Characterization of the mRNA and cloned cDNA specifying the third component of mouse complement.

Eighteen cDNA clones containing inserts specific for the third component of complement (C3) have been derived from high molecular weight mouse liver mRNA. The inserts span 4,600 nucleotides of the C3 coding sequence, including the 3' end of C3 mRNA. The length of C3 mRNA was determined to be 5,100 +/- 200 nucleotides, including a poly(A)-containing tail of mean length 170 nucleotides. From cDNA sequence analysis of the 5'-proximal region of C3 mRNA, the NH2-terminal amino acid sequence of the mature C3 beta chain was predicted to be Ile-Pro-Met-Tyr-Ser-Ile-Ile-Thr-Pro-Asn-Val-Leu-Arg-Leu-Glu. This sequence is in good agreement with the reported amino acid sequences of human and guinea pig C3 beta chains. These data position the C3 beta subunit to the NH2-terminal portion of the precursor C3 molecule (pro-C3) and establish the order of subunits in pro-C3 to be NH2-beta-alpha-COOH. In addition, the cDNA sequence indicates that an NH2-terminal extension peptide precedes the beta chain in pro-C3. The amino acid sequence of the mouse C3a fragment and its flanking regions was determined. The data indicate the presence of four arginine residues located between the COOH terminus of the C3 beta and the NH2 terminus of the C3 alpha subunits in pro-C3. The coding sequences of the amino acids that constitute the internal thioester domain in C3 were determined. Unexpectedly, the glutamyl residue that has been shown to participate in the thioester bond in native C3 was found to be encoded as a glutamine.     

Cloning and sequencing of cDNAs encoding alpha and beta subunits of human pyruvate dehydrogenase.

The cDNAs encoding fragments of the alpha and beta subunits (PDH alpha and PDH beta) of human pyruvate dehydrogenase (PDH, EC 1.2.4.1) were isolated from a HeLa cell cDNA library in the lambda gt11 expression vector by immunoscreening. Phage cDNA fragments were subsequently used to screen a human foreskin fibroblast cDNA library by colony hybridization. Nucleotide sequence analyses of the positive plasmid clones (pHPDA and pHPDB) revealed an insert of 1.36 kilobases (kb) for PDH alpha and one of 1.69 kb for PDH beta, respectively, allowing us to predict the complete amino acid sequences of the precursor and mature proteins of these two subunits. A putative leader sequence of 29 amino acid residues was identified in pHPDA, resulting in a precursor protein of 392 amino acid residues (Mr 43,414) and a mature protein of 363 residues (Mr 40,334). A similar leader sequence of 30 amino acid residues in pHPDB was also identified, resulting in a precursor protein of 359 amino acid residues (Mr 39,046) and a mature protein of 329 residues (Mr 35,911). The amino acid sequences of NH2-terminal regions of the two subunits of human PDH were highly homologous with those of mature porcine PDH. The amino acid sequences of phosphorylation sites determined in PDH alpha of bovine and porcine enzymes were also conserved in the human PDH alpha. Blot analysis of HeLa cell poly(A)+ RNA showed a single mRNA of 1.8 kb for PDH alpha and 1.7 kb for PDH beta, respectively. The precursor proteins of PDH alpha and PDH beta were detected by immunoprecipitation from an 35S-labeled cell-free translation system.     

Prolyl 4-hydroxylase: molecular cloning and the primary structure of the alpha subunit from chicken embryo.

Prolyl 4-hydroxylase (EC 1.14.11.2) is a key enzyme required for the posttranslational hydroxylation of proline residues in collagen. The enzyme is a tetramer composed of two pairs of nonidentical subunits (alpha 2 beta 2). The beta subunit is protein disulfide-isomerase, a ubiquitous enzyme found in the endoplasmic reticulum of many cell types. We report here the amino acid sequence of the alpha subunit. One cDNA clone (alpha 1) was isolated from a chicken embryo cDNA expression library in lambda gt11 by screening with anti-alpha-subunit polyclonal immunoglobulins. This alpha 1 cDNA contains an open reading frame of 1401 base pairs. A comparison of the translation of the nucleotide sequence with protein sequences obtained from the purified chicken alpha-subunit polypeptide verified that alpha 1 cDNA encoded the alpha subunit. Polymerase chain reactions were used to extend the sequence of alpha 1 cDNA toward the 5' end of alpha-subunit mRNA. The mature alpha subunit is composed of 516 amino acids with a calculated molecular mass of 59,373 Da. The compiled amino acid sequence contains two potential glycosylation sites, an observation that agrees with a previous demonstration that the alpha subunit contains two N-linked oligosaccharide chains. Blot hybridization analysis of total chicken embryo RNA detected an mRNA of 3.5 kilobases, a size that closely resembles the size of the cloned cDNA. Since the expression of the alpha subunit is confined to cell types that synthesize and secrete collagens, the regulation of the synthesis of the alpha subunit may play a central role in determining the expression of prolyl 4-hydroxylase activity.     

Isolation and characterization of a cDNA clone for bovine cytochrome c oxidase subunit IV.

We have isolated a cDNA clone for the precursor to subunit IV of bovine cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1). A cDNA library was constructed from poly(A)+ RNA of adult beef liver by insertion of cDNA into the plasmid vector pBR322. Transformants were screened by colony hybridization with two mixtures of [32P]-labeled synthetic oligodeoxyribonucleotides. We screened 20,000 transformants with a mixture of heptadecamers complementary to all 16 possible sequences encoding amino acids 98-103 and obtained two cDNA clones encoding subunit IV amino acid sequences. We determined the DNA sequence of the larger (416 base-pair) insert, which contains the coding sequence for amino acids 1-107 of the mature protein and an NH2-terminal extension (presequence). The deduced amino acid sequence of the mature protein is identical with the previously determined protein sequence: the sequence of the NH2-terminal extension contains a potential initiator methionine at amino acid -22 from the NH2-terminus of the processed protein. The presequence is quite basic and contains several arginines, including one at the processing site. No hydrophobic region analogous to that found in bacterial and eukaryotic signal peptides is present, but there are homologies with other mitochondrial protein presequences, which may include a common signal for their destination and processing.     

Cloning and cDNA sequence of the regulatory subunit of cAMP-dependent protein kinase from Dictyostelium discoideum.

cDNA clones encoding the regulatory subunit of the cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) from Dictyostelium discoideum were isolated by immunoscreening of a cDNA library constructed in the expression vector lambda gt11. High-affinity cAMP-binding activity was detected in extracts from bacteria lysogenized with these clones. Nucleotide sequence analysis of three overlapping clones allowed the determination of a 1195-base-pair cDNA sequence coding for the entire regulatory subunit and containing nontranslated 5' and 3' sequences. The open reading frame codes for a protein of 327 amino acids, with molecular weight 36,794. The regulatory subunit from Dictyostelium shares a high degree of homology with its mammalian counterparts, but is lacking the NH2-terminal domain required for the association of regulatory subunits into dimers in other eukaryotes. On the basis of the comparison of the regulatory subunits from Dictyostelium, yeast, and bovine tissues, a model for the evolution of these proteins is proposed.     

Molecular cloning and characterization of cDNA encoding the GTP-binding protein alpha i and identification of a related protein, alpha h.

We have cloned and characterized cDNA encoding alpha i, the GTP-binding subunit of Gi, a protein that mediates hormonal inhibition of adenylate cyclase and hormonal regulation of other membrane functions. We have also identified cDNA encoding a putative protein, which we have named alpha h, that is highly homologous to alpha i but different from other known GTP-binding proteins. Both cDNAs were isolated from a bovine pituitary library. The cDNA encoding alpha i was identified by finding that the amino acid sequence determined for two tryptic peptides from alpha i agreed exactly with amino acid sequences deduced from the cDNA. We also determined the amino acid sequence of peptides derived from alpha o, a related 39-kDa protein purified from bovine brain. These sequences are approximately 75% identical to the sequence determined for alpha i. Southern blot analysis of bovine genomic DNA, using as probes radiolabeled cDNAs for alpha i, alpha h, and the alpha subunit of a related protein, transducin, showed that each probe recognized different genomic DNA fragments. Our results suggest a further level of complexity in the organization of the G-protein gene family, with multiple G proteins of very similar structural properties likely to be identified as products of distinct genes.     

Human basic fibroblast growth factor gene encodes four polypeptides: three initiate translation from non-AUG codons.

Human basic fibroblast growth factor (bFGF) is an angiogenic polypeptide mitogen present in a wide variety of mesoderm- and neuroectoderm-derived tissues. bFGF cDNA and genomic clones predict a 17.8-kDa (155-amino acid) gene product based on the presence of a single putative translational initiator ATG codon. However, a bFGF protein isolated from human placenta contains two additional amino acids NH2-terminal to the predicted initiator methionine. We report here that the human cell line SK-HEP-1 coexpresses four molecular forms (17.8, 22.5, 23.1, and 24.2 kDa) of bFGF. The 17.8-kDa bFGF protein is translationally initiated at the previously predicted methionine (AUG) codon, whereas the 22.5-, 23.1-, and 24.2-kDa proteins initiate at unusual non-AUG codons. The higher molecular weight forms are colinear NH2-terminal extensions of the 18-kDa bFGF.     

Amyloid plaque core protein in Alzheimer disease and Down syndrome.

We have purified and characterized the cerebral amyloid protein that forms the plaque core in Alzheimer disease and in aged individuals with Down syndrome. The protein consists of multimeric aggregates of a polypeptide of about 40 residues (4 kDa). The amino acid composition, molecular mass, and NH2-terminal sequence of this amyloid protein are almost identical to those described for the amyloid deposited in the congophilic angiopathy of Alzheimer disease and Down syndrome, but the plaque core proteins have ragged NH2 termini. The shared 4-kDa subunit indicates a common origin for the amyloids of the plaque core and of the congophilic angiopathy. There are superficial resemblances between the solubility characteristics of the plaque core and some of the properties of scrapie infectivity, but there are no similarities in amino acid sequences between the plaque core and scrapie polypeptides.     

GTPase of bovine rod outer segments: the amino acid sequence of the alpha subunit as derived from the cDNA sequence.

The sequence of the 350 amino acids in the alpha subunit of GTPase of bovine rod outer segments has been determined. Enriched GTPase mRNA was used to prepare a cDNA library in the expression vector lambda gt11 and several overlapping cDNA clones corresponding to the alpha subunit of the GTPase were identified. The cDNA sequence determined contains 93 nucleotides upstream of the 5' end of the coding region, 1050 nucleotides that specify the amino acid sequence, and 45 nucleotides downstream from the 3' end. The previously described partial amino acid sequences and the sequences at the ADP-ribosylation sites for cholera and pertussis toxins are all confirmed and fitted into the present complete sequence. Homologies are found between the sequence of the alpha subunit and those of other guanine nucleotide-binding proteins, the ras proteins, peptide chain elongation factors EF-Tu and EF-G, and the initiation factor IF2.     

Molecular cloning and nucleotide sequence of rat kidney gamma-glutamyl transpeptidase cDNA.

We have screened a cDNA library (20,000 clones) made from rat kidney poly(A)+ RNA, using an oligonucleotide probe that was a mixture of 14-base DNA oligomers containing all 32 possible sequences coding for residues 32-36 of the gamma-glutamyl transpeptidase (EC 2.3.2.2.) heavy chain. We isolated and sequenced two cDNAs corresponding to the mRNA coding for the entire length of the enzyme precursor. The nucleotide sequence that we obtained (2072 bases) reveals an open reading frame of 1707 nucleotides coding for the common precursor of both enzyme subunits. The amino acid sequence begins with the 21 residues located at the NH2-terminal hydrophobic region of the heavy subunit. We show that this sequence, which is not processed, is the only possible signal peptide in the sequence. Five potential N-glycosylation sites are present in the gamma-glutamyl transpeptidase sequence. Using one of the two cDNA clones as probe, a 2.2-kilobase sequence was detected by blot analysis in rat kidney and human fetal liver RNA.     

Molecular cloning of the alpha subunit of human and guinea pig leukocyte adhesion glycoprotein Mo1: chromosomal localization and homology to the alpha subunits of integrins.

The cell-surface glycoprotein Mo1 is a member of the family of leukocyte cell adhesion molecules (Leu-CAMs) that includes lymphocyte function-associated antigen 1 (LFA-1) and p150,95. Each Leu-CAM is a heterodimer with a distinct alpha subunit noncovalently associated with a common beta subunit. Leu-CAMs play crucial roles in cell-cell and cell-matrix interactions. We describe the isolation and analysis of two partial cDNA clones encoding the alpha subunit of the Leu-CAM Mo1 in humans and guinea pigs. A monoclonal antibody directed against an epitope in the carboxyl-terminal portion of the guinea pig alpha chain was used for immunoscreening a lambda gt11 expression library. The sequence of a 378-base-pair insert from one immunoreactive clone revealed a single continuous open reading frame encoding 126 amino acids including a 26-amino acid tryptic peptide isolated from the purified guinea pig alpha subunit. A cDNA clone of identical size was isolated from a human monocyte/lymphocyte cDNA library by using the guinea pig clone as a probe. The human clone also encoded a 126-amino acid peptide including the sequence of an additional tryptic peptide present in purified human Mo1 alpha chain. RNA gel blots revealed that mature Mo1 alpha chain mRNA is approximately 5 kilobases in guinea pigs and humans. Southern analysis of DNA from hamster-human hybrids localized the human Mo1 alpha chain to chromosome 16, which has been shown to contain the gene for the alpha chain of lymphocyte function-associated antigen 1. A comparison of the Mo1 alpha chain coding region revealed significant homologies with carboxyl-terminal portions of the alpha subunits of fibronectin, vitronectin, and platelet IIb/IIIa receptors. These data suggest that the alpha subunits of Leu-CAMs evolved by gene duplication from a common ancestral gene and strengthen the hypothesis that the alpha subunits of these heterodimeric cell adhesion molecules on myeloid and lymphoid cells, platelets, and fibroblasts are evolutionary related.     

A 125/115-kDa cell surface receptor specific for vitronectin interacts with the arginine-glycine-aspartic acid adhesion sequence derived from fibronectin.

Affinity chromatography was used to identify a cell surface receptor for the adhesive protein vitronectin. Detergent extracts of human osteosarcoma (MG-63) cells were chromatographed on either vitronectin-Sepharose or Sepharose linked to the synthetic peptide Gly-Arg-Gly-Asp-Ser-Pro, which includes the fibronectin cell attachment sequence Arg-Gly-Asp. Two cell surface proteins with apparent molecular mass of 125 and 115 kDa bound to both columns and were specifically eluted with a solution containing the Gly-Arg-Gly-Asp-Ser-Pro peptide. These proteins could be incorporated into phosphatidylcholine liposomes and mediated the specific binding of these liposomes to vitronectin but not to fibronectin. In contrast, liposomes containing a previously identified 140-kDa fibronectin receptor, which interacts with the Arg-Gly-Asp sequence in fibronectin, did not bind to vitronectin. Thus, the fibronectin and vitronectin receptors each recognize the Gly-Arg-Gly-Asp-Ser-Pro peptide but exhibit mutually exclusive reactivities toward fibronectin and vitronectin. These receptors appear to belong to a family of proteins that mediate cell substratum adhesion via related but subtly different specificities.     

A gene encoding the tryptophan synthase beta subunit of Arabidopsis thaliana.

The DNA sequence of a tryptophan synthase gene and the flanking 5' and 3' regions has been determined for Arabidopsis thaliana. The sequence encodes only the beta subunit domain, indicating that alpha and beta subunits are specified by separate genes. The gene contains four introns and encodes 470 amino acid residues. The plant amino acid sequence is highly conserved with respect to corresponding microbial sequences. The NH2-terminal amino acid sequence is characteristic of chloroplast transit peptides. Identity of the sequences of the genomic exons and a cDNA clone and the presence of cellular RNA corresponding in size and 5' sequence to the gene indicate that the gene is expressed. Analysis of Arabidopsis genomic DNA suggests the presence of a second gene for the beta subunit.     

Primary structure and unique expression of the 22-kilodalton light chain of human neutrophil cytochrome b.

Cytochrome b comprising 91-kDa and 22-kDa subunits is a critical component of the membrane-bound oxidase of phagocytes that generates superoxide. This important microbicidal system is impaired in inherited disorders known as chronic granulomatous disease (CGD). Previously we determined the sequence of the larger subunit from the cDNA of the CGD gene, the X chromosome locus affected in "X-linked" CGD. To complete the primary structure of the cytochrome b and to assess expression of the smaller subunit, we isolated cDNA clones for the 22-kDa polypeptide by immunoscreening and confirmed their authenticity by direct N-terminal protein sequencing. Although the deduced amino acid sequence of the 22-kDa subunit is not overtly similar to other known cytochromes, we observed a 31-amino acid stretch of 39% identity with polypeptide I of mitochondrial cytochrome c oxidase centered on a potential heme-coordinating histidine. Similarities in the hydropathy profiles and spacing of histidines of the 22-kDa protein and myoglobin suggest structural motifs in common with other heme-containing proteins that are not readily revealed by primary amino acid sequences. Although RNA for the larger subunit has been found only in cells of the phagocytic lineage, stable RNA encoding the 22-kDa subunit was observed in all cell types. However, the stable 22-kDa protein was detected only in phagocytic cells that were expressing the larger subunit RNA. This observation suggests that the large subunit may play a role in regulating the assembly of the heterodimeric cytochrome b.     

Isolation of a cDNA clone for the human HLA-DR antigen alpha chain by using a synthetic oligonucleotide as a hybridization probe.

We have used a synthetic 20-nucleotide hybridization probe to isolate a cDNA clone encoding the alpha chain of the HLA-DR antigen from a cDNA library constructed from membrane-bound poly(A)+ mRNA. A set of synthetic 11-nucleotide fragments, potentially complementary to the codons for amino acids 11-14 of the HLA-DR alpha chain, were used to prime a cDNA synthesis reaction on various poly(A)+ mRNA templates. Extension of the primers in the presence of a single dideoxynucleotide triphosphate resulted in an 18-nucleotide cDNA product whose sequence corresponded to the NH2-terminal amino acids of the HLA-DR alpha chain. An oligonucleotide was synthesized based on this sequence information and its specificity for HLA-DR alpha mRNA was confirmed by primer extension and blot analysis. The cDNA library made from mRNA from the lymphoblastoid cell line CA-SC was probed with 32P-labeled cDNA synthesized on poly(A)+ mRNA from a B-cell line (CA-SC) or from a T-cell line (Molt-4) to enrich for B-cell-specific clones. A set of cDNA clones that hybridized preferentially with the B-cell probe was screened with the 32P-labeled 20-nucleotide probe. The cDNA clone isolated by this procedure is 1,100 nucleotides long; the nucleotide sequence of the 5' end of the cDNA insert corresponds to the amino acid sequence of the HLA-DR alpha chain. Hybridization of this cDNA clone to genomic blots suggests that the HLA-DR alpha chain is encoded by a single-copy gene. One of the restriction endonucleases used in genomic DNA digests reveals a restriction fragment polymorphism.     

Multiple biological activities are expressed by a mouse interleukin 6 cDNA clone isolated from bone marrow stromal cells.

Interleukin 6 (IL-6) refers to the gene product that was characterized initially as beta 2 interferon/26-kDa protein produced by human fibroblasts and later was found to be identical to B-cell stimulatory factor 2, hybridoma/plasmacytoma growth factor, and probably hepatocyte-stimulating factor. Using the human IL-6 cDNA as a probe, we have isolated functional cDNA clones from mouse bone marrow stromal cell cDNA libraries. Sequence analysis of the mouse cDNA insert revealed significant homology between the human and mouse IL-6 cDNA clones both at the level of nucleotide (65%) and deduced amino acid (41%) sequences. The NH2-terminal sequence of the deduced protein is identical to a partial NH2-terminal sequence determined previously for a hybridoma/plasmacytoma growth factor and a plasmacytoma growth factor isolated from mouse T cells and macrophages, respectively. The mRNA for mouse IL-6 is expressed in IL-1-treated stromal cells and in activated T-cell and macrophage cell lines. Supernatants from COS-7 monkey cells transfected with the cDNA clone have plasmacytoma growth factor, hepatocyte-stimulating factor, and colony-stimulating factor activities, as well as the ability to support the growth of a factor-dependent myeloid cell line, thus revealing an additional biological activity for IL-6.     

cDNA clones coding for the heavy chain of human HLA-DR antigen.

Two cDNA clones, pDRH1 and pDRH2, containing sequences specific for human HLA-DR antigens were isolated from a bank of cDNA clones made from partially purified HLA-DR mRNA from the human lymphoblastoid cell line Maja. The clones were specific for the Mr 34,000 HLA-DR antigen glycoprotein chain. The identity of these clones was established by (i) their ability to hybridize specifically to HLA-DR mRNA in a positive selection assay; (ii) mRNA species hybridizing to the cDNA clones were expressed in B-cell but not in T-cell or fibroblast cell cultures; and (iii) a nucleotide sequence in the longer clone, pDRH2, could be translated into an amino acid sequence that is identical to the limited NH2-terminal amino acid sequence available for the purified HLA-DR antigen Mr 34,000 chain. Analysis of DNA from human, mouse, and human--mouse somatic cell hybrid lines by Southern transfer of restriction endonuclease digests indicated that the HLA-DR heavy chain is encoded in chromosome 6. This finding is compatible with the location of at least one of the HLA-D/DR heavy chain genes within the HLA region. In addition, the sequences coding for HLA-DR heavy chain appear to be present in only one or a few copies in the genome and to be relatively simple in structure.     

Molecular cloning of beta 3 subunit, a third form of the G protein beta-subunit polypeptide.

The signal-transducing guanine nucleotide-binding regulatory (G) proteins are heterotrimers composed of three subunits--alpha, beta, and gamma. Although multiple distinctive forms of the alpha subunit have been described, only two forms of the beta subunits of the G proteins have been identified. To investigate further the structural diversity of the beta subunits, we screened bovine and human retina cDNA libraries and isolated clones encoding three distinct types of G protein beta subunit. One form was identical to previously isolated beta 1-subunit cDNA clones that encode the 36-kDa form of the beta subunit, whereas a second form was identical to previously described beta 2 cDNAs that encode the 35-kDa beta isoform. In addition, we identified another species, designated beta 3 subunit, which encodes a third distinct form of the beta subunit. The beta 3-subunit cDNA corresponds to a 2.0-kilobase mRNA expressed in all tissues and clonal cell lines examined. Nucleotide sequence analysis indicates that the encoded peptide consists of 340-amino acid residues with a Mr of 37,221. The amino acid sequences of the three beta subunits are closely related: 83% identity between beta 1 and beta 3 subunits and 81% identity between beta 2 and beta 3 subunits. By contrast, the 3'-untranslated regions of the three cDNAs show no significant homology. Our data support the hypothesis that a family of beta-subunit polypeptides exists and extend understanding of beta-subunit structure.     

Primary structure of cucumber (Cucumis sativus) ascorbate oxidase deduced from cDNA sequence: homology with blue copper proteins and tissue-specific expression.

cDNA clones for ascorbate oxidase were isolated from a cDNA library made from cucumber (Cucumis sativus) fruit mRNA. The library was screened with synthetic oligonucleotides that encode the NH2-terminal sequence of this enzyme. Nucleotide sequence analysis of the cloned cDNA inserts revealed a 1761-base-pair open reading frame that encoded an NH2-terminal signal peptide of 33 amino acids and a mature enzyme of 554 amino acids (Mr, 62,258). The amino acid sequence deduced from nucleotide sequence analysis agrees with the NH2-terminal amino acid sequence of the purified ascorbate oxidase, as determined by microsequencing methods. Cucumber ascorbate oxidase contained four histidine-rich regions with striking sequence homology to the corresponding parts of the other multicopper oxidases such as Neurospora crassa laccase and human ceruloplasmin and, to some extent, to a low molecular weight copper protein such as plastocyanin. Moreover, these data further support the hypothesis that the small blue copper proteins and the multicopper oxidases have evolved from the same ancestral gene. By RNA blot hybridization analysis, the mRNA for the ascorbate oxidase was found to be abundant in cucumber fruit tissue while expressed at very low levels in leaf and root tissues.