Cloning of a cDNA for a major human protein-tyrosine-phosphatase.

We have isolated a cDNA clone encoding the major protein-tyrosine-phosphatase (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) of human placenta. Degenerate oligonucleotides, based on the amino acid sequence of the protein, were used to amplify an internal fragment of the gene from human placental cDNA by the polymerase chain reaction. This fragment was then used to probe a human placental cDNA library. A 3.3-kilobase (kb) insert was isolated and sequenced. The insert has a single extended open reading frame that predicts a 435 amino acid protein of Mr approximately 50,000. From the amino terminus to residue 321, the deduced amino acid sequence is identical to that previously determined by peptide sequencing [Charbonneau, H., Tonks, N. K., Kumar, S., Diltz, C. D., Harrylock, M., Cool, D. E., Krebs, E. G., Fischer, E. H. & Walsh, K. A. (1989) Proc. Natl. Acad. Sci. USA 86, 5252-5256]; however, the sequence predicts that the protein contains an additional 114 amino acids not present in the reported peptide sequence. In vitro translation of the 3.3-kb insert produces a protein of Mr 56,000, in general agreement with the predicted size. The phosphatase gene appears to be present as a single copy in human genomic DNA and is transcribed into a 3.5-kb message in a variety of tissues.     

Human cDNA clones for an alpha subunit of Gi signal-transduction protein

Two cDNA clones were obtained from a lambda gt11 cDNA human brain library that correspond to alpha i subunits of G signal-transduction proteins (where alpha i subunits refer to the alpha subunits of G proteins that inhibit adenylate cyclase). The nucleotide sequence of human brain alpha i is highly homologous to that of bovine brain alpha i [Nukada, T., Tanabe, T., Takahashi, H., Noda, M., Haga, K., Haga, T., Ichiyama, A., Kangawa, K., Hiranaga, M., Matsuo, H. & Numa, S. (1986) FEBS Lett. 197, 305-310] and the predicted amino acid sequences are identical. However, human and bovine brain alpha i cDNAs differ significantly from alpha i cDNAs from human monocytes, rat glioma, and mouse macrophages in amino acid (88% homology) and nucleotide (71-75% homology) sequences. In addition, the nucleotide sequences of the 3' untranslated regions of human and bovine brain alpha i cDNAs differ markedly from the sequences of human monocyte, rat glioma, and mouse macrophage alpha i cDNAs. These results suggest there are at least two classes of alpha i mRNA.     

Molecular cloning of the mouse cell adhesion molecule uvomorulin: cDNA contains a B1-related sequence.

A clone (F20) containing coding sequences for the cell adhesion molecule uvomorulin was isolated by immunological techniques from cDNA library in the expression vector lambda gt11. The beta-galactosidase-uvomorulin fusion protein was used to affinity purify anti-uvomorulin antibodies. Affinity-purified antibodies recognized uvomorulin from cell lysates of embryonal carcinoma cells and reacted with the cell surface of embryonal carcinoma cells. The 1.8-kilobase cDNA insert hybridized to a single 4.3-kilobase poly(A)+ RNA species found only in cells expressing uvomorulin. Part of the nontranslated 3' sequences of the cloned uvomorulin cDNA is homologous to the interspersed B1 repeat of the mouse genome.     

Identification of genes for the constant region of rabbit T-cell receptor beta chains.

We describe cDNA clones from thymus mRNA of a young rabbit that have sequences highly homologous to the human and murine T-cell receptor beta-chain constant region (C beta). In rabbit, man, and mouse there is a conserved extra cysteine in the constant region that could lead to a free thiol group or alternative disulfide bond formation depending on the locations and total numbers of cysteines in assembled receptor molecules. A cDNA clone (CL ANA 11) with 571 bases 5' of the C beta coding sequence has an open reading frame starting at a methionine codon that encodes 141 amino acids in frame with the C beta sequence. The encoded sequence has no resemblance to known immunoglobulin or beta-chain variable regions or other known proteins. An oligonucleotide probe from the 5' end of the encoded protein hybridizes to an approximately equal to 2-kilobase genomic DNA fragment that contains C beta gene sequences and to an approximately equal to 8-kilobase mRNA species in the thymus mRNA preparation from which the clone was derived. Within the 5' coding sequence there is a stretch of 211 bases containing strings of alternating purines and pyrimidines that may form Z-DNA. The sequence of the last 55 base pairs adjacent to C beta resembles the corresponding segment of mouse cDNA clone 86T3 that contains sequence 5' of the mouse C beta 1 gene. Although the function of a potential protein encoded by the 5' end of CL ANA 11 is unknown, it could play a role in regulation of thymocyte growth and differentiation.     

Isolation and characterization of a human interleukin cDNA clone, homologous to mouse B-cell stimulatory factor 1, that expresses B-cell- and T-cell-stimulating activities.

A cDNA sequence coding for a human interleukin has been isolated from a concanavalin A-activated human T-cell cDNA library based on homology with a mouse interleukin cDNA that expresses B-cell stimulatory factor 1 (BSF-1) activity and T-cell- and mast-cell-stimulating activities. The human cDNA contains a single open reading frame encoding a protein of 153 amino acid residues including a putative signal peptide. Amino acid sequences of the mouse and human polypeptides, deduced from their cDNAs, share extensive homology with the exception of about 40 amino acid residues near the middle portion, which share little homology. Supernatant of COS-7 monkey cells transfected with the human cDNA clone stimulated proliferation of human helper T-cell clones and of anti-IgM-activated human B cells, two properties of mouse BSF-1 on mouse cells. These results indicate that this human cDNA clone encodes a protein structurally and functionally homologous to mouse BSF-1.     

Molecular cloning of a cDNA that encodes the peptide core of a mouse mast cell secretory granule proteoglycan and comparison with the analogous rat and human cDNA.

A cDNA that encodes a mouse secretory granule proteoglycan peptide core was isolated from a cDNA library prepared from nontransformed mouse bone marrow-derived mast cells (BMMC) using as a probe a 280-base-pair fragment of a rat cDNA that encodes the proteoglycan peptide core of rat basophilic leukemia (RBL)-1 cells. Based on the consensus nucleotide sequence and deduced amino acid sequence of the cDNA, the mouse BMMC proteoglycan peptide core is 16.7 kDa and contains a 21-amino acid glycosaminoglycan attachment region consisting of alternating serine and glycine residues. When the predicted amino acid sequence of the mouse BMMC proteoglycan peptide core was compared with the predicted amino acid sequences of the homologous molecules expressed in RBL-1 cells and in human promyelocytic leukemia HL-60 cells, the mouse-derived sequence was more closely homologous to the rat sequence than the human sequence except for the length of the serine-glycine repeat region. The N terminus was found to be a highly conserved region of the molecule in the three species, suggesting that this region is important for the structure, function, and/or metabolism of this family of proteoglycans. Nucleotide sequences within the 5' and 3' untranslated regions of the mouse, rat, and human proteoglycan cDNA were conserved. That similar sequences were also present in the corresponding regions of a cDNA that encodes a rat mast cell protease suggests that particular nucleotide sequences may be important for regulation of expression of those proteins that are destined to reside in secretory granules.     

Isolation and characterization of cDNA clones for human and bovine fibronectins.

A bovine fibronectin (FN) cDNA clone (pFB1) was isolated by screening a cDNA library of calf testis fibroblasts with a synthetic oligonucleotide probe. The probe was a mixture of eight 14-base-long oligonucleotides designed from the amino acid sequence Glu-Cys-Phe-Met-Pro present in the Mr 3,000 COOH-terminal fragment of bovine plasma FN [Petersen, T.E., Thøgersen, H.C., Skorstengaard, K., Vibe-Pedersen, K., Sahl, P., Sottrup-Jensen, L. & Magnusson. S. (1983) Proc. Natl. Acad. Sci. USA 80. 137-141]. pFB1 contained a 1,000 base-pair (bp) insert comprising the complete 3' noncoding sequence (690 bp) and approximately equal to 300 bp of the coding region. The clone pFB1 was used as a radioactive probe in the screening of a human cell line (Hs 578T) cDNA library. Eleven positive cDNA clones were detected, one of which, named pFH1, contained a 2,000-bp insert comprising the complete 3' noncoding region (693 bp) and approximately equal to 1,300 bp of the coding region of human FN. The sequences of the clone pFB1 insert and of the homologous region in clone pFH1 were determined. The nucleotide sequences are 90% homologous. Six amino acid changes were found, clustered in an area connecting two structural domains described in bovine plasma FN. Furthermore, the 204 COOH-terminal amino acid sequence of bovine FN was completed by overlapping two peptide fragments (MrS 3,000 and 23,000). Clone pFH1 was used in estimating the size of human fibronectin mRNA (7,900 bases) through blot hybridization analysis. Southern blot studies suggest that human FN is coded by a unique gene.     

Human c-myb protooncogene: nucleotide sequence of cDNA and organization of the genomic locus.

We have isolated cDNA clones of the human c-myb mRNA that contain approximately 3.4 kilobases of the approximately 3.8-kilobase mRNA sequence. Nucleotide sequence analysis shows that the c-myb mRNA contains an open reading frame of 1920 nucleotides, which could encode a 72-kDa protein. The cDNA nucleotide sequence and the predicted amino acid sequence of the c-myb protein are highly homologous to the corresponding chicken and mouse proteins. In particular, a region toward the NH2 terminus of the protein containing a 3-fold tandem repeat of 51 residues is evolutionarily conserved and is the only region of homology with the Drosophila c-myb protein. This region may represent a functionally important structure, most likely the DNA-binding domain. cDNA clones have been used to isolate genomic clones and to define a preliminary intron/exon organization of the c-myb gene. Identification of 5' and 3' coding and noncoding exons indicates that the human c-myb locus spans a 40-kilobase region.     

Sequence and evolution of the human T-cell antigen receptor beta-chain genes.

We present the nucleotide sequences of the two genomic constant (C)-region gene segments, C beta 1 and C beta 2, encoding the beta chain of the human T-cell antigen receptor. The two C beta genes are organized identically to each other and to the corresponding mouse genes, both having four exons, whose boundaries were confirmed from the sequence of a C beta 2 cDNA clone from the T-cell line MOLT-4. The predicted amino acid sequences of human C beta 1 and C beta 2 differ at only five positions, which suggests that the proteins have very similar functions. This similarity is the result of strong nucleotide-sequence conservation in protein-coding regions, which extends to silent positions. A quantitative analysis of an alignment of the nucleotide sequences of the two human genes shows that whereas the 5' ends (including the first exon) are extremely homologous, the 3' ends are widely divergent, with other regions having intermediate levels of homology. Analysis of published data [Gascoigne, N.R.J., Chien, Y., Becker, D.M., Kavaler, J. & Davis, M.M. (1984) Nature (London) 310, 387-391] shows that the mouse C beta 1 and C beta 2 genes are also virtually identical in their first exons but more divergent in the remaining coding regions. Therefore, partial gene conversion events may have occurred during the evolution of both human and mouse C beta genes.     

Primary structure and cDNA cloning of human fibroblast collagenase inhibitor.

We report the primary structure and cDNA cloning of human fibroblast collagenase inhibitor, a glycoprotein that appears to play a central role in modulating the activity of a number of metalloendoproteases of connective tissue origin including collagenase, gelatinase, and proteoglycanase. Secreted human fibroblast collagenase inhibitor was purified and subjected to automated Edman degradation. The secreted protein consists of 184 amino acid residues; it contains two sites of N-linked oligosaccharide linkage and six disulfide bonds. Synthetic oligonucleotide probes based on selected amino acid sequences of the inhibitor were used to screen a lambda gt10 cDNA library from a human fibroblast line. Two overlapping cDNA clones were characterized to determine the complete coding and noncoding sequences of the specific mRNA. The amino acid sequence deduced from the nucleotide sequence agrees with that determined by protein sequencing. One clone appears to contain the complete 5' end and, in addition, the cDNA sequence predicts a 23-amino acid leader peptide. The other clone represents the 3' end of the mature message and includes a short poly(A)+ tract. This 3' sequence is remarkably similar to a reported cDNA encoding part of the protein derived from mouse fibroblast poly(A)+ RNA. However, this inhibitor has no substantial homology with previously sequenced protease inhibitors.     

cDNA cloning and expression of a metalloproteinase inhibitor related to tissue inhibitor of metalloproteinases.

The purification and characterization of a metalloproteinase inhibitor (MI) from bovine aortic endothelial cells, and the demonstration that it is related to, but distinct from, tissue inhibitor of metalloproteinase (TIMP), have previously been reported [De Clerck, Y. A., Yean, T.-D., Ratzkin, B. J., Lu, H.S. & Langley, K. E. (1989) J. Biol. Chem. 264, 17445-17453]. The cDNA cloning of the bovine MI and its human homolog is now reported. The bovine cDNA cloning used probes designed on the basis of NH2-terminal amino acid sequence of bovine MI. The human cDNA cloning in turn used probes representing parts of the bovine cDNA nucleotide sequence. Both cDNAs encode leader sequences of 26 amino acids and mature protein sequences of 194 amino acids. The amino acid sequences of the mature proteins are 94% identical. The human MI cDNA was expressed in Escherichia coli, and a preparation containing anticollagenase activity was recovered. The amino acid sequence of mature human MI is 38% identical to the sequence for human TIMP, and the 12 cysteines in MI and TIMP are aligned almost identically. Thus MI and TIMP comprise an inhibitor family.     

Isolation and characterization of a cDNA clone for the complete protein coding region of the delta subunit of the mouse acetylcholine receptor.

A mouse cDNA clone has been isolated that contains the complete coding region of a protein highly homologous to the delta subunit of the Torpedo acetylcholine receptor (AcChoR). The cDNA library was constructed in the vector lambda 10 from membrane-associated poly(A)+ RNA from BC3H-1 mouse cells. Surprisingly, the delta clone was selected by hybridization with cDNA encoding the gamma subunit of the Torpedo AcChoR. The nucleotide sequence of the mouse cDNA clone contains an open reading frame of 520 amino acids. This amino acid sequence exhibits 59% and 50% sequence homology to the Torpedo AcChoR delta and gamma subunits, respectively. However, the mouse nucleotide sequence has several stretches of high homology with the Torpedo gamma subunit cDNA, but not with delta. The mouse protein has the same general structural features as do the Torpedo subunits. It is encoded by a 3.3-kilobase mRNA. There is probably only one, but at most two, chromosomal genes coding for this or closely related sequences.     

Fte-1, a v-fos transformation effector gene, encodes the mammalian homologue of a yeast gene involved in protein import into mitochondria.

Revertants were isolated from v-fos-transformed rat-1 cells cotransfected with a human cDNA expression library and a selectable marker (pMEX-neo). Molecular analysis of one clone, R2.2, suggested that the revertant phenotype resulted from the disruption of a transformation effector gene by the integration of the pMEX-neo plasmid. Genomic sequences flanking the plasmid integration site were cloned and used as probes in Northern blot analyses. A probe derived from sequences 5' to the integration site hybridized to a unique 1.2-kilobase mRNA and was used to isolate a 0.9-kilobase cDNA clone (fte-1). The open reading frame of the fte-1 cDNA predicts a highly basic protein that shows a remarkable level of similarity with two genes from Saccharomyces cerevisiae. One of these yeast genes contains an unidentified open reading frame and the other, MFT1, is a gene isolated from a yeast mutant that fails to import a fusion protein into mitochondria [Garrett, J. M., Singh, K. K., Vonder Haar, R. A. & Emr, S. D. (1991) Mol. Gen. Genet. 225, 483-491]. Expression of the fte-1 gene was induced approximately 5-fold in v-fos-transformed fibroblasts, but expression was reduced in clone R2.2 and in several independent revertant clones. Transfection of R2.2 cells with fte-1 expression vectors resulted in the reacquisition of a transformed phenotype. These results demonstrate that the mammalian homologue of a gene implicated in protein import into yeast mitochondria is a v-fos transformation effector gene.     

Human beta-hexosaminidase alpha chain: coding sequence and homology with the beta chain.

We have isolated a cDNA clone, p beta H alpha-5, from an adult human liver library that contains the entire coding sequence of the alpha chain of beta-hexosaminidase. The cDNA insert of p beta H alpha-5 is 1944 base pairs long and contains a 168-base-pair 5' untranslated region, a 186-base-pair 3' untranslated region, and an open reading frame of 1587 base pairs corresponding to 529 amino acids (Mr, 60,697). The first 17-22 amino acids satisfy the requirements of a signal sequence. A striking sequence homology with a published partial amino acid sequence for the beta chain [O'Dowd, B. F., Quan, F., Willard, H. F., Lamhonwah, A. M., Korneluk, R. G., Lowden, J. A., Gravel, R. A. & Mahuran, D. J. (1985) Proc. Natl. Acad. Sci. USA 82, 1184-1188] suggests that both chains may have evolved from a common ancestor. A shorter alpha-chain cDNA was found to hybridize to the long arm of chromosome 15, the known location for the alpha-chain gene. In addition, we isolated another alpha-chain cDNA clone, p beta H alpha-4, from a simian virus 40-transformed human fibroblast library that contained an extra 453-base-pair piece at its 3' end. A probe consisting of this additional sequence hybridized exclusively to a single mRNA species (2.6 kilobases) in mRNA preparations from cultured human fibroblasts. In contrast, p beta H alpha-5 hybridized to both a 2.1-kilobase major and a 2.6-kilobase minor mRNA species in these same mRNA preparations, indicating the presence of two distinct alpha-chain mRNA species differing at the 3' end. Fibroblasts from an Ashkenazi Jewish patient with classic Tay-Sachs disease were deficient in both species of mRNA, confirming their genetic relationship.     

Cloning and in vitro expression of the human selenoprotein, type I iodothyronine deiodinase.

The type I 5' iodothyronine deiodinase (5' DI) catalyzes the deiodination of T4 to the biologically active hormone T3 and accounts for a significant fraction of its production. We have recently cloned the complementary DNA (cDNA) for the rat 5' DI, which contains the rare amino acid selenocysteine, and used this to screen human liver and kidney cDNA libraries to identify a human 5' DI cDNA clone. From these, we constructed a cDNA encoding a functional 5' DI. The 2222 base pair human 5' DI cDNA is approximately 200 nucleotides shorter than the 2.4-kilobase hybridizing band in Northern blots of human liver, kidney, and thyroid, because of missing 5' untranslated sequence and the poly A tail. The deduced amino acid sequence codes for a protein of 28.7 kilodaltons assuming the UGA codon at position 382 encodes selenocysteine, and is highly homologous (88% similarity) to the rat. We transiently expressed the 5' DI in COS-7 cells to establish that it encodes a functional enzyme and to study its kinetics. These show saturable deiodination of rT3 (Ka 0.52 +/- 0.04 mumol/L and Vmax 63.2 +/- 16.4 pmol min-1 mg-1). T4 and gold thioglucose are competitive inhibitors of rT3 deiodination. 6-n-Propylthiouracil (PTU) is an uncompetitive inhibitor (with rT3) and competitive inhibitor (with dithiothreitol) of rT3 deiodination. 6-n-Propylthiouracil inhibits T4 to T3 conversion. Labeling of COS-7 cells transiently transfected with the human 5' DI cDNA with bromoacetyl-125I-T3 demonstrates a 28-kilodalton protein. This indicates that in the human, as well as in the rat messenger RNA, the UGA encodes selenocysteine and translation terminates at the UAA codon at nucleotides 754 to 756. Reverse T3 and gold thioglucose (100 nmol/L) block bromoacetyl-125I-T3 labeling of the transiently expressed human and rat 5' DI proteins. These results demonstrate that the human 5' DI is a selenoprotein, analogous to the rat enzyme. Given the previously demonstrated critical role of the selenium atom in catalyzing deiodination by this protein, we conclude that this trace element is essential for normal thyroid hormone action in man.     

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.