cDNA cloning of porcine transforming growth factor-beta 1 mRNAs. Evidence for alternate splicing and polyadenylation

Most eukaryotic cells encode principally a 2.5-kilobase (kb) transforming growth factor (TGF)-beta 1 mRNA. However, we have found two major TGF-beta 1 RNA species, 3.5 and 2.5 kb long, in porcine tissues. The 3.5-kb species has a longer 3'-untranslated sequence generated by the selection of an alternate polyadenylation site. There is a 117-nucleotide sequence within this unique 3' region, which is similar to the PRE-1 repetitive sequence of unknown function, reported earlier in the porcine genome. We have also cloned and characterized an alternately spliced mRNA species specific for the TGF-beta 1 gene, in which exons IV and V of the corresponding human TGF-beta 1 gene are deleted. The nucleotide sequence of this cDNA clone predicts a putative precursor protein of 256 amino acids; the N-terminal 211 amino acids of this putative protein are identical to the TGF-beta 1 precursor protein (exons I, II, and III of the human TGF-beta 1 gene), but the C-terminal 45 amino acids are distinct, due to a frameshift in the translation of exons VI and VII. In addition we provide data for the existence of other mRNA species generated in a tissue-specific manner either by alternate splicing or by heterogeneous 5' leader sequences.     

Nucleotide sequence of cloned complementary deoxyribonucleic acid for the alpha subunit of bovine pituitary glycoprotein hormones

Recombinant DNA plasmids containing sequences coding for the alpha subunit of the bovine pituitary glycoprotein hormones have been isolated. The nucleotide sequences of three different cDNA clones have been determined. The largest alpha-subunit cDNA clone was found to contain 713 bases including 77 nucleotides from the 5'-untranslated region, 72 nucleotides coding for a precursor segment, 288 nucleotides coding for the mature alpha subunit, and 276 nucleotides from the 3'-untranslated region of the mRNA followed by a poly(A) segment. This cDNA likely represents most of the bovine alpha-subunit mRNA sequence. Nucleotide sequences were obtained from the cDNA inserts of two other alpha-subunit clones, and several differences among the three cDNA sequences have been detected. These differences in nucleotide sequence may represent either individual variation in genomic sequence or cloning artifacts. Comparison of the bovine alpha-subunit cDNA sequence to the sequences of human, rat, and mouse alpha-subunit cDNAs reveals that the bovine sequence has greater than 70% homology with the other cDNAs. The cloned alpha-subunit cDNA should provide a useful probe for further studies of the structure and expression of this interesting gene.     

Identification of a novel transforming growth factor-beta (TGF-beta 5) mRNA in Xenopus laevis

A novel transforming growth factor-beta (TGF-beta) mRNA of about 3.0 kilobases, which encodes a putative protein of 382 amino acids, has been identified in amphibians by cDNA cloning. This mRNA, which we designate as TGF-beta 5, is developmentally regulated and highly expressed beginning at early neurula (stage 14) and in many adult tissues in Xenopus laevis. Following the first methionine, the putative precursor protein has a hydrophobic region, approximately 22 amino acids long, which probably represents a signal sequence, similar to that found in TGF-beta s 1-3. The precursor also has potential sites for glycosylation, integrin binding (RGD), and a tetrabasic amino acid (RKKR) site for potential cleavage of the precursor peptide to a biologically active protein. The putative mature protein consists of 112 amino acids with 9 cysteines and has 76, 66, 69, and 72% identity to TGF-beta s 1-4, respectively.     

Isolation and sequence analysis of the human corticotropin-releasing factor precursor gene.

A human genomic DNA segment containing the gene for the corticotropin-releasing factor precursor has been isolated by screening a gene library with an ovine cDNA probe. The cloned DNA segment has been subjected to restriction endonuclease mapping and nucleotide sequence analysis. Comparison of the nucleotide sequence of the gene with that of the ovine cDNA indicates that an intron of 800 bp is inserted in the segment encoding the 5'-untranslated region of the mRNA. The segment corresponding to the protein-coding and the 3'-untranslated region of the mRNA is uninterrupted. The mRNA and amino acid sequences of the human corticotropin-releasing factor precursor have been deduced from the corresponding gene sequence. The deduced amino acid sequence of human corticotropin-releasing factor exhibits seven amino acid substitutions in comparison with the ovine counterpart.     

Mitochondrial NADH-ubiquinone reductase: complementary DNA sequences of import precursors of the bovine and human 24-kDa subunit

The 24-kDa subunit of mitochondrial NADH-ubiquinone reductase (complex I) is an iron-sulfur protein that is present in the flavoprotein or NADH dehydrogenase II subcomplex. It is a nuclear gene product and is imported into the organelle. A group of human patients with mitochondrial myopathy have been shown to have reduced levels of subunits of complex I in skeletal muscle mitochondria, and in one patient the 24-kDa subunit appears to be absent (Schapira et al., 1988). To investigate the genetic basis of this type of myopathy, cDNA clones have been isolated from a bovine library derived from heart and liver mRNA by hybridization with two mixtures of 48 synthetic oligonucleotides 17 bases in length that were designed on the basis of known protein sequences. The recombinant DNA sequence has been determined, and it encodes a precursor of the mature 24-kDa protein. The N terminus of the mature protein is preceded by a presequence of 32 amino acids that has properties that are characteristic of mitochondrial import sequences. The sequence of the mature protein deduced from the cDNA contains a segment of nine amino acids that was not determined in an earlier partial protein sequence analysis. The bovine clone has been employed as a hybridization probe to identify cDNA clones of the human homologue of the 24-kDa protein. Its DNA sequence has also been determined, and it codes for a protein that is closely related to the bovine protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

The murine transforming growth factor-beta precursor

Transforming growth factor-beta (TGF-beta) is a homodimeric polypeptide which can act, often in cooperation with other growth factors, as a mitogenic factor for a variety of cells. TGF-beta can also exert growth inhibitory activity on many other cell lines. We have isolated cDNAs coding for the murine TGF-beta cDNA precursor. The deduced amino acid sequence localizes the 112-amino acid long TGF-beta monomer to the C terminus of the precursor. Two areas of the precursor exhibit a marked degree of homology to the human counterpart. One of these regions comprises the mature TGF-beta monomer, while the other corresponds to the NH2 terminus of the precursor and suggests an important biological function for this area. Northern hybridization results identify a major 2.5-kilobase TGF-beta mRNA and several minor TGF-beta mRNA species.     

Murine transforming growth factor-beta 2 cDNA sequence and expression in adult tissues and embryos

Murine transforming growth factor-beta 2 (TGF-beta 2) cDNAs were isolated from cDNA libraries derived from a differentiated murine embryonic carcinoma cell line, PCC3. The composite cDNA sequence is 4267 nucleotides long, including a 1217 nucleotides 5'-untranslated sequence, and encodes a murine TGF-beta 2 precursor of 414 amino acids with 96% identity to its human counterpart. Several consensus polyadenylation sequences are present in the 1807 nucleotides 3'-untranslated sequence. Five TGF-beta 2 mRNA species are observed in the developing mouse fetus and they show different patterns of expression during development. TGF-beta 2 mRNA expression was also examined in adult mouse tissues, in which four of the five RNA species were observed. TGF-beta 2 mRNAs were present in all adult mouse tissues examined, except liver, and was most abundant in placenta, the male submaxillary gland and lung. The patterns of expression suggest a physiological role for TGF-beta 2 both in embryonic development and in the maintenance of adult tissues.     

Human F1-ATPase: molecular cloning of cDNA for the beta subunit

F1-ATPase is the major enzyme for ATP synthesis, and its beta subunit is the catalytic site. To date, no full-length cDNA for the eukaryotic F1 gene has been reported. Human F1 was studied because of its importance in medicine and cell biology. Here we report molecular cloning of a full-length cDNA for the human F1 beta subunit and purification of the human F1 beta subunit. The HeLa cell cDNA library constructed in an expression vector gamma gt11 was screened with antiserum against the yeast F1 beta subunit. One of the positive phage DNAs containing the human F1 beta gene and its flanking regions (1.8 kilobase pairs) was sequenced by the dideoxy chain termination method. The open reading frame started from a putative signal presequence, which was rich in both serine and arginine. There was a homologous segment in the signal presequence of human ornithine transcarbamoylase and that of F1 beta. The precursor of F1 beta was expressed in E. coli harboring a plasmid which had been constructed with T5 promotor and the F1 beta cDNA. Both the precursor and mature form of F1 beta were detected in HeLa cells in a pulse-chase experiment. The amino acid sequence of 480 residues (51,568.3 daltons) following the presequence was highly homologous with that of mature beef heart F1 beta (97.5%) and E. coli F1 beta (71.7%), but the codon usage in the human gene was very different from those of reported genes coding for F1 beta of other species.     

A role of the latent TGF-beta 1-binding protein in the assembly and secretion of TGF-beta 1.

Transforming growth factor-beta 1 (TGF-beta 1) is synthesized as latent complexes with high molecular weights. The large latent complex of TGF-beta 1 in platelets is composed of three components, i.e. the mature TGF-beta 1, which is non-covalently associated with a disulphide-bonded complex of the N-terminal remnant of the TGF-beta 1 precursor (TGF-beta 1-latency associated peptide) and the latent TGF-beta 1 binding protein (LTBP). The TGF-beta 1-latency associated peptide is sufficient for the latency of TGF-beta 1, whereas the functions of LTBP remain to be elucidated. In a human erythroleukemia cell line, HEL, the production of the latent form of TGF-beta 1 was induced more than 100-fold by phorbol 12-myristate 13-acetate. Analysis by Northern blotting revealed that both the TGF-beta 1 precursor and LTBP were induced in a coordinated fashion. Analysis by immunoprecipitation using antibodies against LTBP and the TGF-beta 1 precursor dimer revealed that LTBP has a molecular size of 205 kd under reducing conditions in this cell type, i.e. similar to that from cells transfected with cDNA for LTBP, but larger than the platelet form (125-160 kd). Limited tryptic digestion of LTBP in HEL cells and analysis by SDS-PAGE showed protein bands of similar sizes to those of platelet LTBP, suggesting that the difference in molecular sizes of LTBP involves cell-specific processing. The biosynthesis of the latent TGF-beta 1 was studied by pulse-chase analysis. LTBP became covalently associated with the TGF-beta 1 precursor within 15 min after synthesis in this cell line. Secretion of the large latent TGF-beta 1 complex was observed as early as 30 min after the synthesis of LTBP; at the same time, a free form of LTBP not bound to the TGF-beta 1 precursor was seen. In contrast, the TGF-beta 1 precursor remained inside the cells in an unprocessed form for a longer time period and the TGF-beta 1 precursor dimer without LTBP was secreted only very slowly. Furthermore, the results of partial tryptic digestion of this molecule suggested that it contained improper disulphide bonding. These results suggest that LTBP plays a critical role in the assembly and secretion of the latent TGF-beta 1.     

The delta-subunit of ATP synthase from bovine heart mitochondria. Complementary DNA sequence of its import precursor cloned with the aid of the polymerase chain reaction.

The delta-subunit of ATP synthase from bovine heart mitochondria is part of the extrinsic membrane domain, F1-ATPase. The mature protein is 146 amino acids in length and its function is obscure. It is encoded by a nuclear gene and is imported into the organelle. Two mixtures of oligonucleotides 17 bases long, designed on the basis of the known protein sequence, have been synthesized and employed as primers on bovine cDNA in the polymerase chain reaction. By this means a segment of bovine cDNA encoding part of the delta-subunit has been amplified, and this DNA segment has been employed to identify related cDNA clones in a library. These clones encode the mitochondrial import precursor of the delta-subunit; the protein sequence of the mature protein deduced from it is exactly the same as that determined earlier by direct sequence analysis. The clones have also been used to show that both the bovine and human genomes seem to contain a single gene for the delta-subunit.     

Molecular events in the processing of recombinant type 1 pre-pro-transforming growth factor beta to the mature polypeptide.

Recently, the simian type 1 transforming growth factor beta (TGF-beta 1) cDNA was expressed at high levels in Chinese hamster ovary (CHO) cells by dihydrofolate reductase-induced gene amplification (L.E. Gentry, N.R. Webb, G.J. Lim, A.M. Brunner, J.E. Ranchalis, D.R. Twardzik, M.N. Lioubin, H. Marquardt, and A.F. Purchio, Mol. Cell. Biol. 7:3418-3427, 1987). We have now purified and characterized the recombinant proteins released by these cells. Analyses of the precursor proteins by amino acid sequencing identified potentially important proteolytic processing sites. Signal peptide cleavage occurs at the Gly-29-Leu-30 peptide bond of pre-pro-TGF-beta 1, yielding pro-TGF-beta 1 (30 to 390). In addition, proteolytic processing of the precursor to yield mature TGF-beta 1 occurs at the dibasic cleavage site immediately preceding Ala-279, indicating that CHO cells possess the appropriate processing enzyme. Greater than 95% of the biological activity detected in the conditioned medium of the CHO transfectant was due to mature, properly processed growth factor. Highly purified recombinant TGF-beta 1 had the same specific biological activity as natural TGF-beta 1. The concentration of TGF-beta 1 required for half-maximal inhibition of Mv1Lu mink lung epithelial cell growth was approximately 1 to 2 pM. Purified precursor inhibited mink lung cell proliferation at 50 to 60 pM concentrations. The purified precursor preparation was shown to consist of pro-TGF-beta 1 (30 to 390), the pro region of the precursor (30 to 278), and mature TGF-beta 1 (279 to 390) interlinked by at least one disulfide bond with the pro portion of the precursor. These recombinant forms of TGF-beta1 should prove useful for further structural and functional studies.     

Activation of latent TGF-beta by thrombospondin-1: mechanisms and physiology

Regulation of the activation of latent TGF-beta is essential for health as too much or too little TGF-beta activity can have serious, deleterious consequences. The processes that control conversion of the precursor to the biologically active form of TGF-beta in vivo are not well characterized. We have identified a mechanism for the activation of latent TGF-beta that involves binding of the secreted and extracellular matrix protein, thrombospondin-1 (TSP-1), to the latent precursor. Specific sequences in TSP-1 and in the precursor portion (the latency associate peptide-LAP) have been determined to be essential for activation of latent TGF-beta by TSP-1. It is thought that binding of TSP-1 to the latent complex induces a conformational rearrangement of the LAP in such a manner as to prevent the LAP from conferring latency on the mature domain of TGF-beta. A TSP-dependent mechanism of activation may be locally important during wound healing and in post-natal development of epithelial structures. The possible involvement of TSP-1 in TGF-beta activation during several disease processes is also discussed.     

The construction and partial characterization of plasmids containing complementary DNA sequences to human calcitonin precursor polyprotein.

(1) Total poly(A)-containing RNA isolated from human thyroid medullary carcinoma tissue was shown to direct the synthesis in the wheat germ cell-free system of a major (Mr 21000) and several minor forms of human calcitonin precursor polyproteins. Evidence for processing of these precursor(s) by the wheat germ cell-free system is also presented. (2) A small complementary DNA (cDNA) plasmid library has been constructed in the PstI site of the plasmid pAT153, using total human thyroid medullary carcinoma poly(A)-containing RNA as the starting material. (3) Plasmids containing abundant cDNA sequences were selected by hybridization in situ, and two of these (ph T-B3 and phT-B6) were characterized by hybridization--translation and restriction analysis. Each was shown to contain human calcitonin precursor polyprotein cDNA sequences. (4) RNA blotting techniques demonstrate that the human calcitonin precursor polyprotein is encoded within a mRNA containing 1000 bases. (5) The results demonstrate that human calcitonin is synthesized as a precursor polyprotein.     

Cloning, sequence, and expression of bovine interferon-gamma

Bovine interferon-gamma (IFN-gamma) sequences have been isolated by screening a cDNA library with a human IFN-gamma cDNA probe. The cDNA library was constructed from RNA isolated from concanavalin A-stimulated bovine lymph node cells. The open reading frame predicts that the bovine IFN-gamma precursor is composed of 166 amino acids with a predicted m.w. of 19,393. Alignment of the amino acid sequence with human IFN-gamma indicates that mature bovine IFN-gamma is composed of 143 amino acids with a predicted m.w. of 16,858. It has an amino acid homology of 63% with human IFN-gamma, and 47% with murine IFN-gamma. Biologically active bovine IFN-gamma was synthesized in an Escherichia coli expression system.     

Characterization of latent recombinant TGF-beta 2 produced by Chinese hamster ovary cells.

Latent recombinant transforming growth factor-beta 2 (LrTGF-beta 2) complex has been purified from serum-free media conditioned by Chinese hamster ovary cells transfected with a plasmid encoding the TGF-beta 2 (414) precursor. Under neutral conditions, LrTGF-beta 2 had an apparent molecular weight of 130 kDa. The complex contained both mature and pro-region sequences. Acidification of LrTGF-beta 2 resulted in the release of mature 24 kDa TGF-beta 2 from the high molecular weight pro-region-containing complex, suggesting that TGF-beta 2 was non-covalently associated with this complex. These results were confirmed by crosslinking experiments performed on partially purified LrTGF-beta 2. Protein sequence analysis of the purified TGF-beta 2 pro-region indicated that signal peptide cleavage occurred between ser(20) and leu(21). The pro-region, which previously was found to contain mannose-6-phosphate, bound to the mannose-6-phosphate receptor. Proteolytic cleavage of mature TGF-beta 2 from pro-TGF-beta 2 was inhibited by monensin and chloroquine suggesting that binding to this receptor and subsequent transport to acidic vesicles may be involved in the processing of rTGF-beta 2 precursor.