Rat embryonic fibroblast tropomyosin 1. cDNA and complete primary amino acid sequence

A cDNA expression library of approximately 80,000 members was prepared from rat embryonic fibroblast mRNA using the plasmid expression vectors pUC8 and pUC9. Using an immunological screening procedure and 32P-labeled cDNA probes, clones encoding rat embryonic fibroblast tropomyosin 1 (TM-1) were identified and isolated. DNA sequence analysis was carried out to determine the amino acid sequence of the protein. Rat embryonic fibroblast TM-1 was found to contain 284 amino acids and is most homologous to smooth muscle alpha-tropomyosin compared with skeletal muscle alpha- and beta-tropomyosins and platelet beta-tropomyosin. Among the various tropomyosins, two regions where the greatest sequence divergence is evident are between amino acids 185 and 216 and amino acids 258 and 284. Rat embryonic fibroblast TM-1 and chicken smooth muscle alpha-tropomyosin are most closely related from amino acids 185 and 216 compared with skeletal muscle and platelet tropomyosins. In contrast, rat embryonic fibroblast TM-1, smooth muscle alpha-tropomyosin, and platelet tropomyosin are most homologous from amino acids 258 and 284 compared with skeletal muscle tropomyosins. These differences in sequences at the carboxyl-terminal region of the various tropomyosins are discussed in relation to differences in their binding to skeletal muscle troponin and its T1 fragment.     

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 cDNA clones encoding a low molecular weight nonmuscle tropomyosin isoform

cDNA clones encoding rat fibroblast tropomyosin 4 (TM-4) were isolated and characterized. DNA sequence analysis was carried out to determine the sequence of the protein. The derived amino acid sequence revealed that rat fibroblast TM-4 was found to contain 248 amino acids. The amino acid sequence of rat fibroblast TM-4 was compared with two other low molecular weight TM isoforms, equine platelet beta-TM and a human fibroblast TM. Rat TM-4 exhibited 98% sequence identity with the equine platelet TM but only 75% identity with the human fibroblast TM isoform. The high degree of conservation between the rat and equine proteins indicates that they belong to the same isotype of TM. Comparison of the amino acid sequences of the three low molecular TM isoforms along the length of the proteins reveals regions that are strongly conserved and regions that have considerably diverged. In the regions from amino acid residues 1 to 148 and 176 to 221, amino acid substitutes are moderate. The most variant regions in the sequence are in the middle part of the proteins from amino acids 149 to 175 and at the carboxyl-terminal region of the proteins from amino acids 222 to 248. The differences in the sequence of the rat and platelet TMs compared to the human TM may define distinct functional domains among the low molecular weight TMs. In addition, expression of tropomyosin was studied in a variety of tissues and transformed cells. We also demonstrate that at least three separate genes encode tropomyosins expressed in rat fibroblasts.     

Isolation, characterization, and chromosome mapping of a human A-C1 Ha-Ras suppressor gene (HRASLS)

Recently, we cloned a cDNA encoding a novel mouse protein, named A-C1, by differential display between two mouse cell lines, embryonic fibroblast C3H10T1/2 and chondrogenic ATDC5. Mouse A-C1 has homology with a ras-responsive gene, rat Ha-rev107 (Hrasls), and modulates a Ha-ras-mediated signaling pathway. Here, we report a cDNA encoding a human homolog of mouse A-C1. The deduced amino acid sequence of human A-C1 consists of 168 amino acids, and shows 83% identity with that of mouse A-C1. Human A-C1 mRNA was expressed in skeletal muscle, testis, heart, brain, and thyroid in vivo. Moreover, expression of human A-C1 mRNA was detected at a high level in human osteosarcoma-derived U2OS cells in vitro. By FISH analysis the human A-C1 gene (HRASLS) was mapped to human chromosome 3q28--> q29.     

The rat alpha-tropomyosin gene generates a minimum of six different mRNAs coding for striated, smooth, and nonmuscle isoforms by alternative splicing.

Tropomyosin (TM), a ubiquitous protein, is a component of the contractile apparatus of all cells. In nonmuscle cells, it is found in stress fibers, while in sarcomeric and nonsarcomeric muscle, it is a component of the thin filament. Several different TM isoforms specific for nonmuscle cells and different types of muscle cell have been described. As for other contractile proteins, it was assumed that smooth, striated, and nonmuscle isoforms were each encoded by different sets of genes. Through the use of S1 nuclease mapping, RNA blots, and 5' extension analyses, we showed that the rat alpha-TM gene, whose expression was until now considered to be restricted to muscle cells, generates many different tissue-specific isoforms. The promoter of the gene appears to be very similar to other housekeeping promoters in both its pattern of utilization, being active in most cell types, and its lack of any canonical sequence elements. The rat alpha-TM gene is split into at least 13 exons, 7 of which are alternatively spliced in a tissue-specific manner. This gene arrangement, which also includes two different 3' ends, generates a minimum of six different mRNAs each with the capacity to code for a different protein. These distinct TM isoforms are expressed specifically in nonmuscle and smooth and striated (cardiac and skeletal) muscle cells. The tissue-specific expression and developmental regulation of these isoforms is, therefore, produced by alternative mRNA processing. Moreover, structural and sequence comparisons among TM genes from different phyla suggest that alternative splicing is evolutionarily a very old event that played an important role in gene evolution and might have appeared concomitantly with or even before constitutive splicing.     

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.     

Nonmuscle and smooth muscle myosin light chain mRNAs are generated from a single gene by the tissue-specific alternative RNA splicing

We have isolated two cDNA clones for myosin alkali light chain (MLC) mRNA from two respective cDNA libraries of chick gizzard and fibroblast cells by cross-hybridization to the previously isolated cDNA of skeletal muscle MLC. Sequence analysis of the two cloned cDNAs revealed that both of them are homologous to but distinct from the cDNA sequence used as the probe so that they may be classified into members of the MLC family, that they are identical with each other in the 3' and 5' untranslated sequence as well as in the coding sequence with a notable exception of a 39-nucleotide insertion in the fibroblast cDNA, 26 nucleotides of which are used for encoding the C-terminal amino acid sequence, and, therefore, that they encode the identical 142-amino acid sequence with different C-terminals of nine amino acids, each specific for fibroblast and gizzard smooth muscle MLC. The position of the inserted block corresponds exactly to one of the exon-intron junctions in the other MLC genes whose structures have so far been elucidated. DNA blot analysis suggested that the two MLC mRNAs of gizzard (smooth muscle) and fibroblast cells (nonmuscle) are generated from a single gene, probably through alternative RNA splicing mechanisms. RNA blot analysis and S1 nuclease mapping analysis using RNA preparations from fibroblast and gizzard tissues showed that the fibroblast MLC mRNA is expressed predominantly in fibroblast cells, but not, or very scantily if at all, in the gizzard, whereas the reverse is true for the gizzard smooth muscle MLC mRNA.     

The sequence of chick alpha-actinin reveals homologies to spectrin and calmodulin

We have sequenced a cDNA, isolated from a chick embryo fibroblast lambda gt11 library, that encodes all 887 amino acids of alpha-actinin. Sequence from 10 different peptides from chick smooth muscle alpha-actinin was found to match that derived from the cDNA. The deduced protein sequence can be divided into three distinct domains: (a) the N-terminal 240 amino acid contains a highly conserved region (compared with Dictyostelium alpha-actinin) which probably represents the actin-binding domain, (b) amino acids 270-740 contain four repeats of a spectrin-like sequence, and (c) the C-terminal sequence contains two EF-hand Ca2+-binding sites. Each of these sites is defective in at least one oxygen-containing Ca2+-chelating amino acid side chain, suggesting that they are nonfunctional. Southern blots suggest that the alpha-actinin cDNA described here hybridizes to only one gene in chicken. Northern blots reveal only one size class of mRNA in fibroblasts and smooth muscle, but no hybridizing species could be detected in skeletal muscle poly(A+) RNA. The results are consistent with the view that smooth and skeletal muscle alpha-actinins are encoded by separate genes, which are considerably divergent.     

Sequence of rat skeletal muscle phosphoglycerate mutase cDNA

A cDNA clone coding rat skeletal muscle phosphoglycerate mutase was isolated from a rat muscle lambda gt10 cDNA library and its sequence was determined. The deduced protein possesses 252 amino acids and is 94% homologous with respect to human muscle phosphoglycerate mutase. No amino acids changes occur at the active site and structural predictions suggest strong conformational homologies with other enzymes of the mutase family.     

Molecular cloning of cDNA encoding a 55-kDa multifunctional thyroid hormone binding protein of skeletal muscle sarcoplasmic reticulum.

A cDNA clone encoding 55-kDa multifunctional, thyroid hormone binding protein of rabbit skeletal muscle sarcoplasmic reticulum was isolated and sequenced. The cDNA encoded a protein of 509 amino acids, and a comparison of the deduced amino acid sequence with the NH2-terminal amino acid sequence of the purified protein indicates that an 18-residue NH2-terminal signal sequence was removed during synthesis. The deduced amino acid sequence of the rabbit muscle clone suggested that this protein is related to human liver thyroid hormone binding protein, rat liver protein disulfide isomerase, human hepatoma beta-subunit of prolyl 4-hydroxylase and hen oviduct glycosylation site binding protein. The protein contains two repeated sequences Trp-Cys-Gly-His-Cys-Lys proposed to be in the active sites of protein disulfide isomerase. Northern blot analysis showed that the mRNA encoding rabbit skeletal muscle form of the protein is present in liver, kidney, brain, fast- and slow-twitch skeletal muscle, and in the myocardium. In all tissues the cDNA reacts with mRNA of 2.7 kilobases in length. The 55-kDa multifunctional thyroid hormone binding protein was identified in isolated sarcoplasmic reticulum vesicles using a monoclonal antibody specific to the 55-kDa thyroid hormone binding protein from rat liver endoplasmic reticulum. The mature protein of Mr 56,681 contains 95 acidic and 61 basic amino acids. The COOH-terminal amino acid sequence of the protein is highly enriched in acidic residues with 17 of the last 29 amino acids being negatively charged. Analysis of hydropathy of the mature protein suggests that there are no potential transmembrane segments. The COOH-terminal sequence of the protein, Arg-Asp-Glu-Leu (RDEL), is similar to but different from that proposed to be an endoplasmic reticulum retention signal; Lys-Asp-Glu-Leu (KDEL) (Munro, S., and Pelham, H.R.B. (1987) Cell 48, 899-907). This variant of the retention signal may function in a similar manner to the KDEL sequence, to localize the protein to the sarcoplasmic or endoplasmic reticulum. The positively charged amino acids Lys and Arg may thus interchange in this retention signal.     

Cloning and pharmacological characterization of a human bradykinin (BK-2) receptor.

A human BK-2 bradykinin receptor was cloned from the lung fibroblast cell line CCD-16Lu. The cDNA clone encodes a 364 amino acid protein that has the characteristics of a seven transmembrane domain G-protein coupled receptor. The predicted amino acid sequence of the human BK-2 receptor is 81% identical to the smooth muscle rat BK-2 receptor (1). Transfection of the human BK-2 receptor cDNA into COS-7 cells results in the expression of high levels of specific BK binding sites. Saturation binding analysis indicates that the human BK-2 receptor expressed in COS-7 cells binds BK with a KD of 0.13 nM. Pharmacological characterization of the expressed BK receptor is consistent with the cDNA encoding a receptor of the BK-2 subtype. The BK-2 receptor antagonist Hoe 140 (2), D-Arg0[Hyp3, Thi5, D-Tic7, Oic8]BK has a high affinity (IC50 = 65 pM) for the cloned human receptor. The tissue distribution of the human BK-2 receptor was analyzed by competitive PCR with human tissue cDNA and is similar to that determined for the BK-2 receptor in the rat.     

Nonmuscle and muscle tropomyosin isoforms are expressed from a single gene by alternative RNA splicing and polyadenylation.

The molecular basis for the expression of rat embryonic fibroblast tropomyosin 1 and skeletal muscle beta-tropomyosin was determined. cDNA clones encoding these tropomyosin isoforms exhibit complete identity except for two carboxy-proximal regions (amino acids 189 to 213 and 258 to 284) and different 3'-untranslated sequences. The isoform-specific regions delineate the troponin T-binding domains of skeletal muscle tropomyosin. Analysis of genomic clones indicates that there are two separate loci in the rat genome that contain sequences complementary to these mRNAs. One locus is a pseudogene. The other locus contains a single gene made up of 11 exons and spans approximately 10 kilobases. Sequences common to all mRNAs were found in exons 1 through 5 (amino acids 1 to 188) and exons 8 and 9 (amino acids 214 to 257). Exons 6 and 11 are specific for fibroblast mRNA (amino acids 189 to 213 and 258 to 284, respectively), while exons 7 and 10 are specific for skeletal muscle mRNA (amino acids 189 to 213 and 258 to 284, respectively). In addition, exons 10 and 11 each contain the entire 3'-untranslated sequences of the respective mRNAs including the polyadenylation site. Although the gene is also expressed in smooth muscle (stomach, uterus, and vas deferens), only the fibroblast-type splice products can be detected in these tissues. S1 and primer extension analyses indicate that all mRNAs expressed from this gene are transcribed from a single promoter. The promoter was found to contain G-C-rich sequences, a TATA-like sequence TTTTA, no identifiable CCAAT box, and two putative Sp1-binding sites.     

The human P2X4 receptor gene is alternatively spliced

ATP acts as a fast excitatory neurotransmitter by binding to a large family of membrane proteins, P2X receptors, that have been shown to be ligand-gated, non-selective cation channels. We report the cloning of a full-length and alternatively spliced form of the human P2X₄ gene. Clones were identified from a human stomach cDNA library using a rat P2X₄ probe. Nucleotide sequence analysis of positive clones identified the full-length human P2X₄ cDNA, which codes for a 388-residue protein that is highly homologous (82%) to the rat gene, and an alternatively spliced cDNA. In the alternatively spliced cDNA, the 5′-untranslated region and the first 90 amino acids in the coding region of full-length human P2X₄ are replaced by a 35 amino acid coding sequence that is highly homologous with a region of chaparonin proteins in the hsp-90 family. The open reading frames of the full-length and splice variant clones were confirmed by in vitro translation. Northern analysis indicated expression of the full-length P2X₄ message in numerous human tissues including smooth muscle, heart, and skeletal muscles. Alternatively spliced RNAs were identified in smooth muscle and brain by RT–PCR and confirmed by RNAse protection assays using a 710 bp anti-sense RNA probe that spanned the alternatively spliced and native P2X₄ regions. Injection of full-length, but not alternatively spliced, cRNA into Xenopus oocytes resulted in the expression of ATP gated non-selective cation currents.     

Chicken cardiac tropomyosin and a low-molecular-weight nonmuscle tropomyosin are related by alternative splicing

We have isolated and characterized complementary DNAs (cDNAs) encoding chicken cardiac muscle tropomyosin and a low-molecular-weight nonmuscle tropomyosin. The cardiac muscle cDNA (pCHT-4) encodes a 284-amino acid protein that differs from chicken skeletal muscle alpha- and beta-tropomyosins throughout its length. The nonmuscle cDNA (pFT-C) encodes a 248-amino acid protein that is most similar (93-94%) to the tropomyosin class including rat fibroblast TM-4, equine platelet tropomyosin, and human fibroblast TM30pl. The nucleotide sequences of the cardiac and nonmuscle cDNAs are identical from the position encoding cardiac amino acid 81 (nonmuscle amino acid 45) through cardiac amino acid 257 (nonmuscle amino acid 221). The sequences differ both 5' and 3' of this region of identity. These comparisons suggest that the chicken cardiac tropomyosin and low-molecular-weight "platelet-like" tropomyosin are derived from the same genomic locus by alternative splicing. S1 analysis suggests that this locus encodes at least one other tropomyosin isoform.     

Molecular genetic characterization of a developmentally regulated human perinatal myosin heavy chain

We have isolated a human cDNA which corresponds to a developmentally regulated sarcomeric myosin heavy chain. RNA hybridization and DNA sequence analysis indicate that this cDNA, called SMHCP, encodes a perinatal myosin heavy chain isoform. The nucleotide and deduced amino acid sequences of the 3.4-kb cDNA insert show strong homology with other sarcomeric myosin heavy chains. The strongest homology is to a previously described 970-bp cDNA encoding a rat perinatal isoform (Periasamy, M., D. F. Wieczorek, and B. Nadal-Ginard. 1984. J. Biol. Chem. 259:13573-13578). The homology between the analogous human and rat perinatal myosin heavy chain cDNAs is maintained through the highly isoform-specific final 20 carboxyl-terminal amino acids, as well as the 3' untranslated region. Ribonuclease protection studies show that the mRNA encoding this isoform is expressed at high levels in 21-wk fetal skeletal tissue and not in fetal cardiac muscle. In contrast to the rat perinatal isoform, which was not found to be expressed in adult hind-leg tissue, the gene encoding SMHCP continues to be expressed in adult human skeletal tissue, but at lower levels relative to fetal skeletal tissue.     

Cloning of a differentially expressed tropomyosin isoform from cultured rabbit aortic smooth muscle cells

The four known tropomyosin genes have highly conserved DNA and amino acid sequences, and at least 18 isoforms are generated by alternative RNA splicing in muscle and non-muscle cells. No rabbit tropomyosin nucleotide sequences are known, although protein sequences for alpha- and beta-tropomyosin expressed by rabbit skeletal muscle have been described. Subtractive hybridisation was used to select for genes differentially expressed in rabbit aortic smooth muscle cells (SMC), during the change in cell phenotype in primary culture that is characterised by a loss of cytoskeletal filaments and contractile proteins. This led to the cloning of a tropomyosin gene predominantly expressed in rabbit SMC during this change. The full-length cDNA clone, designated "rabbit TM-beta", contains an open reading frame of 284 amino acids, 5' untranslated region (UTR) of 117 base pairs and 3' UTR of 79 base pairs. It is closely related to the beta-gene isoforms in other species, with the highest homology in DNA and protein sequences to the human fibroblast isoform TM-1 (91.7% identity in 1035 bp and 93.3% identity in the entire 284 amino acid sequence of the protein). It differs from rabbit skeletal muscle beta-tropomyosin (81.7% homology at the protein level) mainly in two regions at amino acids 189-213 and 258-283 suggesting alternative splicing of exons 6a for 6b and 9d for 9a. Since this TM-beta gene was the only gene strongly enough expressed in SMC changing phenotype to be observed by the subtractive hybridisation screen, it likely plays a significant role in this process.