Isolation and characterization of related cDNA clones encoding skeletal muscle beta-tropomyosin and a low-molecular-weight nonmuscle tropomyosin isoform.

We have isolated and characterized cDNA clones from chicken cDNA libraries derived from skeletal muscle, body wall, and cultured fibroblasts. A clone isolated from a skeletal muscle cDNA library contains the complete protein-coding sequence of the 284-amino-acid skeletal muscle beta-tropomyosin together with 72 bases of 5' untranslated sequence and nearly the entire 3' untranslated region (about 660 bases), lacking only the last 4 bases and the poly(A) tail. A second clone, isolated from the fibroblast cDNA library, contains the complete protein-coding sequence of a 248-amino-acid fibroblast tropomyosin together with 77 bases of 5' untranslated sequence and 235 bases of 3' untranslated sequence through the poly(A) tract. The derived amino acid sequence from this clone exhibits only 82% homology with rat fibroblast tropomyosin 4 and 80% homology with human fibroblast tropomyosin TM30nm, indicating that this clone encodes a third 248-amino-acid tropomyosin isoform class. The protein product of this mRNA is fibroblast tropomyosin 3b, one of two low-molecular-weight isoforms expressed in chicken fibroblast cultures. Comparing the sequences of the skeletal muscle and fibroblast cDNAs with a previously characterized clone which encodes the smooth muscle alpha-tropomyosin reveals two regions of absolute homology, suggesting that these three clones were derived from the same gene by alternative RNA splicing.     

Isolation and characterization of a cDNA that encodes mouse fibroblast tropomyosin isoform 2.

We isolated and characterized a cDNA clone encoding tropomyosin isoform 2 (TM2) from a mouse fibroblast cDNA library. TM2 was found to contain 284 amino acids and was closely related to the rat smooth and skeletal muscle alpha-TMs and the human fibroblast TM3. The amino acid sequence of TM2 showed a nearly complete match with that of human fibroblast TM3 except for the region from amino acids 189 to 213, the sequence of which was identical to those of rat smooth and skeletal muscle alpha-TMs. These results suggest that TM2 is expressed from the same gene that encodes the smooth muscle alpha-TM, the skeletal muscle alpha-TM, and TM3 via an alternative RNA-splicing mechanism. Comparison of the expression of TM2 mRNA in low-metastatic Lewis lung carcinoma P29 cells and high-metastatic D6 cells revealed that it was significantly less in D6 cells than in P29 cells, supporting our previous observations (K. Takenaga, Y. Nakamura, and S. Sakiyama, Mol. Cell. Biol. 8:3934-3937, 1988) at the protein level.     

Cloning and characterization of a cDNA encoding transformation-sensitive tropomyosin isoform 3 from tumorigenic human fibroblasts.

We isolated a cDNA clone from the tumorigenic human fibroblast cell line HuT-14 that contains the entire protein coding region of tropomyosin isoform 3 (Tm3) and 781 base pairs of 5'- and 3'-untranslated sequences. Tm3, despite its apparent smaller molecular weight than Tm1 in two-dimensional gels, has the same peptide length as Tm1 (284 amino acids) and shares 83% homology with Tm1. Tm3 cDNA hybridized to an abundant mRNA of 1.3 kilobases in fetal muscle and cardiac muscle, suggesting that Tm3 is related to an alpha fast-tropomyosin. The first 188 amino acids of Tm3 are identical to those of rat or rabbit skeletal muscle alpha-tropomyosin, and the last 71 amino acids differ from those of rat smooth muscle alpha-tropomyosin by only 1 residue. Tm3 therefore appears to be encoded by the same gene that encodes the fast skeletal muscle alpha-tropomyosin and the smooth muscle alpha-tropomyosin via an alternative RNA-splicing mechanism. In contrast to Tm4 and Tm5, Tm3 has a small gene family, with, at best, only one pseudogene.     

Isolation and characterization of a cDNA encoding the low molecular weight insulin-like growth factor binding protein (IBP-1).

IGF-I and IGF-II are growth-stimulating peptides with strong mitogenic properties. These polypeptide growth factors circulate in serum bound to specific binding proteins. We report the cloning and complete sequence of a cDNA encoding a low mol. wt IGF-binding protein from a human placenta cDNA library. We propose the designation IGF-binding protein 1 (IBP-1) for the gene and corresponding protein. Expression of the cDNA encoding IBP-1 in COS cells resulted in the synthesis of a 30-kd protein which binds IGF-I and is immunologically indistinguishable from the IGF-binding protein isolated from amniotic fluid or human serum. Northern blotting analysis demonstrated that expression of the IBP-1 gene is highly tissue specific and limited to placental membranes and fetal liver suggesting a rigid control. The IBP-1 gene is a single copy gene, located on chromosome 7. The results obtained suggest that most, if not all, lower mol. wt IGF-binding proteins originate from this gene.     

Nucleotide sequence of a cDNA clone encoding a Drosophila muscle tropomyosin II isoform

A cDNA clone was sequenced that contains the entire coding region for the muscle tropomyosin II isoform from Drosophila. The cDNA clone is 1253 nucleotides (nt) long and contains an 88-nt 5'-leader sequence and a 310-nt 3'-untranslated sequence. The muscle tropomyosin II isoform consists of 285 amino acids and is 60% homologous with the previously reported muscle tropomyosin I isoform Drosophila and 55% homologous with rabbit muscle tropomyosin.     

Isolation and characterization of cDNA clones encoding rat skeletal muscle peptidylarginine deiminase

Various mammalian tissues contain protein-arginine deiminases (EC 3.5.3.15), which convert the arginine residues in normal peptide bonds to the citrulline residues in calcium ion-dependent manners. Here, we describe the complete primary structure of rat skeletal muscle peptidylarginine deiminase deduced from the sequences of its cDNA clones isolated by recombinant DNA technology. We have isolated three overlapping cDNA clones which constitute a 4,507-base pair cDNA sequence including a 2,452-base pair 3'-untranslated region. The coding region consists of 1,995 base pairs encoding 665 amino acid residues. A potential N-linked glycosylation site is present at asparagine-534. The molecular weight of the enzyme calculated from the deduced amino acid sequence is 75,122. Direct repeat sequences resembling the rodent B2 type repetitive sequences appear in the 3'-untranslated region (nucleotides 3,090-3,198 and 3,270-3,391). Northern hybridization demonstrated the presence of its mRNA in poly(A)+ fractions of spinal cord, cerebrum, cerebellum, and submaxillary gland as well as skeletal muscle. The sizes of peptidylarginine deiminase mRNAs in these tissues were estimated to be 4.5-5.0 kilobases. No positive bands were detected on the blots of the corresponding RNA fractions of liver and kidney. Possible similarity of the amino acid sequence of peptidylarginine deiminase to those of other calcium binding proteins is discussed.     

Isolation and characterization of cDNA clones encoding the murine NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase

Forty cDNA clones corresponding to the bifunctional NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase enzyme were isolated from a mouse lambda gt11 library. Two classes of cDNA clones were shown by Northern analysis to correspond to the two mRNA species of 1.7 and 2.0 kilobases present in transformed cells but not in normal tissues and that apparently are derived from alternate polyadenylation signals. The 1050-base pair coding region encodes a protein of 350 amino acids which contains a putative mitochondrial-targeting signal peptide of 34 amino acids following the initiator methionine. The 20 amino acids immediately following the signal peptide correspond exactly to those determined by sequence analysis of the amino terminus of the purified protein. The derived amino acid sequence of the NAD-dependent dehydrogenase-cyclohydrolase shows extensive homology with the corresponding amino-terminal sequence of the trifunctional NADP-dependent dehydrogenase-cyclohydrolase-synthetase enzyme from human cells (approximately 40%), yeast cytosol (approximately 36%), and yeast mitochondria (approximately 45%).     

Isolation and characterization of two cDNA clones encoding ATP-sulfurylases from potato by complementation of a yeast mutant

Sulfur plays an important role in plants, being used for the biosynthesis of amino acids, sulfolipids and secondary metabolites. After uptake sulfate is activated and subsequently reduced to sulfide or serves as donor for sulfurylation reactions. The first step in the activation of sulfate in all cases studied so far is catalyzed by the enzyme ATP-sulfurylase (E.C. 2.7.7.4.) which catalyzes the formation of adenosine-5′-phosphosulfate (APS). Two cDNA clones from potato encoding ATP-sulfurylases were identified following transformation of a Saccharomyces cerevisiae mutant deficient in ATP-sulfurylase activity with a cDNA library from potato source leaf poly(A)⁺ RNA cloned in a yeast expression vector. Several transformants were able to grow on a medium with sulfate as the only sulfur source, this ability being strictly linked to the presence of two classes of cDNAs. The clones StMet3-1 and StMet3-2 were further analyzed. DNA analysis revealed an open reading frame encoding a protein with a molecular mass of 48 kDa in the case of StMet3-1 and 52 kDa for StMet3-2. The deduced polypeptides are 88% identical at the amino acid level. The clone StMet3-2 has a 48 amino acid N-terminal extension which shows common features of a chloroplast transit peptide. Sequence comparison of the ATP-sulfurylase Met3 from Saccharomyces cerevisiae with the cDNA StMet3-1 (StMet3-2) reveals 31% (30%) identity at the amino acid level. Protein extracts from the yeast mutant transformed with the clone StMet3-1 displayed ATP-sulfurylase activity. RNA blot analysis demonstrated the expression of both genes in potato leaves, root and stem, but not in tubers. To the best of the authors' knowledge this is the first cloning and identification of genes involved in the reductive sulfate assimilation pathway from higher plants.     

Isolation and characterization of a cDNA encoding a chick alpha-actinin

We have isolated and sequenced a 2.1-kilobase cDNA encoding 86% of the sequence of alpha-actinin. The cDNA clone was isolated from a chick embryo fibroblast cDNA library constructed in the expression vector lambda gt11. Identification of this sequence as alpha-actinin was confirmed by immunological methods and by comparing the deduced protein sequence with the sequence of several CNBr fragments obtained from adult chicken smooth muscle (gizzard) alpha-actinin. The deduced protein sequence shows two distinct domains, one of which consists of four repeats of approximately 120 amino acids. This region corresponds to a previously identified 50-kDa tryptic peptide involved in formation of the alpha-actinin dimer. The last 19 residues of C-terminal sequence display an homology with the so-called E-F hand of Ca2+-binding proteins. Hybridization analysis reveals only one size of mRNA (approximately 3.5 kilobases) in fibroblasts, but multiple bands in genomic cDNA.     

Isolation and characterization of a cDNA encoding rat cationic trypsinogen

A cDNA encoding rat cationic trypsinogen has been isolated by immunoscreening from a rat pancreas cDNA library. The protein encoded by this cDNA is highly basic and contains all of the structural features observed in trypsinogens. The amino acid sequence of rat cationic trypsinogen is 75% and 77% homologous to the two anionic rat trypsinogens. The homology of rat cationic trypsinogen to these anionic trypsinogens is lower than its homology to other mammalian cationic trypsinogens, suggesting that anionic and cationic trypsins probably diverged prior to the divergence of rodents and ungulates. The most unusual feature of this trypsinogen is the presence of an activation peptide containing five aspartic acid residues, in contrast to all other reported trypsinogen activation peptides which contain four acidic amino acid residues. Comparisons of cationic and anionic trypsins reveal that the majority of the charge changes occur in the C-terminal portion of the protein, which forms the substrate binding site. Several regions of conserved charge differences between cationic and anionic trypsins have been identified in this region, which may influence the rate of hydrolysis of protein substrates.     

Isolation, characterization and expression of cDNA clones encoding a mitochondrial malate translocator from Panicum miliaceum L.

Three cDNA clones that hybridize to a partial rice cDNA that show similarity to bovine mitochondrial 2-oxoglutarate/malate translocator were isolated from leaves of Panicum miliaceum L. (proso millet), an NAD-malic enzyme-type C₄ plant. The nucleotide sequences of the clones resemble each other, and some of the isolated cDNAs contained extra sequences that seemed to be introns. The predicted proteins encoded by the cDNAs have 302 amino acids and molecular weights of 32211 and 32150. The hydrophobic profile of the amino acid sequence predicted the existence of six transmembrane -helices that is a common property of members in the mitochondrial transporter family. The predicted amino acid sequence showed the highest similarity with that of the 2-oxoglutarate/malate translocator from mammalian mitochondria. An expression plasmid containing the coding region of the cDNAs was used to over-express recombinant protein with a C-terminal histidine tag Escherichia coli, which was affinity-purified. The antibody against the recombinant protein cross-reacted with proteins of 31–32 kDa in the membrane fraction from P. miliaceum mitochondria, but not with the chloroplast fraction. The recombinant protein reconstituted in liposomes efficiently transported malate, citrate, and 2-oxoglutarate.