Primary structure of pregnancy zone protein. Molecular cloning of a full-length PZP cDNA clone by the polymerase chain reaction

A full-length cDNA clone of the human pregnancy zone protein (PZP) was cloned from the hepatocellular carcinoma cell line Hep3B. Based on the exon sequences of the PZP gene (Devriendt et al. (1989) Gene 81, 325-334; Marynen et al., unpublished data), primer pairs were designed to amplify six overlapping fragments of the PZP cDNA. The obtained cDNA is 4609 bp long and contains an open reading frame coding for 1482 amino acids, including a signal peptide of 25 amino acid residues. Comparison with the published partial PZP amino acid sequence (Sottrup-Jensen et al. (1984) Proc. Natl. Acad. Sci. USA 81, 7353-7357) and the PZP genomic sequences confirmed the identity as a PZP cDNA. 71% of the corresponding amino acid residues in PZP and human alpha 2-macroglobulin (alpha 2M) are identical and all cysteine residues are conserved. A typical internal thiol ester site and a bait domain were identified. A Pro/Thr polymorphism was identified at amino acid position 1180, and an A/G nucleotide polymorphism at bp 4097.     

Antisera against the 3-17 sequence of rat G alpha i recognize only a 40 kDa G-protein in brain

To obtain antisera specific for the GTP-binding protein Gi alpha we immunized rabbits against a synthetic peptide derived from the N-terminal (3-17) sequence predicted from the rat Gi alpha cDNA clone published by Itoh et al. (1986) (Proc. Natl. Acad. Sci. USA 83, 3776-3780). Western-blot analysis of bovine brain G-proteins purified and resolved by hydrophobic chromatography and of rat striatal membranes, indicate that this antiserum does not recognize 41 kDa alpha i or 39 kDa alpha o. However, it reacts with a 40 kDa alpha-subunit. The data suggest that the sequence deduced from the rat G alpha i cDNA corresponds to a G40 alpha protein and that N-terminus directed antisera are useful tools to discriminate between two different G alpha i-like types of G-proteins present in mammalian brain.     

Frameshift and nonsense mutations in a human genomic sequence homologous to a murine UDP-Gal:beta-D-Gal(1,4)-D-GlcNAc alpha(1,3)-galactosyltransferase cDNA

We have previously isolated a murine UDP-Gal:beta-D-Gal(1,4)-D-GlcNAc alpha(1,3)-galactosyltransferase (alpha(1,3)-GT) cDNA (Larsen, R. D., Rajan, V. P., Ruff, M. M., Kukowska-Latallo, J., Cummings, R. D., and Lowe, J. B. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8227-8231). This enzyme constructs the terminal alpha(1,3)-galactosyl linkage within the epitope Gal alpha 1----3Gal. This epitope is expressed by New World monkeys and many nonprimate mammals but generally not by Old World primates, anthropoid apes, or man. To investigate the molecular basis for the apparent species-specific absence of this enzyme and its oligosaccharide product, we have sequenced a human genomic DNA fragment homologous to the murine alpha(1,3)-GT cDNA. This fragment contains a 703-nucleotide region that shares 82% identity with a region of the murine cDNA encoding part of the enzyme's catalytic domain. The human sequence, however, has suffered deletion of single nucleotides at two separate positions, relative to the murine sequence. These frameshift mutations disrupt the translational reading frame that would otherwise maintain a 76% amino acid sequence identity between the human sequence and the murine alpha(1,3)-GT. Moreover, nonsense mutations exist within this disrupted reading frame that would truncate the human polypeptide, relative to the murine enzyme. We therefore propose that this human sequence represents a pseudogene and cannot determine expression of Gal alpha 1----3Gal epitopes on human cells.     

Molecular cloning and nucleotide sequences of cDNAs specific for rat liver ribosomal proteins S17 and L30

cDNA clones coding for rat liver ribosomal proteins S17 and L30 have been isolated by positive hybridization-translation assay from a cDNA library prepared from 8-9S poly(A)+RNA from free polysomes of regenerating rat liver. The cDNA clone specific for S17 protein (pRS17-2) has a 466-bp insert with the poly(A) tail. The complete amino acid (aa) sequence of S17 protein was deduced from the nucleotide sequence of the cDNA. S17 protein consists of 134 aa residues with an Mr of 15 377. The N-terminal aa sequence of S17 protein determined by automatic Edman degradation is consistent with the sequence data. The aa sequence of S17 shows strong homology (76.9%) to that of yeast ribosomal protein 51 [Teem and Rosbash, Proc. Natl. Acad. Sci. USA 80 (1983) 4403-4407] in the two-thirds N-terminal region. The cDNA clone specific for L30 protein (pRL30) has a 394-bp insert. The aa sequence of L30 protein was deduced from the nucleotide sequence of the cDNA. The protein consists of 114 aa residues with an Mr of 12 652. When compared with the N-terminal aa sequence of rat liver L30 protein [Wool, Annu. Rev. Biochem. 48 (1979) 719-754], pRL30 was found not to contain the initiation codon and 5'-noncoding region. The cDNA showed twelve silent changes in the coding region, one point mutation and one base deletion in the 3'-noncoding region, compared with mouse genomic DNA for L30 protein [Wiedemann and Perry, Mol. Cell Biol. 4 (1984) 2518-2528].     

Isolation of cDNA clones of human argininosuccinate lyase and corrected amino acid sequence

In the present study, we isolated clones of human argininosuccinate lyase (ASL) cDNA from a liver cDNA library using a clone of rat ASL cDNA and analyzed human ASL cDNA nucleotide sequence. The results reveal that the sequence of human ASL cDNA published by O'Brien et al. in 1986 [Proc. Natl. Acad. Sci USA 83, 7211-7215] had one-base deletions at three independent positions in the coding regions near the COOH-terminus, which caused frame-shift variations in the amino acid sequence. Amino acid sequencing of peptides prepared from purified human liver ASL showed our predicted amino acid sequence to be correct.     

Structural analysis of the sequence coding for an inducible 26-kDa protein in human fibroblasts

When human fibroblast cells were stimulated with poly(I) X poly(C) in the presence of cycloheximide for the production of interferon-beta (IFN-beta), a 26-kDa protein could be immunoprecipitated by antiserum raised against partially purified human IFN-beta [Content, J., De Wit, L., Pierard, D., Derynck, R., De Clercq, E. & Fiers, W. (1982) Proc. Natl Acad. Sci. USA 79, 2768-2772]. In our hands this 26-kDa protein showed no antiviral activity. Other investigators have, however, reported the presence in the same conditions of a second type of IFN, a so-called beta 2 species [Weissenbach, J., Chernajovsky, Y., Zeevi, M., Shulman, L., Soreq, H., Nir, U., Wallach, D., Perricaudet, M., Tiollais, P. & Revel, M. (1980) Proc. Natl Acad. Sci. USA 77, 7152-7156] of which the mRNA structure and protein characteristics strongly suggests identity with the 26-kDa product. In this paper we describe the nucleotide sequence of the 26-kDa cDNA and part of the corresponding genomic clone. The cDNA clones were isolated from a library made with mRNA from induced human fibroblasts. As, however, the information thus obtained was still incomplete, genomic clones were isolated from a total human DNA library. In this way, the entire region coding for the 26-kDa protein was established, as well as the neighbouring sequences including the inducible promoter area. From the deduced polypeptide sequence a number of characteristics of the 26-kDa protein can be explained. It turns out that the 26-kDa protein gene and the so-called 'IFN-beta 2' gene are identical. However, extensive homology searches indicate that the 26-kDa protein does not show statistically significant sequence homology with any known interferon species. Hence, the question of whether the 26-kDa product represents a novel IFN species remains open.     

Nucleotide sequence and expression of the human gene encoding apolipoprotein H (beta 2-glycoprotein I).

Human apolipoprotein H (ApoH), also called beta 2-glycoprotein I, is a 50-kDa serum glycoprotein whose function is not clearly defined. We have cloned and sequenced ApoH cDNAs both from human liver and from a human hepatoma cell line (HepG2). Both cDNA sequences predict a protein 345 amino acids (aa) in length. This sequence includes a 19-aa hydrophobic, N-terminal signal sequence which is not present in the mature protein [Lozier et al., Proc. Natl. Acad. Sci. USA 81 (1984) 3640-3644]. It differs from this previously reported aa sequence at two positions, both of which strengthen the conservation among the four short consensus repeats within the ApoH molecule. COS-1 cells transiently transfected with the ApoH cDNA in a eukaryotic expression vector produced a single species of ApoH mRNA and secreted in the ApoH protein. The level of ApoH mRNA expressed by HepG2 cells is downregulated by incubation with inflammatory mediators, implying that ApoH is a negative acute-phase protein.     

Chromosomal locations of members of a family of novel endogenous human retroviral genomes.

Human cellular DNA contains two distinguishable families of retroviral related sequences. One family shares extensive nucleotide sequence homology with infectious mammalian type C retroviral genomes (T. I. Bonner, C. O'Connell, and M. Cohen, Proc. Natl. Acad. Sci. USA 79:4709-4713, 1982; M. A. Martin, T. Bryan, S. Rasheed, and A. S. Khan, Proc. Natl. Acad. Sci. USA 78:4892-4896, 1981). The other family contains major regions of homology with the pol genes of infectious type A and B and avian type C and D retroviral genomes (R. Callahan, W. Drohan, S. Tronick, and J. Schlom, Proc. Natl. Acad. Sci. USA 79:5503-5507, 1982; I. M. Chiu, R. Callahan, S. R. Tronick, J. Schlom, and S. A. Aaronson, Science 223:364-370, 1984). Analysis of the human recombinant clone HLM-2 has shown that the pol gene in the latter family is located within an endogenous proviral genome (R. Callahan, I. M. Chiu, J. F. H. Wong, S. R. Tronick, B. A. Roe, S. A. Aaronson, and J. Schlom, Science 228:1208-1211, 1985). We show that the proviral genome in HLM-2 and the related recombinant clone HLM-25 are located, respectively, on human chromosomes 1 and 5. Other related proviral genomes are located on chromosomes 7, 8, 11, 14, and 17.     

Cloning and sequence determination of a cDNA encoding Aspergillus nidulans calmodulin-dependent multifunctional protein kinase.

A partial cDNA encoding Aspergillus nidulans calmodulin-dependent multifunctional protein kinase (ACMPK) was isolated from a lambda ZAP expression library by immunoselection using monospecific polyclonal antibodies to the enzyme. The sequence of both strands of the cDNA (CMKa) was determined. The deduced amino acid (aa) sequence contained all eleven consensus domains found in serine/threonine protein kinases [Hanks et al., Science 241 (1988) 42-52], as well as a putative calmodulin-binding domain. The cDNA contained an intron, lacked an in-frame start codon, and was not polyadenylated. A full-length copy of CMKa was subsequently isolated from a lambda gt10 library of A. nidulans cDNA using a restriction fragment of the first clone as a probe. It contained an in-frame start codon, an open reading frame (ORF) of 1242 bp and was polyadenylated. The ORF encoded a protein of 414 aa residues with an M(r) of 46,895 and an isoelectric point pI = 6.4. These values are in good agreement with that observed for the native enzyme [Bartelt et al., Proc. Natl. Acad. Sci. USA 85 (1988) 3279-3283]. When aligned to optimize homology, 29% of the predicted aa sequence of ACMPK is identical to that of the alpha-subunit of rat brain calmodulin-dependent protein kinase II. ACMPK shares 40 and 44% identity in aa sequence with YCMK1 and YCMK2, respectively, two Ca2+/calmodulin-dependent protein kinases recently cloned from Saccharomyces cerevisiae [Pausch et al., EMBO J. 10 (1991) 1511-1522]. Results of Southern analysis of restriction digests of genomic DNA indicate that ACMPK is encoded by a single-copy gene.     

Identification of the G-protein alpha-subunit encoded by alpha o2 cDNA as a 39 kDa pertussis toxin substrate

A novel form of the Go alpha-subunit (alpha o2) has been identified by molecular cloning (Hsu et al., J. Biol. Chem. 265, 11220-11226, 1990). An antibody was generated against a synthetic peptide corresponding to a region of the protein encoded by alpha o2 cDNA. The antibody reacted with an apparently single 39 kDa protein in membrane preparations of rodent brain and with a 39 kDa pertussis toxin substrate in membranes of rodent neuroendocrine and pituitary cells. A previously produced antibody raised against a region common to proteins encoded by alpha o2 cDNA and the previous cloned alpha o1 cDNA (Itoh et al., Proc. Natl. Acad. Sci. USA 83, 3776-3780, 1986) recognized proteins of 39 and 40 kDa in preparations of bovine, porcine and rodent brain and pertussis toxin substrates of 39 and 40 kDa in membranes of rodent neuroendocrine and pituitary cells. We conclude that the 39 kDa Go alpha subunit is encoded by alpha o2 cDNA.     

A human cDNA encoding the small subunit of RNA polymerase II transcription factor SIII

A full-length cDNA encoding a human homolog of the 15-kDa subunit (p15) of RNA polymerase II elongation factor SIII was isolated and sequenced. Comparison of the open reading frames of the human p15 cDNA and the previously characterized rat p15 cDNA [Garrett et al., Proc. Natl. Acad. Sci. USA 91 (1994) 5237-5241] indicates that they encode identical proteins and are 93% conserved in nucleotide sequence.     

Probable cloning artefacts previously interpreted as unusual leader sequences of rodent ornithine decarboxylase mRNAs--a cautionary tale

Messenger RNAs that have structurally unusual 5' leaders attract interest and provoke conjecture. Cloning and sequencing of two rodent ornithine decarboxylase (ODC) cDNAs, those for mouse [Kahana and Nathans, Proc. Natl. Acad. Sci. USA 82 (1985) 1673-1677] and, recently as published in this journal, for rat [Van Kranen et al., Gene 60 (1987) 145-155], have indicated the presence of such features. In both cases, the leader is unusually long and contains multiple AUG start codons preceding that which encodes the N terminus of the protein. In addition, the leader of the rat clone contains a 54-nt perfect inverted repeat. Because ODC expression appears to be regulated translationally, functional implications immediately suggest themselves. Certain unusual features of the mouse cDNA have proven artefactual [Brabant et al., Proc. Natl. Acad. Sci. USA 85 (1988) 2200-2204; Katz and Kahana, J. Biol. Chem. 263 (1988) 7604-7609]. It is likely that the putative leader sequence of rat ODC cDNA also resulted from a cloning artefact.     

The primary structure of the VLA-2/collagen receptor alpha 2 subunit (platelet GPIa): homology to other integrins and the presence of a possible collagen-binding domain

VLA-2 (also called gpIa/IIa on platelets) is a collagen receptor with a unique alpha subunit and a beta subunit common to other adhesion receptors in the VLA/integrin family. Multiple cDNA clones for the human VLA-2 alpha 2 subunit have been selected from a lambda gtll library by specific antibody screening. The 5,374-bp nucleotide sequence encoded for 1,181 amino acids, including a signal peptide of 29 amino acids followed by a long extracellular domain (1,103 amino acids), a transmembrane domain, and a short cytoplasmic segment (22 amino acids). Direct sequencing of purified alpha 2 protein confirmed the identity of the 15 NH2-terminal amino acids. Overall, the alpha 2 amino acid sequence was 18-25% similar to the sequences known for other integrin alpha subunits. In particular, the alpha 2 sequence matched other integrin alpha chains in (a) the positions of 17 of its 20 cysteine residues; (b) the presence of three metal-binding domains of the general structure DXDXDGXXD; and (c) the transmembrane domain sequence. In addition, the alpha 2 sequence has a 191-amino acid insert (called the I-domain), previously found only in leukocyte integrins of the beta 2 integrin family. The alpha 2 I-domain was 23-41% similar to domains in cartilage matrix protein and von Willebrand factor, which are perhaps associated with collagen binding. The NH2-terminal sequence reported here for alpha 2 does not match the previously reported alpha 2 NH2-terminal sequence (Takada, Y., J. L. Strominger, and M. E. Hemler. 1987. Proc. Natl. Acad. Sci. USA. 84:3239-3243). Resolution of this discrepancy suggests that there may be another VLA heterodimer that resembles VLA-2 in size but has a different amino acid sequence.     

Cloning and sequence analysis of the human brain beta-adrenergic receptor. Evolutionary relationship to rodent and avian beta-receptors and porcine muscarinic receptors

Two cDNA clones, lambda-CLFV-108 and lambda-CLFV-119, encoding for the beta-adrenergic receptor, have been isolated from a human brain stem cDNA library. One human genomic clone, LCV-517 (20 kb), was characterized by restriction mapping and partial sequencing. The human brain beta-receptor consists of 413 amino acids with a calculated Mr of 46480. The gene contains three potential glucocorticoid receptor-binding sites. The beta-receptor expressed in human brain was homology with rodent (88%) and avian (52%) beta-receptors and with porcine muscarinic cholinergic receptors (31%), supporting our proposal [(1984) Proc. Natl. Acad. Sci. USA 81, 272 276] that adrenergic and muscarinic cholinergic receptors are structurally related. This represents the first cloning of a neurotransmitter receptor gene from human brain.     

The nontranscribed chicken calmodulin pseudogene cross-hybridizes with mRNA from the slow-muscle troponin C gene.

A chicken calmodulin pseudogene with no introns was previously shown to hybridize under stringent conditions with an mRNA species present in skeletal and cardiac muscles, yet it would not hybridize to calmodulin mRNA (J. P. Stein, R. P. Munjaal, L. Lagace', E. C. Lai, B. W. O'Malley, and A. R. Means, Proc. Natl. Acad. Sci. USA 80:6485-6489, 1983). Using the pseudogene as a probe, we isolated a full-length cDNA corresponding to this mRNA from a chicken breast muscle library and showed by sequence analysis that it encodes slow-muscle troponin C and not the pseudogene product. Hybridization between the calmodulin pseudogene and slow-muscle troponin C cDNA is due to a short region of high homology in those nucleotides that encode helices B and C of troponin C and calmodulin. Genomic Southern analysis showed the calmodulin pseudogene and the gene for slow-muscle troponin C to exist as distinct single copies.