Characterization of new members of the pregnancy-specific β1-glycoprotein family

Three cDNAs encoding members of the pregnancy-specific 1-glycoprotein (PSG) family were isolated from human term placental cDNA library. All three cDNAs encode proteins with similar domain structure. There is a leader sequence of 34 amino acids followed by an N-domain of 109 amino acids. Immediately after the N-domain are one or two copies of a repeating A-domain of 93 amino acids, a B-domain of 85 amino acids and a C-domain of variable size. The proteins are highly hydrophilic. However, one of them has an 81-amino acid C-domain which is very hydrophobic and could potentially serve as a membrane attachment site. The putative cell-cell recognition tripeptide, Arg-Gly-Asp, is present in the N-domain of two of the proteins. Partial sequence of one of the cDNAs has been found in HeLa cells while cDNAs highly homologous to two of the cDNAs have been found in the fetal liver. Functional roles of the PSG proteins basing on their structure are proposed.Abbreviations PSG Pregnancy-Specific 1-Glycoprotein, according to nomenclature recommended at the ISOBM XVII Meeting, 1989 [31] - CEA Carcinoembryonic Antigen - bp base-pair - kb kilo-base-pair - nt nucleotide - aa amino acid - UTR Untranslated Region - RGD Arg-Gly-Asp The nucleotide sequence of two of the cDNAs presented in this article have been submitted to GenBank under the accession numbers M37102 (hPS91) and M37103 (hPS133). The nucleotide sequence of hPS176 has also been submitted. No accession number is available yet.     

Mouse macrophage β subunit (CD11b) cDNA for the CR3 complement receptor/Mac-1 antigen

The cDNA for the common \_Mac-1 subunit (CD11b) of the mouse LFA-1/Mac-1/p150,95 group of leukocyte cell adhesion receptors, formally designated integrin \₂, has been cloned and sequenced. Clones were isolated from cDNA libraries made from J774 macrophage and WEHI-3B myelomonocytic tumor cells which express this subunit as a component of the macrophage activation antigen 1 (Mac-1), also known as complement receptor type 3 (CR3). This subunit is expressed as a single, abundant mRNA species approximately 2.7 kilobase (kb) in size. The 2422 base pair (bp) cDNA sequence obtained codes for a 771 amino acid protein organized with leader, extracellular, transmembrane, and cytoplamic domains of 23, 680, 23, and 46 amino acids, respectively, yielding an 82700 mature protein of 747 amino acids. The mouse \_Mac-1 subunit is highly similar to its human counterpart with an overall sequence identity of 81% and identical positioning of 5 out of 6 potential N-linked glycosylation sites, as well as 56 Cys residues that are organized in repeating motifs characteristic of integrin \ subunits. The most highly conserved regions are the transmembrane and cytoplasmic domains where only 4 out of 69 amino acids differ, indicating that the functions associated with this domain in Mac-1-mediated processes, such as iC3b-triggered phagocytosis, have been evolutionarily conserved.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M31039. Offprint requests to: P. M. Hogarth.     

Human 2-5A synthetase: characterization of a novel cDNA and corresponding gene structure.

The enzyme 2-5A synthetase is induced in cultured cells in response to interferon (IFN) treatment. A lambda gt10 cDNA library of mRNA from IFN-induced Daudi lymphoblastoid cells was screened with oligonucleotide probes. Several overlapping cDNAs were isolated and shown to be derived from the human synthetase gene using filter selection and oocyte microinjection assays. The nucleotide sequence of one of these, cDNA 8-2, extended the 2-5A synthetase sequence already described 72 bp in the 5' direction but was found to differ significantly in coding sequence at the 3' end. The longest cDNA isolated (6-2) was approximately 1.4 kb. By Northern hybridization analysis single mRNAs of 1.7 kb were detected in Daudi and T98G (glioblastoma) cells. However, in HeLa cells, four mRNAs ranging in size from 1.5 to 3.5 kb were found, one of which differed at the 3' end. Analysis of both phage and cosmid genomic clones and comparison with genomic DNA indicate that there is a single gene for 2-5A synthetase, comprising at least six exons and five introns, which can undergo a novel form of alternative RNA processing depending on cell type.     

Complete cDNA sequence of chicken vigilin, a novel protein with amplified and evolutionary conserved domains.

The complete cDNA (4375 bp), coding for a new protein called vigilin, was isolated from chicken chondrocytes. The cDNA shows an open reading frame of 1270 amino acids which are organized in 14 tandemly repeated homologous domains. Each domain consists of two subdomains, one with a conserved sequence motif of 35 amino acids (subdomain A) and another one with a presumptive alpha-helical structure of 21-33 amino acids (subdomain B). 149 amino acids at the N-terminus and 71 amino acids at the C-terminus of vigilin do not show the characteristic domain structure. No sequence characteristic of a signal peptide has been found, which argues for an intracellular localisation of vigilin. Vigilin is highly expressed in freshly isolated chicken chondrocytes but little in chondrocytes after prolonged time in culture. Vigilin mRNA exists in two size species, 4.4 kb and 6.5 kb in length due to the usage of different polyadenylation sites. Comparison of the vigilin sequence with data bases showed a remarkable similarity to protein HX from Saccharomyces cerevisiae [Delahodde, A., Becam, A. M., Perea, J. & Jacq, C. (1986) Nucleic Acids Res. 14, 9213-9214]. The yeast protein consists of eight homologous domains with 11 conserved amino acid residues within a set of 35 amino acids. The N-terminal and C-terminal regions of vigilin and protein HX do not reveal any sequence similarity. These results, together with the demonstration of the characteristic vigilin sequence motif in a human cDNA clone, suggest that the repeats represent evolutionary conserved autonomous domains within a family of proteins found in yeast, chicken and man.     

Isolation of human cDNAs for asparagine synthetase and expression in Jensen rat sarcoma cells.

Asparagine synthetase cDNAs containing the complete coding region were isolated from a human fibroblast cDNA library. DNA sequence analysis of the clones showed that the message contained one open reading frame encoding a protein of 64,400 Mr, 184 nucleotides of 5' untranslated region, and 120 nucleotides of 3' noncoding sequence. Plasmids containing the asparagine synthetase cDNAs were used in DNA-mediated transfer of genes into asparagine-requiring Jensen rat sarcoma cells. The cDNAs containing the entire protein-coding sequence expressed asparagine synthetase activity and were capable of conferring asparagine prototrophy on the Jensen rat sarcoma cells. However, cDNAs which lacked sequence for as few as 20 amino acids at the amino terminal could not rescue the cells from auxotrophy. The transferant cell lines contained multiple copies of the human asparagine synthetase cDNAs and produced human asparagine synthetase mRNA and asparagine synthetase protein. Several transferants with numerous copies of the cDNAs exhibited only basal levels of enzyme activity. Treatment of these transferant cell lines with 5-azacytidine greatly increased the expression of asparagine synthetase mRNA, protein, and activity.     

Cloning and nucleotide sequence of a full-length cDNA for human liver gamma-glutamylcysteine synthetase.

We have cloned and sequenced a full-length cDNA for human liver gamma-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in glutathione biosynthesis. The cDNA consists of 2634 bp containing an open reading frame encoding a protein of 367 amino acids and having a calculated M(r) = 72,773. The nucleotide sequence of the cDNA for human liver GCS shares an 84% overall similarity with the composite rat GCS sequence deduced from three overlapping partial cDNAs (Yan and Meister, JBC 265: 1588-1593, 1990). The deduced amino acid sequences are 94% similar. Comparison of Northern blots of total RNA isolated from rat kidney or liver with that from human kidney revealed the GCS mRNA to be larger in the human tissue (approximately 4.0 kb vs. approximately 3.7 kb). (The sequence for the human liver GCS cDNA has been assigned accession number M90656 in GenBank/EMBL databases.     

Human ERCC5 cDNA-cosmid complementation for excision repair and bipartite amino acid domains conserved with RAD proteins of Saccharomyces cerevisiae and Schizosaccharomyces pombe.

Several human genes related to DNA excision repair (ER) have been isolated via ER cross-species complementation (ERCC) of UV-sensitive CHO cells. We have now isolated and characterized cDNAs for the human ERCC5 gene that complement CHO UV135 cells. The ERCC5 mRNA size is about 4.6 kb. Our available cDNA clones are partial length, and no single clone was active for UV135 complementation. When cDNAs were mixed pairwise with a cosmid clone containing an overlapping 5'-end segment of the ERCC5 gene, DNA transfer produced UV-resistant colonies with 60 to 95% correction of UV resistance relative to either a genomic ERCC5 DNA transformant or the CHO AA8 progenitor cells. cDNA-cosmid transformants regained intermediate levels (20 to 45%) of ER-dependent reactivation of a UV-damaged pSVCATgpt reporter plasmid. Our evidence strongly implicates an in situ recombination mechanism in cDNA-cosmid complementation for ER. The complete deduced amino acid sequence of ERCC5 was reconstructed from several cDNA clones encoding a predicted protein of 1,186 amino acids. The ERCC5 protein has extensive sequence similarities, in bipartite domains A and B, to products of RAD repair genes of two yeasts, Saccharomyces cerevisiae RAD2 and Schizosaccharomyces pombe rad13. Sequence, structural, and functional data taken together indicate that ERCC5 and its relatives are probable functional homologs. A second locus represented by S. cerevisiae YKL510 and S. pombe rad2 genes is structurally distinct from the ERCC5 locus but retains vestigial A and B domain similarities. Our analyses suggest that ERCC5 is a nuclear-localized protein with one or more highly conserved helix-loop-helix segments within domains A and B.     

Molecular cloning, cDNA structure and predicted amino acid sequence of bovine 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4 isomerase

We have used our recently characterized human 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) cDNA as probe to isolate cDNAs encoding bovine 3 beta-HSD from a bovine ovary lambda gtll cDNA library. Nucleotide sequence analysis of two overlapping cDNA clones of 1362 bp and 1536 bp in length predicts a protein of 372 amino acids with a calculated molecular mass of 42,093 (excluding the first Met). The deduced amino acid sequence of bovine 3 beta-HSD displays 79% homology with human 3 beta-HSD while the nucleotide sequence of the coding region shares 82% interspecies similarity. Hybridization of cloned cDNAs to bovine ovary poly(A)+ RNA shows the presence of an approximately 1.7 kb mRNA species.     

cDNA sequences for human von Willebrand factor reveal five types of repeated domains and five possible protein sequence polymorphisms

A human umbilical vein endothelial cell cDNA library in lambda gt11 was screened with two previously described cDNA inserts for human von Willebrand factor. Among 16 positive isolates, two that hybridized with a probe corresponding to the amino terminus of von Willebrand factor were sequenced. Together, these four cDNA inserts span 6.5 kilobases of the von Willebrand factor mRNA sequence, completely specifying the 2050 amino acids of the subunit of mature, secreted von Willebrand factor and 24 residues of a precursor peptide. Approximately 77% of the sequence is contained in five types of repeated domains. Domain A consists of 193-220 amino acids and is present in three tandem copies between residues 497 and 1111. Domain B contains 25-35 amino acids and is present in three copies between residues 1533 and 1636. Domain C consists of 116-119 amino acids and is duplicated between residues 1637 and 1899. In contrast to the essentially contiguous repetition of domains A-C, the two copies of domains D and E are each separated by 804 and 1383 amino acids, respectively. Domain D1 contains 289 amino acids between residues 79 and 367, while domain D2 consists of 270 amino acids between residues 1171 and 1440. Domain E1 consists of 46 amino acids between residues 25 and 70, and domain E2 consists of 46 amino acids between residues 1453 and 1498. The triplicated A domains are notably poor in Cys content, while the remaining domains are Cys-rich. The A domains appear to be homologous to a 225-residue segment of complement factor B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of 69-kd and 100-kd forms of 2-5A synthetase in interferon-treated human cells by specific monoclonal antibodies.

Recently, the existence of 40-kd and 46-kd 2-5A synthetases in interferon-treated cells has been confirmed by cloning and characterization of cDNA corresponding to these small size enzymes. By the use of specific monoclonal antibodies, we describe here two forms of high mol. wt 2-5A synthetases of 69 and 100 kd in human cells. The monoclonal antibodies immunoprecipitate either a 69- or a 100-kd 2-5A synthetase. These purified 2-5A synthetases in immune complex preparations are active, i.e. addition of poly(I).poly(C) and ATP results in the synthesis of 2-5A. Both 2-5A synthetases are composed of several subspecies with similar isoelectric points in the range of 7-8 but have different subcellular localizations: 100-kd synthetase is recovered from the microsomal pellet whereas 69-kd synthetase is found to be associated with cell membranes as well as with the microsomal pellet. Different types of interferon-treated human cells express both or either forms of these enzymes. The 69- and 100-kd 2-5A synthetases were also identified by electrophoretic transfer immunoblot analysis using rabbit polyclonal antibodies against a synthetic peptide common on both 46- and 40-kd 2-5A synthetases. These results indicate that small and large size isozymes share a common peptide sequence.     

Cloning, analysis, and bacterial expression of human farnesyl pyrophosphate synthetase and its regulation in Hep G2 cells

A partial length cDNA encoding farnesyl pyrophosphate synthetase (hpt807) has been isolated from a human fetal liver cDNA library in lambda gt11. DNA sequence analysis reveals hpt807 is 1115 bp in length and contains an open reading frame coding for 346 amino acids before reaching a stop codon, a polyadenylation addition sequence, and the first 14 residues of a poly(A+) tail. Considerable nucleotide and deduced amino acid sequence homology is observed between hpt807 and previously isolated rat liver cDNAs for farnesyl pyrophosphate synthetase. Comparison with rat cDNAs suggests that hpt807 is about 20 bp short of encoding the initiator methionine of farnesyl pyrophosphate synthetase. The human cDNA was cloned into a prokaryotic expression vector and Escherichia coli strain DH5 alpha F'IQ was transformed. Clones were isolated that express an active fusion protein which can be readily observed on protein gels and specifically stained on immunoblots with an antibody raised against purified chicken farnesyl pyrophosphate phosphate synthetase. These data confirm the identity of hpt807 as encoding farnesyl pyrophosphate synthetase. Slot blot analyses of RNA isolated from Hep G2 cells show that the expression of farnesyl pyrophosphate synthetase mRNA is regulated. Lovastatin increases mRNA levels for farnesyl pyrophosphate synthetase 2.5-fold while mevalonic acid, low-density lipoprotein, and 25-hydroxycholesterol decrease mRNA levels to 40-50% of control values.     

Molecular cloning and nucleotide sequence of cDNA encoding the rat kidney S-adenosylmethionine synthetase.

We previously reported the isolation of a cDNA encoding the liver-specific isozyme of rat S-adenosylmethionine synthetase from a lambda gt11 rat liver cDNA library. Using this cDNA as a probe, we have isolated and sequenced cDNA clones for the rat kidney S-adenosylmethionine synthetase (extrahepatic isoenzyme) from a lambda gt11 rat kidney cDNA library. The complete coding sequence of this enzyme mRNA was obtained from two overlapping cDNA clones. The amino acid sequence deduced from the cDNAs indicates that this enzyme contains 395 amino acids and has a molecular mass of 43,715 Da. The predicted amino acid sequence of this protein shares 85% similarity with that of rat liver S-adenosylmethionine synthetase. This result suggests that kidney and liver isoenzymes may have originated from a common ancestral gene. In addition, comparison of known S-adenosylmethionine synthetase sequences among different species also shows that these proteins have a high degree of similarity. The distribution of kidney- and liver-type S-adenosylmethionine synthetase mRNAs in kidney, liver, brain, and testis were examined by RNA blot hybridization analysis with probes specific for the respective mRNAs. A 3.4-kilobase (kb) mRNA species hybridizable with a probe for kidney S-adenosylmethionine synthetase was found in all tissues examined except for liver, while a 3.4-kb mRNA species hybridizable with a probe for liver S-adenosylmethionine synthetase was only present in the liver. The 3.4-kb kidney-type isozyme mRNA showed the same molecular size as the liver-type isozyme mRNA. Thus, kidney- and liver-type S-adenosylmethionine synthetase isozyme mRNAs were expressed in various tissues with different tissue specificities.