Showing papers on "Complementary DNA published in 1983"

Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli

Abstract: Bacterial clones containing human tissue-type plasminogen activator (t-PA) cDNA sequences were identified in a cDNA library prepared using gel-fractionated mRNA from human melanoma cells. A plasmid containing the Escherichia coli trp promoter and the cDNA sequence coding f or the 527-amino acid mature t-PA protein was constructed for expression in E. coli. A polypeptide was produced having the fibrinolytic properties characteristic of authentic human t-PA.

Isolation and characterization of full-length cDNA clones for human alpha-, beta-, and gamma-actin mRNAs: skeletal but not cytoplasmic actins have an amino-terminal cysteine that is subsequently removed.

Abstract: cDNA clones encoding three classes of human actins have been isolated and characterized. The first two classes (gamma and beta, cytoplasmic actins) were obtained from a cDNA library constructed from simian virus 40-transformed human fibroblast mRNA, and the third class (alpha, muscle actin) was obtained from a cDNA library constructed from adult human muscle mRNA. A new approach was developed to enrich for full-length cDNAs. The human fibroblast cDNA plasmid library was linearized with restriction enzymes that did not cut the inserts of interest; it was then size-fractionated on gels, and the chimeric molecules of optimal length were selected for retransformation of bacteria. When the resulting clones were screened for actin-coding sequences it was found that some full-length cDNAs were enriched as much as 50- to 100-fold relative to the original frequency of full-length clones in the total library. Two types of clones were distinguished. One of these clones encodes gamma actin and contains 100 base pairs of 5' untranslated region, the entire protein coding region, and the 3' untranslated region. The second class encodes beta actin, and the longest such clone contains 45 base pairs of 5' untranslated region plus the remainder of the mRNA extending to the polyadenylic acid tail. A third class, obtained from the human muscle cDNA library, encodes alpha actin and contains 100 base pairs of 5' untranslated region, the entire coding region, and the 3' untranslated region. Analysis of the DNA sequences of the 5' end of the clones demonstrated that although beta- and gamma-actin genes start with a methionine codon (MET-Asp-Asp-Asp and MET-Glu-Glu-Glu, respectively), the alpha-actin gene starts with a methionine codon followed by a cysteine codon (MET-CYS-Asp-Glu-Asp-Glu). Since no known actin proteins start with a cysteine, it is likely that post-translational removal of cysteine in addition to methionine accompanies alpha-actin synthesis but not beta- and gamma-actin synthesis. This observation has interesting implications both for actin function and actin gene regulation and evolution.

Structure and expression of a cloned cDNA for human interleukin-2

Abstract: A cDNA coding for human interleukin-2 (IL-2) has been cloned from a cDNA library prepared from partially purified IL-2 mRNA. The DNA sequence codes for a polypeptide which consists of 153 amino acids including a putative signal sequence. A biologically active polypeptide, characteristic of human IL-2, was produced when the cDNA was fused to a simian virus 40 promoter sequence and used to transfect cultured monkey COS cells.

A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells.

Abstract: This paper describes a plasmid vector for cloning cDNAs in Escherichia coli; the same vector also promotes expression of the cDNA segment in mammalian cells. Simian virus 40 (SV40)-derived DNA segments are arrayed in the pcD vector to permit transcription, splicing, and polyadenylation of the cloned cDNA segment. A DNA fragment containing both the SV40 early region promoter and two introns normally used to splice the virus 16S and 19S late mRNAs is placed upstream of the cDNA cloning site to ensure transcription and splicing of the cDNA transcripts. An SV40 late region polyadenylation sequence occurs downstream of the cDNA cloning site, so that the cDNA transcript acquires a polyadenylated 3' end. By using pcD-alpha-globin cDNA as a model, we confirmed that the alpha-globin transcript produced in transfected cells is initiated correctly, spliced at either of the two introns, and polyadenylated either at the site coded in the cDNA segment or at the distal SV40 polyadenylation signal. A cDNA clone library constructed with mRNA from SV40-transformed human fibroblasts and this vector (about 1.4 X 10(6) clones) yielded full-length cDNA clones that express hypoxanthine-guanine phosphoribosyltransferase (Jolly et al., Proc. Natl. Acad. Sci. U.S.A., in press).

Easy identification of cDNA clones.

Abstract: A set of six cloning vectors, pUR 278, 288, 289, 290, 291, 292 is presented. These vectors have the cloning sites, BamHI, SalI, PstI, XbaI and HindIII, in all frames at the 3' end of the lacZ gene. Insertion of cDNA in the proper cloning sites leads to a fusion protein of active beta-galactosidase and the peptide encoded by the cDNA. A simple immunoenzymatic assay can be used to identify clones in such a cDNA library.

Activation of ki-ras2 gene in human colon and lung carcinomas by two different point mutations

Abstract: Kirsten (Ki)-ras cDNA clones were prepared from human lung and colon carcinoma cell lines expressing an activated c-Ki-ras2 gene. DNA sequence analysis and transfection studies indicate that different point mutations at the same codon can activate the gene; that most human c-Ki-ras2 mRNA uses sequences from a fourth coding exon distinct from that of its viral counterpart; and that at least one cell line is functionally homozygous for the activated gene.

Nucleotide sequence of epidermal growth factor cDNA predicts a 128,000-molecular weight protein precursor

Abstract: Epidermal growth factor (EGF) has a profound effect on the differentiation of specific cells in vivo, and has been shown to be a potent mitogenic factor for a variety of cultured cells, of both ectodermal and mesodermal origin (see ref. 1 for review). This 53-amino acid polypeptide of known sequence2 contains six cysteine residues3, which are thought to form three intrachain disulphide bonds4. Urogastrone, a polypeptide bearing anti-gastric secretory activity isolated from human urine5, which is presumably synthesized in submandibular and Brunner's glands6,7, shares extensive sequence homology (70%) with EGF and may represent the human EGF equivalent. Here we present the sequence of a mouse EGF cDNA clone, which suggests that EGF is synthesized as a large protein precursor of 1,168 amino acids. Our data indicate that the discrepancy between EGF levels in male and female mouse submaxillary glands (MSGs) is due to different EGF mRNA levels in these tissues, and suggest that precursor EGF processing may differ from that described previously for other polypeptide hormones.

Human β -nerve growth factor gene sequence highly homologous to that of mouse

Abstract: Nerve growth factor (NGF) is thought to have a profound effect on the development and maintenance of sympathetic and embryonic sensory neurones (see refs 1–3 for review). NGF activity isolated from the male mouse submaxillary gland (MSG) consists of three types of subunits, α, β and γ, which specifically interact to form a 7S, ∼130,000-molecular weight (Mr) complex. The 7S complex contains two identical 118-amino acid β-chains, which are solely responsible for the nerve growth-stimulating activity of NGF. While NGF is found in almost all vertebrates4, most research has focused on murine NGF, as the mouse male submaxillary gland contains higher levels of this polypeptide than other tissues4. Even so, β-NGF comprises only ∼0.1% of the protein in this small gland, which has made the study of this polypeptide difficult. The amino acid sequence of the mouse NGF β-chain has been determined5 and some information has been obtained regarding the size of a mouse precursor molecule6, pro-β-NGF, but little was known about the structure and relatedness of β-NGF from other vertebrates. Here we describe the isolation of mouse β-NGF complementary DNA (cDNA) and present its nucleotide sequence, which predicts a prepro-β-NGF molecule of Mr 27,000 (27K) and a pro-β-NGF molecule of Mr 25K. We have used the mouse β-NGF cDNA clone to isolate the human β-NGF gene, the coding regions of which are highly homologous to the mouse prepro-β-NGF nucleotide and amino acid sequences.

Isolation and expression of an altered mouse dihydrofolate reductase cDNA

Abstract: We have constructed a cDNA library from a murine cell line expressing high levels of a dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) that displays an abnormally low affinity for methotrexate. From this library we have isolated a cDNA clone similar to, but distinguishable from, a cDNA clone previously demonstrated to encode the wild-type enzyme. Analysis of the nucleotide sequence of this cDNA clone allows us to predict that the altered dihydrofolate reductase differs from the wild-type enzyme at a single amino acid, reflecting the substitution of an arginine for a leucine residue in a region of the polypeptide thought to form a hydrophobic pocket essential for inhibitor binding. To confirm that this substitution was responsible for the altered properties of the enzyme, we genetically localized the region of the cDNA that specified resistance to methotrexate by in vitro recombination. These results reveal that a single nucleotide change in the codon specifying amino acid 22 of the enzyme was sufficient to alter the methotrexate sensitivity of the enzyme. We demonstrate that this altered gene can be employed as a dominant selectable marker in cultured cells expressing normal levels of wild-type dihydrofolate reductase.

Cloning and sequencing of a deoxyribonucleic acid copy of glyceraldehyde-3-phosphate dehydrogenase messenger ribonucleic acid isolated from chicken muscle.

Abstract: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purified from the breast muscles of 3-week-old chickens and used to raise a specific antiserum in rabbits. This antiserum was coupled to an in vitro translation assay to monitor the purification of GAPDH mRNA. RNA was isolated from identical breast muscles and consecutively fractionated with several techniques to yield a preparation of GAPDH mRNA which was at least 50% pure. Double-stranded cDNA was made against this purified RNA, inserted into pBR322, and used to transform Escherichia coli. Recombinants were screened by colony filter hybridization with a cDNA probe made against the purified RNA. The hybridization-positive clone with the largest insert, pGAD-28, was then characterized by using pGAD-28-cellulose to select complementary RNA from total poly(A) RNA and then translating the hybridization-selected RNA in vitro. The single translation product was shown to be GAPDH by (1) comigration with pure GAPDH on sodium dodecyl sulfate-polyacrylamide gels, (2) precipitation with specific anti-GAPDH antiserum, (3) cyanylation fingerprinting, and (4) AMP-agarose affinity chromatography. pGAD-28 was mapped with several restriction enzymes and then sequenced by the method of Maxam and Gilbert [Maxam, A. M., & Gilbert, W. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 560]. The 1261-nucleotide insert was found to contain 29 nucleotides of noncoding sequence at the 5' end, the entire coding region, and 230 nucleotides of the 3'-noncoding region including a poly(A) addition signal (AATAAA) and the first five residues of the poly(A) tail.

Primary structures of beta- and delta-subunit precursors of Torpedo californica acetylcholine receptor deduced from cDNA sequences.

Abstract: The nicotinic acetylcholine receptor (AChR) from fish electric organ and mammalian skeletal muscle is the best characterized neurotransmitter receptor (reviewed in refs 1–3). The AChR from the electroplax of the ray Torpedo californica consists of five subunits present in a molar stoichiometry of α2βγδ (refs 4–6); the apparent molecular weights of the α-, β-, γ- and δ-subunits are 40,000 (40K), 50K, 60K and 65K, respectively7–11. Knowledge of the primary structures of these constituent polypeptides would facilitate the understanding of the molecular mechanism underlying the function of the neurotransmitter receptor. Recently, we have cloned cDNA for the α-subunit precursor of the T. californica AChR and have deduced the primary structure of this polypeptide from the nucleotide sequence of the cloned cDNA12. Here we report the cloning and nucleotide analysis of cDNAs for the AChR β- and δ-subunit precursors. The primary structures of the two polypeptides deduced from the cDNA sequences reveal conspicuous amino acid sequence homology among these and the α-subunits. The three subunits contain several highly conserved regions which may be essential for the receptor function or inter-summit interaction.

Cloning and expression of murine immune interferon cDNA.

Abstract: The murine immune interferon (IFN-gamma) gene was cloned and expressed under control of the simian virus 40 early promoter in the monkey COS-1 cell line. A protein is secreted from these cells having the biological, antigenic, and biochemical characteristics of natural murine IFN-gamma. Cloned murine IFN-gamma cDNAs were obtained by using RNA from both mitogen-induced murine spleens and the transfected COS cells, and both code for identical proteins. The mature murine IFN-gamma encoded is 136 amino acids long, 10 amino acids shorter than human IFN-gamma. The nucleotide homology between the murine and human IFN-gamma genes is 60-65%, whereas the encoded proteins are only 40% homologous. Murine IFN-gamma cDNA was expressed in Escherichia coli under trp promoter control.

Structure of a Mouse Submaxillary Messenger RNA Encoding Epidermal Growth Factor and Seven Related Proteins

Abstract: The structure of the messenger RNA (mRNA) encoding the precursor to mouse submaxillary epidermal growth factor (EGF) was determined from the sequence of a set of overlapping complementary DNA's (cDNA). The mRNA is unexpectedly large, about 4750 nucleotide bases, and predicts the sequence of preproEGF, a protein of 1217 amino acids (133,000 molecular weight). The EGF moiety (53 amino acids) is flanked by polypeptide segments of 976 and 188 amino acids at its amino and carboyxl termini, respectively. The amino terminal segment of the precursor contains seven peptides with sequences that are similar but not identical to EGF.

Molecular Cloning of Exo–Cellobiohydrolase I Derived from Trichoderma Reesei Strain L27

Abstract: The molecular cloning and characterization of the gene encoding exo–cellobiohydrolase I (CBHI) of Trichoderma reesei strain L27 is reported. Two adjacent HindIII genomic fragments of 1.16 kb and 2.3 kb were identified using differential hybridization techniques and were sub–cloned into plasmid pBR322. The identification of the gene encoding CBHI was determined by hybrid selection and confirmed by DNA sequence analysis. There are two introns in the genomic DNA that were identified by comparing the coding sequence with the published amino acid sequence1 and confirmed by sequencing of cDNA clones. Both introns were found to contain a 10 bp sequence, CAGCT–GACTG, that is homologous to a sequence necessary for splicing of introns in yeast2.

Sequence of cDNA encoding human insulin-like growth factor I precursor.

Abstract: Somatomedins (SM) or insulin-like growth factors (IGF) constitute a heterogeneous group of peptides with important growth-promoting effects in vitro as well as in vivo. Amino acid sequences have been determined for only two of them, IGF-I and IGF-II, which are highly homologous. IGF-I, which is identical with SM-C, is composed of 70 amino acid residues and IGF-II contains 73 amino acids and may be identical with SM-A. Other peptides with different charge properties but with similar SM-like or insulin-like behaviour in biological and receptor assays, have been described but have not yet been fully characterized. The liver is known to be a major site of production of these peptides, but many other tissues--especially in the fetus--may synthesize them as well. We report here the nucleotide sequence of a human liver cDNA encoding the complete amino acid sequence of IGF-I. The IGF-I coding region is flanked by sequences encoding an amino-terminal peptide of at least 25 amino acid residues and a carboxyl-terminal peptide of 35 amino acids. This provides evidence that IGF-I is synthesized as a precursor protein and that formation of IGF-I from this precursor requires proteolytic processing at both ends.

Cloning and sequence analysis of calf cDNA and human genomic DNA encoding α -subunit precursor of muscle acetylcholine receptor

Abstract: The nicotinic acetylcholine receptor (AChR) from fish electric organ is well characterized and is known to consist of five subunits present in a molar stoichiometry of α2βγδ (reviewed in refs 1–3). The mammalian skeletal muscle AChR is thought to have a similar subunit structure4–6. We have recently elucidated the primary structures of the α-, β-, γ- and δ-subunit precursors of the Torpedo californica AChR by cloning and sequencing cDNAs for these polypeptides7–9; cDNA sequences for the γ-subunit precursor of the T. californica AChR10 and the α-subunit precursor of the Torpedo marmorata AChR11,12 have also been reported by other groups. The four subunits exhibit conspicuous sequence homology and are similar in hydrophilicity profile and predicted secondary structure, thus being most probably oriented in a pseudosymmetric fashion across the membrane. The transmembrane topology of the subunit molecules and the locations of functionally important regions, such as the acetylcholine binding site and the transmembrane segments which may be involved in the ionic channel, have been proposed. We have now cloned cDNA for the α-subunit precursor of the calf skeletal muscle AChR and a human genomic DNA segment containing the corresponding gene. Nucleotide sequence analysis of the cloned DNAs has revealed the primary structures of the calf and human AChR α-subunit precursors, which exhibit marked sequence homology with their Torpedo counterpart. The protein-coding sequence of the human AChR α-subunit precursor gene is divided by eight introns into nine exons, which seem to correspond to different structural and functional domains of the subunit precursor molecule.

Isolation and sequence analysis of the human corticotropin-releasing factor precursor gene.

Abstract: A human genomic DNA segment containing the gene for the corticotropin-releasing factor precursor has been isolated by screening a gene library with an ovine cDNA probe. The cloned DNA segment has been subjected to restriction endonuclease mapping and nucleotide sequence analysis. Comparison of the nucleotide sequence of the gene with that of the ovine cDNA indicates that an intron of 800 bp is inserted in the segment encoding the 5'-untranslated region of the mRNA. The segment corresponding to the protein-coding and the 3'-untranslated region of the mRNA is uninterrupted. The mRNA and amino acid sequences of the human corticotropin-releasing factor precursor have been deduced from the corresponding gene sequence. The deduced amino acid sequence of human corticotropin-releasing factor exhibits seven amino acid substitutions in comparison with the ovine counterpart.

Gene expression in rat brain

Abstract: 191 randomly selected cDNA clones prepared from rat brain cytoplasmic poly (A)+ RNA were screened by Northern blot hybridization to rat brain, liver and kidney RNA to determine the tissue distribution, abundance and size of the corresponding brain mRNA. 18% hybridized to mRNAs each present equally in the three tissues, 26% to mRNAs differentially expressed in the tissues, and 30% to mRNAs present only in the brain. An additional 26% of the clones failed to detect mRNA in the three tissues at an abundance level of about 0.01%, but did contain rat cDNA as demonstrated by Southern blotting; this class probably represents rare mRNAs expressed in only some brain cells. Therefore, most mRNA expressed in brain is either specific to brain or otherwise displays regulation. Rarer mRNA species tend to be larger than the more abundant species, and tend to be brain specific; the rarest, specific mRNAs average 5000 nucleotides in length. Ten percent of the clones hybridize to multiple mRNAs, some of which are expressed from small multigenic families. From these data we estimate that there are probably at most 30,000 distinct mRNA species expressed in the rat brain, the majority of which are uniquely expressed in the brain.

Molecular cloning of human interleukin 2 cDNA and its expression in E. coli

Abstract: A recombinant plasmid containing human interleukin 2 (IL2) cDNA was identified in a cDNA library constructed from mRNA derived from PHA-TPA induced splenocytes. Using this cDNA as a hybridization probe, a DNA fragment containing the IL2 gene was isolated from a collection of hybrid phages derived from human genomic DNA. A unique reading frame was identified from the nucleotide sequence derived from these plasmids coding for a polypeptide of 153 amino acids and containing a putative signal sequence of 20 amino acids. A mature polypeptide starting with either Met-Ala-Pro or Met-Pro was expressed in E. coli under control of the E. coli trp promoter or using a combination of the phage lambda PL promoter and a ribosome binding site derived from phage Mu. The bacterial IL2 polypeptide had a molecular weight of 15,000 daltons and accounted for more than 10% of the total E. coli proteins in fully induced cells; it was biologically active in the T-cell specific DNA synthesis assay, even after recovery from a SDS-containing polyacrylamide gel.

Nucleotide sequences of complementary deoxyribonucleic acids for the pro alpha 1 chain of human type I procollagen. Statistical evaluation of structures that are conserved during evolution.

Abstract: Nucleotide sequences were determined for two cloned cDNAs encoding for over three-fourths of the pro alpha 1 (I) chain of type I procollagen from man. Comparison with previously published data on amino acid sequences of the alpha 1 (I) chain of type I collagen made it possible to examine mutations in the transcribed products of the gene which have occurred during the evolution of man, calf, rat, mouse, and chick. Comparison of the nucleotide sequences with the corresponding sequences of cDNAs from chick [Fuller, F., & Boedtker, H. (1981) Biochemistry 20, 996] and with cDNAs for the pro alpha 2(I) chain from man [Bernard, M.P., Myers, J. C., Chu, M.-L., Ramirez, F., Eikenberry, E. F., & Prockop, D. J. (1983) Biochemistry 22, 1139] demonstrated that selective pressure during evolution for 250 million or more years acted more strongly on the structure of the pro alpha 1 (I) chain than on the pro alpha 2(I) chain. To improve the reliability of the comparison, the nucleotide sequences were examined with a modification of previous procedures for evaluating mutations in replacement sites and silent sites. The corrected divergence for replacement sites between the alpha 1 (I) chains was 6 +/- 0.8% whereas it was 15 +/- 1.9% for the alpha 2(I) chains. The C-propeptide domain of the pro alpha 1 (I) chain was also highly conserved with a corrected divergence at replacement sites of 5 +/- 0.9%, a value that was not distinguishable from the value previously found for the C-propeptide of the pro alpha 2(I) chain. Therefore, a large part of the structure of both C-propeptides appears to be under selective pressure. Inspection of changes in the C-propeptide of the pro alpha 1 (I) chain suggested that there was a highly conserved region around the carbohydrate attachment site similar to the highly conserved region of 37 amino acids previously found in the C-propeptide of the pro alpha 2(I) chain. Two statistical tests, however, were unable to confirm nonrandom distribution of changes in the C-propeptide of the pro alpha 1(I) chain. The same tests established the presence of a nonrandom distribution in nucleotide changes of the C-propeptide of the pro alpha 2(I) chain. The 3'-noncoding region of the cDNA for pro alpha 1(I) of human type I procollagen showed no homology with the same region in the chick.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of clones that encode chicken tropomyosin by direct immunological screening of a cDNA expression library.

Abstract: A cDNA library of approximately equal to 9,000 members has been prepared from chicken smooth muscle mRNA by using the plasmid expression vector pUC8. Addition of Sal I and EcoRI linkers at different stages during the preparation of the cDNA resulted in a population of molecules, most of which had EcoRI linkers at the end of the cDNA that corresponded to the 5' end of the template mRNA and Sal I linkers at the end that corresponded to the 3' end of the mRNA. The cDNA molecules then were inserted into a EcoRI-Sal I-cut plasmid vector, pUC8, that contains the transcriptional and translational start sequences from the lacZ gene upstream of the EcoRI site. The sequential addition of the linkers to the cDNA ensured that most of the cDNAs were inserted into pUC8 in the proper orientation for expression. The colonies were replica plated onto nitrocellulose filters and lysed in situ with chloroform vapor. The library was screened for colonies producing products immunologically related to chicken tropomyosin by incubating the filters first with a rabbit antitropomyosin antibody and second with a 125I-labeled goat anti-rabbit IgG. Two colonies were detected that reacted specifically with the antisera. Plasmids from both clones have been partially subjected to sequence analysis; both plasmids contain cDNAs that encode tropomyosin. These protocols are potentially useful for the identification of cDNA clones for genes expressed at low levels from large cDNA expression libraries.

Molecular cloning of the gene for human anti-haemophilic factor ix

Abstract: It has been a problem to find an alternative, less time-consuming, and more reliable source of factor IX, a polypeptide which is essential to the human blood-clotting process and necessary for the treatment of patients with Christmas disease The invention is an important step towards solving the problem by way of genetic engineering, in that it provides recombinant DNA containing a DNA sequence occurring in the human factor IX genome It includes recombinant DNA comprising substantially the whole sequence of human factor IX genome inserted in a cloning vehicle and transformed into a host such as Ecoli It is conveniently characterised by a 129 or 203- nucleotide long sequence (J-J' and J'-J'' in Figure 9) Other fragments of the sequence have also been cloned and the invention includes DNA molecules comprising part or all of the human factor IX DNA The invention also includes cDNA derived from human factor IX RNA Uses of the invention include the provision of an intermediate of value in the genetic engineering of a factor IX polypeptide precursor and thence manufacture of the factor IX polypeptide, and in making probes for use in diagnosing the presence of normal or abnormal factor IX DNA in patients with Christmas disease

Complete mRNA coding sequence of the acetylcholine binding alpha-subunit of Torpedo marmorata acetylcholine receptor: a model for the transmembrane organization of the polypeptide chain

Abstract: A 1,350-base-pair-long cDNA clone, named p alpha-2, was isolated by hybridization to the previously characterized clone p alpha-1 and found to be specific for the alpha-subunit of the Torpedo marmorata acetylcholine receptor. The nucleotide sequences of both cDNA inserts were analyzed and the sequence of the complete coding region and part of the 5' and 3' untranslated regions of the alpha-chain mRNA was determined. The complete amino acid sequence of the alpha-chain precursor is presented and used to develop a model for the transmembrane organization of the polypeptide.

Characterization of the complementary deoxyribonucleic acid and gene coding for human prothrombin.

Abstract: The DNA sequences of a complementary deoxyribonucleic acid (cDNA) and a portion of the gene coding for human prothrombin have been determined. The cDNA was 2005 base pairs in length and was found to code for part of a leader sequence of 36 amino acids, 579 amino acids present in the mature protein, a stop codon, a noncoding region of 97 base pairs, and a poly(A) tail of 27 base pairs. It is proposed that the leader sequence consists of a signal sequence and a pro sequence for the mature protein that circulates in plasma. The 10 glutamic acid residues that are present in the amino-terminal region of prothrombin and are converted to gamma-carboxyglutamic acid in the mature protein are coded by only the GAG codon. The cDNA for prothrombin was also employed as a probe for screening a human fetal liver genomic DNA library. One of the strongly positive phage containing a human DNA insert of 5 kilobases was mapped with restriction endonucleases and sequenced. This DNA contained approximately half of the gene for human prothrombin and included six introns and five exons coding for amino acid residues 144-448. The two largest intervening sequences in the genomic DNA contained two copies each of AluI repetitive DNA.