Cloning and characterization of a cDNA coding for mouse placental alkaline phosphatase.

Mouse alkaline phosphatase [ALP; orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] was partially purified from placenta. Data obtained by immunoblotting analysis suggested that the primary structure of this enzyme has a much greater homology to that of human and bovine liver ALPs than to the human placental isozyme. Therefore, a full-length cDNA encoding human liver-type ALP was used as a probe to isolate the mouse placental ALP cDNA. The cloned mouse cDNA is 2459 base pairs long and is composed of an open reading frame encoding a 524-amino acid polypeptide that contains a putative signal peptide of 17 amino acids. Homology at the amino acid level of the mouse placental ALP is 90% to the human liver isozyme but only 55% to the human placental counterpart. RNA blot hybridization results indicate that the mouse placental ALP is encoded by a gene identical to the gene expressed in mouse liver, kidney, and teratocarcinoma stem cells. This gene is therefore evolutionarily highly conserved in mouse and human.     

Nucleotide and amino acid sequences of human intestinal alkaline phosphatase: close homology to placental alkaline phosphatase.

A cDNA clone for human adult intestinal alkaline phosphatase (ALP) [orthophosphoric-monoester phosphohydrolase (alkaline optimum); EC 3.1.3.1] was isolated from a lambda gt11 expression library. The cDNA insert of this clone is 2513 base pairs in length and contains an open reading frame that encodes a 528-amino acid polypeptide. This deduced polypeptide contains the first 40 amino acids of human intestinal ALP, as determined by direct protein sequencing. Intestinal ALP shows 86.5% amino acid identity to placental (type 1) ALP and 56.6% amino acid identity to liver/bone/kidney ALP. In the 3'-untranslated regions, intestinal and placental ALP cDNAs are 73.5% identical (excluding gaps). The evolution of this multigene enzyme family is discussed.     

Molecular cloning of mouse placental lactogen cDNA.

We have isolated a cDNA clone for the 23-kDa mouse placental lactogen II (mPL-II) from a phage lambda gt11 expression library containing cDNA synthesized from BALB/c placental RNA. Translation in vitro of placental mRNA selected by hybridization to the mPL-II cDNA clones yields a 26-kDa polypeptide that is the size of the expected precursor protein and that is immunoprecipitated with anti-mPL-II antiserum. The mPL-II cDNA clones hybridize to a 1.0-kilobase placental-specific mRNA. This mRNA, found in the fetal portion of the placenta, appears as early as day 10 of gestation and increases to a maximal level by day 12. The mPL-II cDNA nucleotide sequence has been determined. This sequence contains an open reading frame encoding a polypeptide of 222 amino acids with the amino-terminal 31 amino acids forming the signal sequence for secretion. The predicted secreted protein has 51% amino acid homology with mouse prolactin.     

Molecular cloning of a functional bovine interleukin 2 cDNA.

A cDNA clone of the bovine interleukin 2 (IL-2) gene has been isolated and demonstrated to be functional in the production of secreted bovine IL-2 protein when transfected into monkey cells. The bovine IL-2 clone is 791 base pairs in length and contains an open reading frame of 474 base pairs coding for a bovine IL-2 precursor polypeptide of 158 amino acids with an estimated molecular weight of 17,884. The putative hydrophobic leader or signal sequence of the precursor protein is 23 amino acid residues long, suggesting that, after removal by processing, the mature secreted bovine IL-2 protein contains 135 amino acids and has a molecular weight of 15,464. Comparisons of both the nucleotide sequence and the predicted amino acid sequence of bovine IL-2 with those of the human and mouse IL-2 show extensive regions of sequence conservation between the species, interspersed with other regions of less similarity. The 3' untranslated region of the bovine IL-2 gene shares as much, if not greater, sequence homology with the 3' untranslated regions of the human and mouse genes as do the transcribed coding regions of these genes, suggesting an involvement of this region in regulation. In particular, a tandemly repeated sequence, (TATT)n, found in the 3' untranslated tail of the bovine IL-2 clone is also found in the 3' untranslated region of the other known interleukin and interferon genes, as well as in similar regions of many other inducible genes of the lymphoid and immune response systems, suggesting a cell or tissue-specific regulatory function for these evolutionarily conserved sequences.     

Structure and expression of rat osteosarcoma (ROS 17/2.8) alkaline phosphatase: product of a single copy gene.

Alkaline phosphatase [ALP; orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] is a ubiquitous enzyme of unknown function expressed at high levels in cells of mineralizing tissues. To study the structure, function, and expression of ALP, a full-length cDNA of rat ALP (2415 bases) was isolated from a ROS 17/2.8 osteosarcoma cell lambda gt10 cDNA library. The predicted amino acid sequence spans 524 residues and includes an N-terminal signal peptide of 17 amino acids, the phosphohydrolase active site, a rather hydrophilic backbone with five potential N-glycosylation sites, and a short hydrophobic C-terminal sequence. ALP negative CHO cells transfected with an expression vector containing the ALP coding sequences express ALP. The rat bone, liver, and kidney ALP shows remarkable 90% homology with the corresponding human enzyme, the most divergent region being the C-terminal hydrophobic domain through which the enzyme may be anchored to the plasma membrane. The rat ALP also shows 50% homology with the human placental and intestinal ALP and 25% homology with the Escherichia coli ALP. The amino acids involved in catalysis show nearly complete homology among all known ALP sequences, suggesting that these enzymes evolved from a common ancestral gene. The rat ALP cDNA pRAP 54, used as a hybridization probe in RNA blot analysis of several tissues that express ALP, revealed the presence of an ALP mRNA of approximately equal to 2500 bases. Furthermore, hybridization patterns derived from Southern blot analysis of rat chromosomal DNA offered molecular evidence that the ALP expressed in ROS 17/2.8 osteosarcoma and various rat tissues, excluding the intestine, is the product of the same single copy gene.     

Two distinct genes for ADP/ATP translocase are expressed at the mRNA level in adult human liver.

Several clones hybridizing with a bovine ADP/ATP translocase cDNA were isolated from an adult human liver cDNA library in the vector pEX1. DNA sequence analysis revealed that these clones encode two distinct forms of translocase. In particular, two clones specifying the COOH-end-proximal five-sixths of the protein exhibit a 9% amino acid sequence divergence and totally dissimilar 3' untranslated regions. One of these cDNAs is nearly identical in sequence to an ADP/ATP translocase clone (hp2F1) recently isolated from a human fibroblast cDNA library [Battini, R., Ferrari, S., Kaczmarek, L., Calabretta, B., Chen, S. & Baserga, R. (1987) J. Biol. Chem. 262, 4355-4359], with three amino acid changes and a few differences in the 3' untranslated region. Another clone isolated from the pEX1 library contains a reading frame encoding the remaining, NH2-end-proximal, 37 amino acids of the translocase. This sequence differs significantly (14% amino acid sequence divergence) from the corresponding segment of hp2F1, and the 5' untranslated regions of the two clones are totally dissimilar. RNA transfer hybridization experiments utilizing the clones isolated from the pEX1 library revealed the presence in HeLa cells of three distinct mRNA species. The pattern of hybridization and the sizes of these mRNAs suggest a greater complexity of organization and expression of the ADP/ATP translocase genes in human cells than indicated by the analysis of the cDNA clones.     

Complete cDNA and derived amino acid sequence of human factor V.

cDNA clones encoding human factor V have been isolated from an oligo(dT)-primed human fetal liver cDNA library prepared with vector Charon 21A. The cDNA sequence of factor V from three overlapping clones includes a 6672-base-pair (bp) coding region, a 90-bp 5' untranslated region, and a 163-bp 3' untranslated region within which is a poly(A) tail. The deduced amino acid sequence consists of 2224 amino acids inclusive of a 28-amino acid leader peptide. Direct comparison with human factor VIII reveals considerable homology between proteins in amino acid sequence and domain structure: a triplicated A domain and duplicated C domain show approximately equal to 40% identity with the corresponding domains in factor VIII. As in factor VIII, the A domains of factor V share approximately 40% amino acid-sequence homology with the three highly conserved domains in ceruloplasmin. The B domain of factor V contains 35 tandem and approximately 9 additional semiconserved repeats of nine amino acids of the form Asp-Leu-Ser-Gln-Thr-Thr/Asn-Leu-Ser-Pro and 2 additional semiconserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues.     

Nucleotide sequence of cDNA encoding human alpha 2-macroglobulin and assignment of the chromosomal locus.

Six alpha 2-macroglobulin (alpha 2M) cDNA clones were isolated from a human liver cDNA library by using synthetic oligonucleotides as hybridization probes. One of these, p alpha 2M1, carries a 4.6 kilobase-pair insert, which was sequenced. The insert contains the coding sequences for the mature alpha 2M polypeptide (1451 amino acids) and for a 23-amino acid signal peptide at the NH2 terminus of the precursor pro-alpha 2M. At the 3' end of the insert a poly(A) addition signal A-A-T-A-A-A and part of the poly(A) tail of the messenger RNA were found. The protein sequence deduced from the nucleotide sequence agrees with the published alpha 2M amino acid sequence for all except three residues. The alpha 2M locus was assigned to human chromosome 12 by Southern blot analysis with DNA from a panel of mouse/human somatic cell hybrids, using alpha 2M cDNA as a hybridization probe.     

The amino acid sequence of a glutamic acid-rich protein from bovine retina as deduced from the cDNA sequence.

cDNA clones encoding a glutamic acid-rich protein were isolated from a bovine retina cDNA expression library. The cDNA sequence contained an open reading frame of 1770 base pairs encoding a protein of 590 amino acids (64,509 Da) and untranslated regions of 60 and 490 base pairs at the 5' and 3' ends, respectively. The cDNA hybridized to a 2.4-kilobase retinal mRNA. The amino acid sequence derived from the cDNA sequence contains a glutamic acid-rich domain in which 68 of 109 amino acids are glutamic acid. In addition, this domain contains four repeats of a peptide of 11 amino acids and two repeats of a peptide of 26 amino acids. A polyclonal antibody raised against a decapeptide corresponding to the undecapeptide repeat sequence reacted with a protein in an extract of bovine rod outer segments, whose molecular mass, 65 kDa, corresponded to that of the above glutamic acid-rich protein. The retinal glutamic acid-rich protein showed homology with glutamic acid-rich proteins from bovine brain and the C-terminal region of mammalian neurofilaments.     

Cloning and characterization of human liver cDNA encoding a protein S precursor.

Human liver cDNA encoding a protein S precursor was isolated from two cDNA libraries by two different techniques. Based upon the frequency of positive clones, the abundance of mRNA for protein S is approximately 0.01%. Blot hybridization of electrophoretically fractionated poly(A)+ RNA revealed a major mRNA approximately 4 kilobases long and two minor forms of approximately 3.1 and approximately equal to 2.6 kilobases. One of the cDNA clones contains a segment encoding a 676 amino acid protein S precursor, as well as 108 and 1132 nucleotides of 5' and 3' noncoding sequence, respectively, plus a poly(A) region at the 3' end. The cDNAs are adenosine plus thymidine-rich (60%) except for the 5' noncoding region, where 78% of the nucleotides are guanosine or cytosine. The protein precursor consists of a 41 amino acid "leader" peptide followed by 635 amino acids corresponding to mature protein S. Comparison of the mature protein region with homologous vitamin K-dependent plasma proteins shows that it is composed of the following domains: an amino-terminal gamma-carboxyglutamic acid-rich region of 37 amino acids; a 36 amino acid linker region rich in hydroxy amino acids; four epidermal growth factor-like segments, each approximately 45 amino acids long; and a 387 amino acid carboxyl-terminal domain of unrecognized structure and unknown function.     

Isolation of a full-length cDNA insert encoding human aromatase system cytochrome P-450 and its expression in nonsteroidogenic cells.

The isolation and cloning of a full-length cDNA insert complementary to mRNA encoding human aromatase system cytochrome P-450 is reported. The insert contains an open reading frame encoding a protein of 503 amino acids. This gene is clearly a member of the cytochrome P-450 gene superfamily, because the sequence contains regions of marked homology to those of other members, notably a putative membrane-spanning region, I helix, Ozols, and heme-binding regions. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to human placental aromatase system cytochrome P-450, as detected by immunoblot analysis, and catalyzed the aromatization of androstenedione, testosterone, and 16 alpha-hydroxyandrostenedione. This activity was inhibited by the known aromatase inhibitors, 4-hydroxyandrostenedione and econazole. Thus the several steps involved in the aromatization reaction appear to be catalyzed by a single polypeptide chain, which can metabolize the three major physiological substrates.     

Hunter syndrome: isolation of an iduronate-2-sulfatase cDNA clone and analysis of patient DNA.

Iduronate 2-sulfatase (IDS, EC 3.1.6.13) is required for the lysosomal degradation of heparan sulfate and dermatan sulfate. Mutations causing IDS deficiency in humans result in the lysosomal storage of these glycosaminoglycans and Hunter syndrome, an X chromosome-linked disease. We have isolated and sequenced a 2.3-kilobase cDNA clone coding for the entire sequence of human IDS. Analysis of the deduced 550-amino acid IDS precursor sequence indicates that IDS has a 25-amino acid amino-terminal signal sequence, followed by 8 amino acids that are removed from the proprotein. An internal proteolytic cleavage occurs to produce the mature IDS present in human liver shown to contain a 42-kDa polypeptide N-terminal to a 14-kDa polypeptide. The IDS sequence has strong sequence homology with other sulfatases (such as sea urchin arylsulfatase, human arylsulfatases A, B, and C, and human glucosamine 6-sulfatase), suggesting that the sulfatases comprise an evolutionarily related family of genes that arose by gene duplication and divergent evolution. The arylsulfatases have a greater homology with each other than with the non-arylsulfatases (IDS and glucosamine 6-sulfatase). The IDS cDNA detected RNA species of 5.7, 5.4, 2.1, and 1.4 kilobases in human placental RNA and revealed structural alterations and gross deletions of the IDS gene in many of the clinically severe Hunter syndrome patients studied.     

Characterization of a cDNA coding for human factor VII.

Factor VII is a precursor to a serine protease that is present in mammalian plasma. In its activated form, it participates in blood coagulation by activating factor X and/or factor IX in the presence of tissue factor and calcium. Clones coding for factor VII were obtained from two cDNA libraries prepared from poly(A) RNA from human liver and Hep G2 cells. The amino acid sequence deduced from the cDNAs indicates that factor VII is synthesized with a prepro-leader sequence of 60 or 38 amino acids. The mature protein that circulates in plasma is a single-chain polypeptide composed of 406 amino acids. The amino acid sequence analysis of the protein and the amino acid sequence deduced from the cDNAs indicate that factor VII is converted to factor VIIa by the cleavage of a single internal bond between arginine and isoleucine. This results in the formation of a light chain (152 amino acids) and a heavy chain (254 amino acids) that are held together by a disulfide bond. The light chain contains a gamma-carboxyglutamic acid (Gla) domain and two potential epidermal growth factor domains, while the heavy chain contains the serine protease portion of the molecule. Factor VII shows a high degree of amino acid sequence homology with the other vitamin K-dependent plasma proteins.     

Isolation of the gene and hypothalamic cDNA for the common precursor of gonadotropin-releasing hormone and prolactin release-inhibiting factor in human and rat.

Cloned cDNAs encoding the precursor protein for gonadotropin-releasing hormone (Gn-RH) and prolactin release-inhibiting factor (PIF) were isolated from libraries derived from human and rat hypothalamic mRNA. Nucleotide sequence analyses predict precursor proteins of 92 amino acids for both species and show identity between the human placental and human hypothalamic precursor proteins. Whereas the Gn-RH peptide structure is completely conserved in human and rat, the PIF domain of the precursor displays 70% interspecies homology. Genomic analyses revealed the presence of a single Gn-RH-PIF gene in human and rat containing sequences corresponding to the cDNA distributed across four exons.     

Complete primary structures of two major murine serum amyloid A proteins deduced from cDNA sequences.

cDNA clones encoding two major mouse serum amyloid A proteins, SAA1 and SAA2, were isolated from a liver cDNA library of the lipopolysaccharide-stimulated BALB/c mouse, and their nucleotide sequences were determined. The insert of the SAA2 cDNA clone contained 607 nucleotides with a 5' untranslated region of 36 nucleotides, a signal peptide region corresponding to 19 amino acids, a mature protein region corresponding to 103 amino acids, and a 3' untranslated region of 202 nucleotides. The SAA1 cDNA insert contained 549 nucleotides specifying a part of a signal peptide region, a mature protein region, and a 3' untranslated region. A comparison of the nucleotide and deduced amino acid sequences of SAA1 cDNA with that of SAA2 cDNA showed a high degree of homology: 95% nucleotide sequence homology in the coding region (91% amino acid sequence homology) and 90% homology in the 3' untranslated region. One of nine amino acid differences between SAA1 and SAA2 predicted from the cDNA sequences was located in a putative proteolytic cleavage site for amyloid A protein formation: SAA2 had the Thr-Met sequence in this site, while SAA1 had the Thr-Ile sequence. This suggests that SAA1, which does not deposit as amyloid A protein, is also potentially susceptible to putative proteolytic enzymes. In addition, as compared with mouse SAA2, human SAA1, monkey and mink amyloid A protein, mouse SAA1 had two unique substitutions, which may play a role in differential deposition of mouse SAA isotypes in amyloid tissues.     

Molecular cloning of cDNA encoding Sm autoantigen: derivation of a cDNA for a B polypeptide of the U series of small nuclear ribonucleoprotein particles.

The Sm snRNPs play a central role in the processing of pre-mRNA. Anti-Sm antibodies, the diagnostic hallmark of systemic lupus erythematosus, target the B'/B and D polypeptides of these snRNPs. We have used patient autoantibodies to clone a cDNA from a human fibroblast cDNA library that encodes the full length of a polypeptide identical with, or closely related to, polypeptide B. This cDNA is comprised of 1139 bases and contains an open reading frame of 855 nucleotides that is capable of encoding 285 amino acids. The first 223 amino acids at the NH2 terminus exhibit nearly complete homology with polypeptide N, a newly recognized brain- and heart-specific component of Sm snRNPs. The derived amino acid sequence for B differs from that of the N polypeptide primarily by a 50-amino acid insert 12 residues upstream from the homologous COOH termini of these polypeptides. The structural differences in these cDNAs for B and N may regulate tissue-specific alternative splicing mechanisms for mRNA. In addition, these clones make it possible to map in fine detail the most characteristic autoimmune responses of systemic lupus erythematosus.