Structure of the gene for human coagulation factor V.

Activated factor V (Va) serves as an essential protein cofactor for the conversion of prothrombin to thrombin by factor Xa. Analysis of the factor V cDNA indicates that the protein contains several types of internal repeats with the following domain structure: A1-A2-B-A3-C1-C2. In this report we describe the isolation and characterization of genomic DNA coding for human factor V. The factor V gene contains 25 exons which range in size from 72 to 2820 bp. The structure of the gene for factor V is similar to the previously characterized gene for factor VIII. Based on the aligned amino acid sequences of the two proteins, 21 of the 24 intron-exon boundaries in the factor V gene occur at the same location as in the factor VIII gene. In both genes, the junctions of the A1-A2 and A2-A3 domains are each encoded by a single exon. In contrast, the boundaries between domains A3-C1 and C1-C2 occur at intron-exon boundaries, which is consistent with evolution through domain duplication and exon shuffling. The connecting region or B domain of factor V is encoded by a single large exon of 2820 bp. The corresponding exon of the factor VIII gene contains 3106 bp. The 5' and 3' ends of both of these exons encode sequences homologous to the carboxyl-terminal end of domain A2 and the amino-terminal end of domain A3 in ceruloplasmin. There is otherwise no homology between the B domain exons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cloning and sequencing of cDNA for the rat plasminogen activator inhibitor-1

A cDNA encoding rat plasminogen activator-inhibitor (PAI-1) has been isolated from an HTC rat hepatoma cell cDNA library constructed in phage lambda gt10. The cDNA contains 118 bp of 5'-untranslated sequence, 1206 bp encoding a 402-amino acid (aa) protein and 1747 bp of 3'-untranslated sequence. The protein-coding sequence and the derived amino acid sequence share 82% and 81% identity, respectively, with human PAI-1 cDNA and protein. The rat cDNA encodes a preprotein with a 23-aa leader peptide and a predicted N-terminal serine for the mature protein. Three of four potential N-glycosylation acceptor sites as well as the active site of rat PAI-1 are identical to the human protein. The 3'-untranslated region contains a number of unusual regions, including 80 bp of tandemly repeated GpA dinucleotides, a 115-bp stretch which shares greater than 90% sequence identity with a region within the 3'-untranslated cDNA of human PAI-1, and two 70-bp stretches of highly T-rich sequence located close to the 3'-terminus of the cDNA.     

Nucleotide sequence of a cDNA for branched chain acyltransferase with analysis of the deduced protein structure

Nucleotide sequence was determined for a 1.6-kilobase human cDNA putative for the branched chain acyltransferase protein of the branched chain alpha-ketoacid dehydrogenase complex. Translation of the sequence reveals an open reading frame encoding a 315-amino acid protein of molecular weight 35,759 followed by 560 bases of 3'-untranslated sequence. Three repeats of the polyadenylation signal hexamer ATTAAA are present prior to the polyadenylate tail. Within the open reading frame is a 10-amino acid fragment which matches exactly the amino acid sequence around the lipoate-lysine residue in bovine kidney branched chain acyltransferase, thus confirming the identity of the cDNA. Analysis of the deduced protein structure for the human branched chain acyltransferase revealed an organization into domains similar to that reported for the acyltransferase proteins of the pyruvate and alpha-ketoglutarate dehydrogenase complexes. This similarity in organization suggests that a more detailed analysis of the proteins will be required to explain the individual substrate and multienzyme complex specificity shown by these acyltransferases.     

Isolation and sequence determination of cDNA clones for porcine and human lipoamide dehydrogenase. Homology to other disulfide oxidoreductases

A 2.3-kilobase cDNA clone encoding lipoamide dehydrogenase was isolated from a porcine adrenal medulla library in the vector pCD by screening with four synthetic oligonucleotide probes corresponding to amino acid sequence from tryptic peptides of porcine lipoamide dehydrogenase. A 450-bp fragment of the porcine cDNA was used to screen a human small cell lambda gt10 library at reduced stringency. Overlapping human cDNA clones of various lengths were isolated, the largest of which was again 2.3 kilobases in length. Sequencing of both porcine and human cDNAs revealed a short 5'-untranslated region followed by 1530-bp of coding region and 700 bp of 3'-untranslated region preceding a poly(A) tail. The porcine cDNA displayed coding regions corresponding to the known tryptic peptides and a 35-amino acid leader sequence involved in targeting of the protein to the mitochondria. The human lipoamide dehydrogenase cDNA is 96% identical to the porcine at the amino acid level. Alignment of the deduced amino acid sequence of human lipoamide dehydrogenase with human erythrocyte glutathione reductase and mercuric reductase from Tn501 revealed extensive homologies throughout the primary sequence, suggesting that secondary and tertiary structure is also similar among these three enzymes.     

Full-length sequence of the cDNA for human erythroid beta-spectrin

Spectrin is the major molecular consituent of the red cell membrane skeleton. We have isolated overlapping human erythroid beta-spectrin cDNA clones and determined 6773 base pairs of contiguous nucleotide sequence. This includes the entire coding sequence of beta-spectrin. The sequence translates into a 2137 amino acid, 246-kDa peptide. beta-Spectrin is found to consist of three distinct domains. Domain I, at the N terminus, is a 272-amino acid region lacking resemblance to the spectrin repetitive motif. Sequences in this region exhibit striking sequence homology, at both nucleotide and amino acid levels, to the N-terminal "actin-binding" domains of alpha-actinin and dystrophin. Between residues 51 and 270 there is 55% amino acid identity to human dystrophin, with only four single amino acid gaps in alignment. Domain II consists of 17 spectrin repeats. Several sequence variations are observed in typical repeat structure. Homology to alpha-actinin extends beyond domain I into the N-terminal portion of domain II. Domain III, 52 amino acid residues at the C terminus, does not adhere to the spectrin repeat motif. Combining knowledge of spectrin primary structure with previously reported functional studies, it is possible to make several inferences regarding structure/function relationships within the beta-spectrin molecule.     

Cloning and sequencing of a human pancreatic tumor mucin cDNA

A monospecific polyclonal antiserum against deglycosylated human pancreatic tumor mucin was used to select human pancreatic mucin cDNA clones from a lambda gt11 cDNA expression library developed from a human pancreatic tumor cell line. The full-length 4.4-kilobase mucin cDNA sequence included a 72-base pair 5'-untranslated region and a 307-base pair 3'-untranslated region. The predicted amino acid sequence for this cDNA revealed a protein of 122,071 daltons containing 1,255 amino acid residues of which greater than 60% were serine, threonine, proline, alanine, and glycine. Approximately two-thirds of the protein sequence consisted of identical 20-amino acid tandem repeats which were flanked by degenerate tandem repeats and nontandem repeat sequences on both the amino-terminal and carboxyl-terminal ends. The amino acid sequence also contained five putative N-linked glycosylation sites, a putative signal sequence and transmembrane domain, and numerous serine and threonine residues (potential O-linked glycosylation sites) outside and within the tandem repeat position. The cDNA and deduced amino acid sequence of the pancreatic mucin sequence was over 99% homologous with a mucin cDNA sequence derived from breast tumor mucin, even though the native forms of these molecules are quite distinct in size and degree of glycosylation.     

Molecular cloning of DNA complementary to bovine growth hormone mRNA

We have cloned DNA complementary to mRNA coding for bovine growth hormone (bGH). Double-stranded DNA complementary to bovine pituitary mRNA was inserted into the Pst I site of plasmid pBR322 by the dC x dG tailing technique and amplified in E. coli x 1776. A recombinant plasmid containing bGH cDNA ws identified by hybridization to cloned rat growth hormone cDNA. It contains the entire coding and 3'-untranslated regions and 31 bases in the 5'-untranslated region. Nucleotide sequence analysis determined the sequence of the 26-amino acid signal peptide and confirmed the published amino acid sequence of the secreted hormone at all but 2 residues. Codon usage is nonrandom, with 81.7% of the codons ending in G or C. The nucleotide sequence of bGH mRNA is 83.9% homologous with rat GH mRNA and 76.5% homologous with human GH mRNA, while the respective amino acid sequence homologies are 83.5% and 66.8%.     

Cloning of human muscle phosphofructokinase cDNA

Three overlapping cDNA clones for human muscle phosphofructokinase (HMPFK) covering the complete coding sequence were isolated. The sequence included a poly(A) tail, a 399 bp 3'-untranslated region, a 2337 bp coding region for 779 amino acid residues and a part of the 5'-untranslated region. Homologies between HMPFK and rabbit muscle phosphofructokinase (RMPFK) were 96% of the amino acids and 89% of the nucleotides in the coding region. Like RMPFK, HMPFK also possessed the internal homology between C- and N-halves in its primary structure. Cloning of HMPFK cDNA will help to identify the molecular defect in patients with glycogenosis type VII (HMPFK deficiency).     

Primary structure of the leukocyte function-associated molecule-1 alpha subunit: an integrin with an embedded domain defining a protein superfamily

The leukocyte function-associated molecule 1 (LFA-1, CD11a/CD18) is a membrane glycoprotein which functions in cell-cell adhesion by heterophilic interaction with intercellular adhesion molecule 1 (ICAM-1). LFA-1 consists of an alpha subunit (Mr = 180,000) and a beta subunit (Mr = 95,000). We report the molecular biology and protein sequence of the alpha subunit. Overlapping cDNAs containing 5,139 nucleotides were isolated using an oligonucleotide specified by tryptic peptide sequence. The mRNA of 5.5 kb is expressed in lymphoid and myeloid cells but not in a bladder carcinoma cell line. The protein has a 1,063-amino acid extracellular domain, a 29-amino acid transmembrane region, and a 53-amino acid cytoplasmic tail. The extracellular domain contains seven repeats. Repeats V-VII are in tandem and contain putative divalent cation binding sites. LFA-1 has significant homology to the members of the integrin superfamily, having 36% identity with the Mac-1 and p150,95 alpha subunits and 28% identity with other integrin alpha subunits. An insertion of approximately 200 amino acids is present in the NH2-terminal region of LFA-1. This "inserted/interactive" or I domain is also present in the p150,95 and Mac-1 alpha subunits but is absent from other integrin alpha subunits sequenced to date. The I domain has striking homology to three repeats in human von Willebrand factor, two repeats in chicken cartilage matrix protein, and a region of complement factor B. These structural features indicate a bipartite evolution from the integrin family and from an I domain family. These features may also correspond to relevant functional domains.     

Isolation of a full-length cDNA encoding mouse aromatase P450

A full-length cDNA clone for aromatase P450 has been isolated from a pregnant mouse ovarian cDNA library. The insert of this clone (2394 bp) contains a 1509-bp open reading frame encoding 503 amino acid residues together with a 46-bp 5'-untranslated stretch and an 839-bp 3'-untranslated region to which a poly(A) tract is attached. Northern blot analysis of ovarian RNA from pregnant mice reveals a major mRNA band of 2.5 kb with a minor band of 2.1 kb. Comparison of mouse aromatase P450 with that of rat, human, and chicken shows 91, 81, and 69% identity in the nucleotide sequence and 92, 79, and 69% identity in the deduced amino acid sequence, respectively. The membrane-spanning domain of mouse aromatase P450 is estimated to be an extremely hydrophobic segment located within the N-terminal region of the molecule. Furthermore, a highly conserved heme-binding domain is noticed.     

Drosophila substrate adhesion molecule: sequence of laminin B1 chain reveals domains of homology with mouse

Laminin, a substrate adhesion molecule in vertebrates, is a large glycoprotein complex in basement membranes that promotes cell adhesion, cell migration, and neurite outgrowth. Here we report on the cloning of the genes encoding the three subunits of Drosophila laminin. Sequence analysis of cDNA clones encoding the Drosophila B1 chain reveals a multidomain structure similar to that of its mouse homolog. The Drosophila sequence has only 25% amino acid identity with the mouse sequence in domains I, II, and IV. However, in one of the putative collagen-binding regions (domain VI) and the two cysteine-rich domains of EGF-like repeats (domains III and V), the amino acid identity between these two evolutionarily distant species jumps to 55%. Moreover, the number, length, and unique amino acid sequences of each of the 13 EGF-like repeats are highly conserved between Drosophila and mouse, suggesting that each may serve a unique function in protein-protein interactions.     

The complete cDNA and polypeptide sequences of human erythroid alpha-spectrin.

Overlapping human erythroid alpha-spectrin cDNA clones were isolated from lambda gt11 libraries constructed from cDNAs of human fetal liver and erythroid bone marrow. The composite 8001-base pair (bp) cDNA nucleotide sequence contains 187-bp 5'- and 528-bp 3'-untranslated regions and has a single long open reading frame of 7287 bp that encodes a polypeptide of 2429 residues. As previously described (Speicher, D. W., and Marchesi, V. T. (1984) Nature 311, 177-180), spectrin is composed largely of homologous 106-amino acid repeat units. From the amino acid sequence deduced from the cDNA, alpha-spectrin can be divided into 22 segments. Segments 1-9 and 12-19 are homologous and can therefore be considered repeats; the average number of identical residues in pairwise comparisons of these repeats is 22 out of 106, or 21%. Of these 17 repeats, 11 are exactly 106 amino acids in length, whereas five others differ from this length by a single residue. Segments 11, 20, and 21, although less homologous, appear to be related to the more highly conserved repeat units. The very N-terminal 22 residues, segment 10, which is atypical both in length and sequence, and the C-terminal 150 residues in segment 22 appear to be unrelated to the conserved repeat units. The sequence of the erythroid alpha-spectrin polypeptide chain is compared to that of human alpha-fodrin and chicken alpha-actinin to which it is related. alpha-Spectrin is more distantly related to dystrophin.     

Cloning of cDNAs encoding human interphotoreceptor retinoid-binding protein (IRBP) and comparison with bovine IRBP sequences

We have determined the sequence of the human interphotoreceptor retinoid-binding protein mRNA from three separately isolated cDNAs. The sequence is 4.28 kb long and encodes a protein of 1247 amino acids (aa) including a putative signal peptide and propeptide. The sequence is shorter (by about 1.67 kb) than the bovine mRNA with the major difference in the lengths located in the 3'-untranslated region. We suggest that this resulted from an insertion in the bovine gene or a large deletion from the human gene. The insertion/deletion is flanked on either side by sequences that are similar in the bovine and human sequences. Like the bovine polypeptide, the deduced protein sequence from the human cDNA contains a fourfold repeat, with each repeat containing about 300 aa. Among the four repeats, the identity is about 30-40%. The identity between the complete bovine and human polypeptide sequences is 84%. The identity between the nucleotide sequences is 83% (excluding the major insertion/deletion). Comparison with the bovine gene indicates that the human sequence may lack about 5-10 bp at the 5' end of the cDNA; it, however, includes a poly(A) tail at the 3' end. Thus, the human sequence is virtually full length, is similar to the bovine sequence, and contains a striking fourfold repeat.     

Cloning of the cDNA encoding neural adhesion molecule F3 from bovine brain

We cloned a bovine cDNA encoding the neural adhesion molecule F3 and analyzed its nucleotide sequence. The coding region consisted of 3054 bp encoding 1018 amino acid (aa) residues. The M_r calculated from the deduced as sequence was 113 383. Bovine F3 had 93, 94 and 77% as identity with the mouse, human and chicken homologs, respectively. Bovine F3, similar to those of chicken and human, was devoid of two as residues (Ile-Thr) in the sixth immunoglobulin type C2-like domain, as compared with the mouse homolog. Parts of bovine F3 protein were overproduced in Escherichia coli. The antibodies raised against the recombinant proteins in rabbits reacted specifically with F3. F3 protein was detected in cerebellum, cerebrum and spinal cord in Western blot analysis.     

Characterization of the cDNA coding for mouse prothrombin and localization of the gene on mouse chromosome 2

A series of overlapping cDNAs coding for mouse prothrombin (coagulation factor II) have been isolated and the composite DNA sequence has been determined. The complete prothrombin cDNA is 1,987 bp in length [excluding the poly(A) tail] and codes for 18 bp of 5' untranslated sequence, an open reading frame coding for 618 amino acids, a stop codon, and a 3' untranslated region of 112 bp followed by a poly(A) tail. The translated amino acid sequence predicts a molecular weight of 66,087, which includes 10 residues of gamma-carboxyglutamic acid. There are five potential N-linked glycosylation sites. Mouse prothrombin is 81.4% and 77.3% identical to the human and bovine proteins, respectively. Comparison of the cDNA coding for mouse prothrombin to the human and bovine cDNAs indicates 79.9% and 76.5% identity, respectively. Amino acid residues important for the structure and function of human prothrombin are conserved in the mouse and bovine proteins. In the adult mouse and rat, prothrombin is primarily synthesized in the liver, where is constitutes 0.07% of total mRNA as determined by solution hybridization analysis. The genetic locus for mouse prothrombin, Cf-2, has been mapped using an interspecies backcross and DNA fragment differences between the two species. The prothrombin locus lies on mouse chromosome 2, 1.8 +/- 1.3 map units proximal to the catalase locus. The gene order in this region is Cen-Acra-Cf-2-Cas-1-A-Tel. This localization extends the proximal boundary of the known region of homology between mouse chromosome 2 and human chromosome 11p from Cas-1 about 2 map units toward the centromere.     

Isolation of a cDNA for human muscle 6-phosphofructokinase

A cDNA for human muscle 6-phosphofructokinase (EC.2.7.1.11) has been isolated from a human fibroblast cDNA library made using the Okayama-Berg procedure. The cDNA isolated as a Bam H1 fragment of the pcD recombinant, pO4, is approximately 2000 bp in length. It represents approximately 1350 bp of the C-terminus coding sequence of the enzyme, approximately 500 bp of the 3'-untranslated region and approximately 150 bp of the vector sequences. The identity of the pO4 cDNA was established by the observation of a high degree of homology (approximately 95%) between the deduced amino acid sequence with the published protein sequence of rabbit muscle 6-phosphofructokinase, and the assignment of the sequence to human chromosome 1 (the known location of PFKM) by using somatic cell hybrids. Based on immunochemical evidence, we had previously predicted not only a remarkable structural conservation of the vertebrate muscle PFK, but also partial structural identity among all three vertebrate PFK isozymes. The pO4 cDNA is, therefore, expected to permit isolation of cDNAs for muscle and non-muscle PFKs from a wide variety of vertebrate species.