistent with the presence in lower and higher eukaryotes of homologous Pi carrier proteins. This is the first report of the isolation, sequencing, and characterization of a full length cDNA coding for a protein involved in energy-coupled Pi transport.     

Cloning and characterization of a cDNA coding for the alpha-subunit of a stimulatory G protein from Schistosoma mansoni.

Guanine nucleotide-binding proteins (G proteins) mediate signals between serotonin receptors and adenylate cyclase in Schistosoma mansoni. A bovine Gs alpha cDNA probe was used to isolate a cDNA clone, SG12, encoding the entire alpha-subunit of a G protein of S. mansoni. The cDNA is 1897 base pairs long, contains an open reading frame of 1137 base pairs, and codes for a deduced protein of 379 amino acids. The putative protein encoded by the clone has an exact amino acid match with bovine Gs alpha of 65% and a 78% match when conserved amino acid substitutions are considered. In contrast, the exact and conserved matches of the schistosome alpha-subunit with bovine Gi are 41 and 61%, respectively. A comparison of the deduced amino acid sequence of SG12 with a variety of different G alpha proteins indicates that all the major structural features characteristic of a Gs alpha protein are present in the S. mansoni gene. The schistosome clone contains the putative site for ADP-ribosylation by cholera toxin found in Gs alpha but does not contain the ADP-ribosylation site for pertussis toxin present in Gi alpha. The amino acids are completely conserved at the GTP-binding sites. On a Northern blot, the cDNA hybridizes to a major band of 3.1 kilobases in RNA from adult schistosomes. The message appears to be absent in miracidia and cercariae, but a faint 3.1-kilobase band is visible in the early schistosomule stage preceding adulthood. This evidence, when added to previous biochemical data, indicates that the expression of this gene is developmentally controlled.     

Cloning and sequencing of cDNA that encodes goat growth hormone

The cDNA that encodes goat growth hormone (gGH) was isolated from a goat pituitary cDNA library. The cDNA, about 880 base pairs long, had a coding sequence, 5'- and 3'-untranslated regions and a poly(A) chain. The cDNA could encode a polypeptide of 217 amino acids. The amino acid sequence homology between gGH and the sequences of bovine GH, rat GH and human GH was 99, 83 and 66%, respectively. By Northern blot hybridization, we found that the possible gGH gene is transcribed in the goat pituitary.     

Characterization of a complementary deoxyribonucleic acid coding for human and bovine plasminogen

A cDNA library was constructed in pBR322 from bovine liver mRNA that was enriched for plasminogen mRNA by polysome immunoprecipitation. A 32P-labeled single-stranded cDNA was then prepared from the enriched bovine mRNA and employed as a probe to screen the cDNA library. The screening was carried out by testing for clones that protect the hybridized 32P-labeled cDNA from S1 nuclease digestion. The longest clone that was found was 581 base pairs in length and coded for the C-terminal 107 amino acids of bovine plasminogen, a 3' noncoding region of 246 nucleotides and a poly(A) tail. The bovine cDNA clone was then used as a probe to screen a human liver cDNA library of 18 000 recombinants. Six isolates were found to contain human plasminogen sequences. The longest clone consisted of 1851 base pairs corresponding to amino acid residues 272-790, followed by a 3' noncoding region of 227 base pairs and a poly(A) tail. Restriction fragments of the human cDNA were then used as probes to screen a human genomic DNA library present in a Charon 4A lambda phage library. Approximately 50 isolates from 10(6) recombinants were identified that hybridized to varying degrees with the cDNA probe. Among these, 10 corresponding to the gene for human plasminogen have been analyzed, and 3 that overlap have been shown to extend from kringle 3 through the 3' noncoding region of the gene. A 160 base pair exon with flanking splice junctions was then characterized and shown to encode for the first half of plasminogen kringle 4, including amino acid residues 346-399.     

Recombinant bovine dihydrofolate reductase produced by mutagenesis and nested PCR of murine dihydrofolate reductase cDNA

Recent reports of the slow-tight binding inhibition of bovine liver dihydrofolate reductase (bDHFR) in the presence of polyphenols isolated from green tea leaves has spurred renewed interest in the biochemical properties of bDHFR. Earlier studies were done with native bDHFR but in order to validate models of polyphenol binding to bDHFR, larger quantities of bDHFR are necessary to support structural studies. Bovine DHFR differs from its closest sequence homologue, murine DHFR, by 19 amino acids. To obtain the bDHFR cDNA, murineDHFR cDNA was transformed by a series of nested PCRs to reproduce the amino acid coding sequence for bovine DHFR. The bovine liver DHFR cDNA has an open reading frame of 561 base pairs encoding a protein of 187 amino acids that has a high level of conservation at the primary sequence level with other DHFR enzymes, and more so for the amino acid residues in the active site of the mammalian DHFR enzymes. Expression of the bovine DHFR cDNA in bacterial cells produced a stable recombinant protein with high enzymatic activity and kinetic properties similar to those previously reported for the native protein.     

The relationship between rat major acute phase protein and the kininogens

The rat major acute phase protein (alpha 1-MAP) is a cysteine protease inhibitor. The stoichiometry of the interaction between the inhibitor and enzyme was shown to be 1:2. A cDNA clone specific for rat alpha 1-MAP was isolated from a cDNA library prepared from an inflamed rat liver RNA template. The 1458-base pair insert was sequenced and positively identified by alignment with a partial amino acid sequence obtained by radiosequence analysis of the primary translation product for alpha 1-MAP. Complete sequence analysis determined the alpha 1-MAP cDNA coded for the entire protein with the exception of the first four amino acids of the signal peptide, all of which were identified by radiosequencing. The coding sequence spans 1282 nucleotides, followed by 115 base pairs of a 3' untranslated region. Two putative active sites, suggested by the enzyme-inhibitor ratio, have been identified by analysis of internal duplications of the alpha 1-MAP sequence and the alignment of these regions with the sequences of several low molecular weight cysteine protease inhibitors. A computer homology analysis of the protein sequence revealed a 59.3% overall identity between rat alpha 1-MAP and bovine low molecular weight (LMW) kininogen. The homology included the signal peptide regions. LMW kininogen is a precursor of bradykinin. alpha 1-MAP does contain a bradykinin sequence; the flanking amino acids are different, however. Evidence for the expression of the LMW and a high molecular weight kininogen from the same gene, and the high degree of homology between these proteins and the rat acute phase protein suggest that all three proteins belong to a precisely regulated gene family.     

The amino acid sequence of the human RNA polymerase II 33-kDa subunit hRPB 33 is highly conserved among eukaryotes

We have cloned and sequenced a cDNA of 1766 base pairs in length encoding the 275 amino acids of hRPB 33, the third largest subunit of human RNA polymerase II. The DNA was isolated by screening of a human lambda gt11 cDNA library with oligonucleotides designed on the basis of the amino acid residue analysis of the bovine material. The hRPB 33 amino acid sequence is highly conserved between Saccharomyces cerevisiae and human. Overall, 45% of the amino acid residues are identical with the yeast homologue RPB 3, and 65% of the amino acids are identical in the two major conserved regions at residues 0-103 and 151-197. hRPB 33 is also homologous to yeast RPC 5. The amino acid sequence of hRPB 33 showed no obvious homology with bacterial RNA polymerase or with any of its sigma factors.     

Human thrombomodulin: complete cDNA sequence and chromosome localization of the gene

A human umbilical vein endothelial cell cDNA library in lambda gt11 was screened for expression of thrombomodulin antigens with affinity-purified rabbit polyclonal anti-thrombomodulin immunoglobulin G (IgG) and mouse monoclonal anti-human thrombomodulin IgG. Among 7 million recombinant clones screened, 12 were recognized by both antibodies. Two of these, lambda HTm10 and lambda HTm12, were shown to encode thrombomodulin by comparison of the amino acid sequence deduced from the nucleotide sequence to the amino acid sequence determined directly from tryptic peptides of thrombomodulin. Thrombomodulin mRNA was estimated to be 3.7 kilobases in length by Northern blot analysis of endothelial cell and placental poly(A)+ RNA. Thrombomodulin mRNA was not detected in human brain, HepG2 hepatoma cells, or the monocytic U937 cell line. Additional cDNA clones were selected by hybridization with the 1.2-kilobase insert of lambda HTm10. One isolate, lambda HTm15, contained a 3693 base pair cDNA insert with an apparent 5'-noncoding region of 146 base pairs, an open reading frame of 1725 base pairs, a stop codon, a 3'-noncoding region of 1779 base pairs, and a poly(A) tail of 40 base pairs. The cDNA sequence encodes a 60.3-kDa protein of 575 amino acids. The predicted protein sequence includes a signal peptide of approximately 21 amino acids, an amino-terminal ligand-binding domain of approximately 223 amino acids, an epidermal growth factor (EGF) homology region of 236 amino acids, a serine/threonine-rich segment of 34 amino acids, a membrane-spanning domain of 23 amino acids, and a cytoplasmic tail of 38 amino acids. The EGF-homology region consists of six tandemly repeated EGF-like domains.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide sequences of the cDNA and an intronless pseudogene for human lactate dehydrogenase-A isozyme

Eight cDNA clones for lactate dehydrogenase-A isozyme (LDH-A) were isolated from a human fibroblast cDNA library, characterized, and no sequence heterogeneity was found. Four cDNA clones appear to contain nearly full-length cDNA inserts and the complete nucleotide sequence of 1710 base pairs consists of the protein-coding sequence (999 base pairs), the 5' (97 base pairs) and 3' (565 base pairs) untranslated regions and poly(dA) tail (49 base pairs). The predicted amino acid sequence of the human LDH-A polypeptide shows 92% homology (27 differences out of 331 amino acids compared) with that of the pig LDH-A subunit determined by direct protein sequencing [Kiltz et al. (1977) Hoppe-Seyler's Z. Physiol. Chem. 358, 123-127]. Human genomic clones containing an LDH-A pseudogene were isolated and the nucleotide sequence of 1635 base pairs from an intronless pseudogene was determined. The presence of two termination codons, two deletions of three nucleotides each and the replacement of three arginine residues at the active site (nos 98, 105 and 168) by other amino acids renders its coding region incapable of producing a functional LDH-A protein. A comparison between human LDH-A cDNA and the pseudogene sequences reveals 12.9% differences (114 transitions, 65 transversions and 36 deletions/insertions). Further, only four out of the 25 dCpdG dinucleotides present in the cDNA sequence remain unchanged, although the sequences possess 87.1% homology.     

The nuclear-encoded human NADH:ubiquinone oxidoreductase NDUFA8 subunit: cDNA cloning, chromosomal localization, tissue distribution, and mutation detection in complex-I-deficient patients

We report the cloning of the cDNA sequence of the nuclear-encoded NDUFA8 subunit of NADH: ubiquinone oxidoreductase, the first mitochondrial respiratory chain complex. The NDUFA8 open reading frame (ORF) includes 519 bp and encodes 172 amino acids (Mr=20.1 kDa). The human cDNA sequence shows 86.2% identity with the bovine sequence, whereas the human NDUFA8 amino acid sequence is 87.8% similar to its bovine PGIV protein counterpart. Both human and bovine NDUFA8 contain a conserved cysteine motif. Polymerase chain reaction analysis of rodent/human somatic cell hybrids maps the human NDUFA8 gene to chromosome 9. A multiple tissue blot has revealed the highest NDUFA8 mRNA expression in human heart, skeletal muscle, and fetal heart. Mutation analysis of the NDUFA8 fibroblast cDNA in 20 patients with an isolated enzymatic complex I deficiency in cultured skin fibroblasts has revealed two polymorphisms, one within the ORF and the other in the 3’ untranslated region of the NDUFA8 cDNA sequence. The allelic frequency of both polymorphisms was similar in controls and complex-I-deficient patients. Received: 17 April 1998 / Accepted: 22 July 1998     

A novel cDNA clone encoding a prolactin-like protein that lacks the two C-terminal cysteine residues isolated from bovine placenta

A new prolactin-like cDNA clone, bPLP-IV, was isolated from a bovine placental cDNA library and the complete nucleotide sequence was determined. The bPLP-IV encodes a protein consisting of 237 amino acids, which is related to, but different from seven other known bovine prolactin-like proteins including two placental lactogens. The predicted amino acid sequence of the bPLP-IV shows over 52% identity to other known members of bovine prolactin-like proteins, 48% to bovine prolactin, 40% to both two bovine placental lactogens and only 22% to bovine growth hormone. The bPLP-IV protein has a unique feature in its primary structure, lacking the two C-terminal cysteine residues which are completely conserved in all other known members of prolactin-growth hormone-placental lactogen gene family. The expression of bPLP-IV in developing bovine placenta was apparently stage-specific, being maximal in the full-term placenta.     

Molecular cloning of a cDNA for the catalytic subunit of rabbit muscle phosphorylase phosphatase

A cDNA coding for the catalytic subunit of phosphorylase phosphatase (phosphatase C-I/phosphatase-1c) was cloned from a rabbit muscle cDNA library by screening with oligonucleotide probes. Ten clones were analyzed. The full cDNA sequence of 1395 base pairs contained an open reading frame of 990 base pairs flanked by 3' and 5' noncoding regions of 84 and 321 base pairs, respectively. The DNA sequence (and deduced amino acid sequence) of this cDNA is distinctly different from that of a clone of 1492 base pairs previously reported. Our cDNA is essentially identical to the 1492-base pair clone from residue 182 in the 3' direction, but it is completely different in the 5' direction. Consequently, the amino acid sequence deduced from our cDNA differs by 14 amino acids in the amino terminal from that previously reported and extends for an additional 19 amino acids. Probes to the divergent and common region of our cDNA clone hybridized to an mRNA of the same size by Northern blotting. Thus the cDNA we have isolated appears to code for an isoform of the catalytic subunit of phosphorylase phosphatase.     

Nucleotide sequence of a full-length complementary DNA clone and amino acid sequence of human phenylalanine hydroxylase

A full-length human phenylalanine hydroxylase complementary DNA (cDNA) clone was isolated from a human liver cDNA library, and the nucleotide sequence encoding the entire enzyme was determined. The cDNA clone contains an inserted DNA fragment of 2448 base pairs, including 19 base pairs of poly(A) at the 3' end. The first methionine codon occurs at nucleotide position 223, followed by an open reading frame of 1353 base pairs, encoding 451 amino acids. Translation of the nucleotide sequence in the open reading frame predicts the amino acid sequence of human phenylalanine hydroxylase. The human protein shows a 96% amino acid sequence homology with the corresponding rat enzyme. The determination of the complete primary structure for phenylalanine hydroxylase represents the first among mixed-function oxidases.     

Primary amino acid sequence of bovine dopamine beta-hydroxylase

Fifty-eight tryptic and Staphylococcus aureus V8 protease generated peptides from bovine dopamine beta-hydroxylase were isolated by reverse-phase high pressure liquid chromatography and sequenced. These peptide sequences were compared with the deduced amino acid sequences of bovine and human dopamine beta-hydroxylase obtained from the cloned cDNAs. Bovine peptide sequences had five differences with the sequence derived from the bovine cDNA, and four of the changes could be accounted for by a single base change in the DNA. N-terminal sequence analysis of the bovine enzyme indicated that it contained two N termini, one of which is 3 amino acids longer than the other and begins with the sequence Ser-Ala-Pro. The amino acid sequences deduced from the bovine and human cDNAs are 19 and 25 amino acids longer, respectively, and these additional amino acids represent leader peptide sequences. Two bovine peptide sequences contained glycosylation sites and gave positive tests for carbohydrate residues, and two others contained the consensus sequence for a glycosylation site but were negative in the carbohydrate test. The bovine enzyme contains 6 Trp, as compared with 7 in the bovine cDNA and 8 in the human cDNA. The protein and bovine cDNA contain 24 Tyr each, as compared with 26 in the human cDNA. These numbers indicate that the true epsilon 1% 280 = 8.95, and, therefore, that it is 28% lower than the previously determined value. The data also identify 5 His-containing regions that may be involved in Cu2+ coordination at the active site.