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)     

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)     

cDNA sequence and chromosomal localization of the mouse parvalbumin gene, Pva

In the homozygous condition, the mutation adr (arrested development of righting response) of the mouse causes a myotonia and a drastic reduction of the Ca2+-binding protein parvalbumin (PV) in fast muscles. Using a rat PV probe, a mouse cDNA clone was isolated from a lambda gt11 wild-type fast-muscle library and its nucleotide sequence was determined. The protein coding and the 3' nontranslated regions of the mouse gene show extensive homology with the rat PV gene. The result of Southern blot hybridization is consistent with a single copy gene for parvalbumin. Restriction fragment length polymorphisms (RFLPs) between Mus musculus domesticus (e.g. C57BL/6) and Mus spretus (SPE) were detected with the enzymes Eco RI, Pst I, and Sst I. The restriction fragment patterns of DNA samples from 65 individual offspring of (C57BL/6 x SPE)F1 x C57BL/6 backcrosses were tested with the PV probe and matched, for linkage detection, to pre-existing patterns established with various RFLP probes on the same samples. A co-distribution of PV-RFLPs with Pvt-1 and Mlvi-2, which had been localized on chromosome 15, was detected. Thus, the structural gene for PV, designated Pva, maps to chromosome 15 of the mouse whereas the adr mutation shows no linkage with markers on this chromosome. Gene locus homology between chromosome 15 of the mouse and chromosome 22 of man (which carries the human PV gene) is discussed.     

Human apolipoprotein A-II: nucleotide sequence of a cloned cDNA, and localization of its structural gene on human chromosome 1

[3H]yohimbine, a potent and selective alpha 2-adrenergic antagonist was used to label alpha-adrenoceptors in intact human lymphocytes. Binding of [3H]yohimbine was rapid (t1/2 1.5 -2.0 min) and readily reversed by 10 microM phentolamine (t1/2 = 5 - 6 min) and of high affinity (Kd = 3.7 +/- 0.86 nM). At saturation, the total number of binding sites was 19.9 +/- 5.3 fmol/10(7) lymphocytes. Adrenergic agonists competed for [3H]yohimbine binding sites with an order of potency: clonidine greater than (-) epinephrine greater than (-) norepinephrine greater than (+) epinephrine much greater than (-) isoproterenol; adrenergic antagonists with a potency order of yohimbine greater than phentolamine greater than prazosin. These results indicate the presence of alpha 2-adrenoceptors in human lymphocytes.     

Schistosoma mansoni tropomyosin: cDNA characterization, sequence, expression, and gene product localization

We have defined the polypeptide pattern of 3-hr Schistosoma mansoni schistosomula on nonequilibrium two-dimensional gels (NEPHGE). An acidic group of polypeptides with a molecular weight of about 40 kDa and a pI value of around 5.0 (numbered 48/59/53) were identified as antigens on Western blots probed with chronic human infection sera or vaccinated mouse sera. Polypeptides 48/49/53 from silver-stained NEPHGE gels produced antisera that were specific as demonstrated by Western blot analysis and immunoprecipitations of in vitro translation products. A cDNA clone (clone 1) from a S. mansoni adult worm pBR322 library was isolated by using cDNA probes made from size-fractionated mRNA and defined as encoding polypeptide 49 by hybridization selection of the mRNA which was in vitro translated and immunoprecipitated with specific mouse antiserum. A lambda gt 11 expression clone which contained an insert close to the full length mRNA was isolated from a S. mansoni cercariae library. The complete sequence of the mRNA was determined by sequencing the insert of this clone as well as primer extension of total RNA. The only open reading frame coding for 284 amino acids in the 1316 nucleotide sequence showed a 44.76 to 55.44% homology with the amino acid sequences of 18 different tropomyosins from various species. Computer-predicted secondary structure of schistosome tropomyosin was mainly alpha-helix which was very similar to other tropomyosins. Northern analysis showed the mRNA to be about 1.5 kb in size and detectable at much higher levels in the adult worm stage as compared to the cercariae and the egg stages. Western blot analysis likewise showed that greater amounts of tropomyosin were detected in extracts from adult worm stage as compared to extracts from cercariae and egg stages. Immunocytochemical analysis shows that tropomyosin is strongly associated with the tegument of adult worms. The restriction digestion pattern given by genomic Southern analysis suggests the existence of introns and/or multiple gene copies. Thus polypeptide 49, an immunodominant antigen, represents schistosome tropomyosin.     

The human glucagon receptor encoding gene: structure, cDNA sequence and chromosomal localization

Characterization of the human glucagon-receptor-encoding gene (GGR) should provide a greater understanding of blood glucose regulation and may reveal a genetic basis for the pathogenesis of diabetes. A cDNA encoding a complete functional human glucagon receptor (GGR) was isolated from a liver cDNA library by a combination of polymerase chain reaction and colony hybridization. The cDNA encodes a receptor protein with 80% identity to rat GGR that binds [¹²⁵I] glucagon and transduces a signal leading to increases in the concentration of intracellular cyclic adenosine 3′,5′-monophosphate. Southern blot analysis of human DNA reveals a hybridization pattern consistent with a single GGR locus. In situ hybridization to metaphase chromosome preparations maps the GGR locus to chromosome 17q25. Analysis of the genomic sequence shows that the coding region spans over 5.5 kb and is interrupted by 12 introns.     

Human indolethylamine N-methyltransferase: cDNA cloning and expression, gene cloning, and chromosomal localization.

Indolethylamine N-methyltransferase (INMT) catalyzes the N-methylation of tryptamine and structurally related compounds. We recently cloned and characterized the rabbit INMT cDNA and gene as a step toward cloning the cDNA and gene for this enzyme in humans. We have now used a PCR-based approach to clone a human INMT cDNA that had a 792-bp open reading frame that encoded a 263-amino-acid protein 88% identical in sequence to rabbit INMT. Northern blot analysis of 35 tissues showed that a 2.7-kb INMT mRNA species was expressed in most tissues. When the cDNA was expressed in COS-1 cells, the recombinant enzyme catalyzed the methylation of tryptamine with an apparent K(m) value of 2.9 mM. The human cDNA was then used to clone the human INMT gene from a human genomic BAC library. The gene was 5471 bp in length, consisted of three exons, and was structurally similar to the rabbit INMT gene as well as genes for nicotinamide N-methyltransferase and phenylethanolamine N-methyltransferase in several species. All INMT exon-intron splice junctions conformed to the "GT-AG" rule, and no canonical TATA or CAAT sequences were present within the 5'-flanking region of the gene. Human INMT mapped to chromosome 7p15.2-p15.3 on the basis of both PCR analysis and fluorescence in situ hybridization. Finally, two possible single nucleotide polymorphisms were identified within exon 3, both of which altered the encoded amino acid. The cloning and expression of a human INMT cDNA, as well as the cloning, structural characterization, and mapping of its gene represent steps toward future studies of the function and regulation of this methyltransferase enzyme in humans.     

Human epidermal growth factor precursor: cDNA sequence, expression in vitro and gene organization.

Complementary DNA clones encoding the human kidney epidermal growth factor (EGF) precursor have been isolated and sequenced. They predict the sequence of a 1,207 amino acid protein which contains EGF flanked by polypeptide segments of 970 and 184 residues at its NH2- and COOH-termini, respectively. The structural organization of the human EGF precursor is similar to that previously described for the mouse protein and there is 66% identity between the two sequences. Transfection of COS-7 cells with the human EGF precursor cDNA linked to the SV40 early promoter indicate that it can be synthesized as a membrane protein with its NH2-terminus external to the cell surface. The human EGF precursor gene is approximately 110 kilobase pairs and has 24 exons. Its exon-intron organization revealed that various domains of the EGF precursor are encoded by individual exons. Moreover, 15 of the 24 exons encode protein segments that are homologous to sequences in other proteins. Exon duplication and shuffling appear to have played an important role in determining the present structure of this protein.     

Human embryonic myosin heavy chain cDNA. Interspecies sequence conservation of the myosin rod, chromosomal locus and isoform specific transcription of the gene

A 3.6 kilobase cDNA clone coding for the human embryonic myosin heavy chain has been isolated and characterized from an expression library prepared from human fetal skeletal muscle. The derived amino acid sequence for the entire rod part of myosin shows 97% sequence homology between human and rat and a striking interspecies sequence conservation among the charged amino acid residues. The single copy gene is localized to human chromosome 17 and its expression in fetal skeletal muscle is developmentally regulated. The sequence information permits the design of isoform-specific probes for studies on the structure of the gene and its role in normal and defective human myogenesis.     

Human dioxin-inducible cytosolic NAD(P)H:menadione oxidoreductase. cDNA sequence and localization of gene to chromosome 16

NAD(P)H:menadione oxidoreductase (NMOR1) is a flavoprotein that catalyzes the two-electron reduction of various redox dyes and quinones. It has been proposed that this enzyme may have a protective effect against cancer caused by quinones and their metabolic precursors. We show that tetrachlorodibenzo-p-dioxin (TCDD) treatment of the human hepatoblastoma cell line Hep-G2 produces a 5-fold induction of NMOR activity. Several overlapping human NMOR1 cDNAs were isolated from a human liver lambda gt 11 expression library, and their composite sequence corresponds to an mRNA of 2448 nucleotides containing a continuous open reading frame encoding a protein of 274 residues (molecular weight, 30,880). The corresponding human NMOR1 mRNA has an unusually long 3'-untranslated region (1679 base pairs) with four potential polyadenylation signals (I-IV) at positions 986, 1460, 1838, and 2419 and a single copy of human Alu repetitive sequence between polyadenylation sites II and III. Southern blot analysis of human genomic DNA suggests the presence of a single NMOR1 gene approximately 10 kilobases (KB) in length. The use of three of the aforementioned polyadenylation signals is likely to account for the three different species (2.7, 1.7, and 1.2 kb) of mRNA hybridizing to NMOR1 cDNA in Hep-G2 cells. Indeed several partial cDNA clones were isolated that corresponded to the mRNA derived by use of the proximal polyadenylation signal. Interestingly, the longest (2.7 kb) mRNA species was induced severalfold by TCDD, whereas the other two mRNAs (1.7 and 1.2 kb) were induced to a much lesser extent by TCDD treatment. The human NMOR1 cDNA and protein are 83 and 85% similar to rat liver cytosolic NMOR1 cDNA and protein, respectively. Southern analysis of DNA from 54 human x mouse and 39 human x hamster somatic cell hybrids shows that the NMOR1 gene resides on human chromosome 16.     

Human serum amyloid P component. cDNA isolation, complete sequence of pre-serum amyloid P component, and localization of the gene to chromosome 1

Complementary DNA clones corresponding to the human serum amyloid P component (SAP) were isolated, and the complete nucleotide and derived amino acid sequence of preSAP was determined. PreSAP biosynthesis is directed by a 1.1-kilobase mRNA. Synthesis and postsynthetic processing of preSAP in Xenopus oocytes result in secretion of a protein with mobility similar to native purified SAP when analyzed by sodium dodecyl sulfate gel electrophoresis. The human SAP gene is on chromosome 1, probably closely linked to the gene for C-reactive protein which encodes the related acute phase reactant found in human plasma.     

Linkage studies in a new X-linked myopathy, suggesting exclusion of DMD locus and tentative assignment to distal Xq.

We here report linkage studies in a family suffering from a recently described hereditary muscle disease named X-linked myopathy with excessive autophagy (XMEA). Significant lod scores excluding linkage to the Duchenne-Becker muscular dystrophy locus were found. Several other loci on the short and long arms of the X chromosome produced negative lod scores, whereas probe DX13-7 defining locus DXS15 showed no recombinants and a lod score of z = 0.903 at theta = .0. Further studies should be done to determine whether the gene for XMEA is (1) located at Xq and (2) caused by a mutation of the Emery-Dreifuss muscular dystrophy gene, which has been assigned to the same region.     

Human thymidine kinase gene: molecular cloning and nucleotide sequence of a cDNA expressible in mammalian cells.

A cDNA containing the entire coding region of the human thymidine kinase gene has been molecularly cloned. The cDNA is under the control of a simian virus 40 promoter and is expressible in mammalian cells. The complete nucleotide sequence of the human thymidine kinase cDNA has been determined. The cDNA is 1,421 base pairs in length and has a large open reading frame of 702 base pairs capable of specifying a protein with a molecular weight of 25,504. Genomic Southern blotting experiments show that sequences homologous to the human thymidine kinase cDNA are conserved among many vertebrates, including prosimians (lemur), tree shrews, rats, mice, and chickens. Direct comparison of the nucleotide sequences of the human thymidine kinase cDNA and the chicken thymidine kinase gene reveals ca. 70% overall homology. This homology is extended further at the amino acid sequence level, with greater than 74% amino acid residues matched between the human and chicken thymidine kinase proteins.     

Human esterase D gene: complete cDNA sequence,genomic structure,and application in the genetic diagnosis of human retinoblastoma

Summary The gene encoding human esterase D (EsD), a member of the nonspecific esterase family, is a useful genetic marker for retinoblastoma (RB) and Wilson's disease. Previously we identified a cDNA clone from this gene and determined its chromosomal location. In this report, we present the complete cDNA sequence of the human EsD gene. A long open reading frame encoded a predicted protein of 282 amino acids with molecular weight of 30 kD. A computer-assisted search of a protein sequence data base revealed homology with two other esterases, acetylcholinesterase of Torpedo and esterase-6 of Drosophila. Homologous region were centered around presumptive active sites, suggesting that the catalytic domains of the esterases are conserved during evolution. Three genomic clones of this gene were also isolated and characterized by restriction mapping. At least ten exons were distributed over a 35-kb (kilobase pair) region; each exon contained an average of 100 basepairs (bp). A polymorphic site for Apa I, located within an intron of the esterase D gene, can be used to identify chromosome 13 carrying defective RB alleles within retinoblastoma families.