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.     

The phosphoenolpyruvate carboxylase gene family of Sorghum: promoter structures, amino acid sequences and expression of genes

Two different members of the phosphoenolpyruvate carboxylase(PEPC)-encoding multigene family (clones lambda CP21 and lambda CP46) have been isolated from a Sorghum vulgare lambda EMBL4 genomic library. The use of the 3'-noncoding regions to probe Northern blots of RNA from roots, etiolated leaves and green leaves indicated that lambda CP21 and lambda CP46 encode the C3- and C4-type leaf PEPC isoforms, respectively. The lambda CP21 clone is expressed in the three tissues and is not light-regulated, whereas lambda CP46 is only expressed in greening leaves. The nucleotide sequence of the 5'-flanking DNA (520 bp) has been determined for both genes. For lambda CP46, several direct repeats were located in this region with similarities to sequences found in other light-regulated genes, but not in lambda CP21. The deduced amino acid sequences of the two S. vulgare PEPC proteins are 75% identical.     

Structure and expression of fibulin-2, a novel extracellular matrix protein with multiple EGF-like repeats and consensus motifs for calcium binding

A new protein, fibulin-2, was predicted from sequence analysis of cDNA clones obtained from a mouse fibroblast library. This protein consists of a 1195-residue polypeptide preceded by a 26-residue signal peptide. The COOH-terminal region of 787 amino acids contained three anaphylatoxin-related segments (domain I), 11 EGF-like repeats (domain II), 10 of which had a consensus motif for calcium-binding, and a 115- residue globular domain III. Except for two additional EGF-like repeats, this COOH-terminal region showed 43% sequence identity with the previously described fibulin-1 (BM-90). The NH2-terminal 408 residues, unique to fibulin-2, showed no sequence homology to other known proteins and presumably form two additional domains that differ in their cysteine content. Recombinant fibulin-2 was produced and secreted by human cell clones as a disulfide-bonded trimer. Rotary shadowing visualized the protein as three 40-45 nm long rods which are connected at one end in a globe-like structure. No significant immunological cross-reaction could be detected between fibulin-1 and fibulin-2. Production of the fibulin-2 was demonstrated by Northern blots and radioimmunoassay in fibroblasts but not in several tumor cell lines. Together with the observation that the serum level of fibulin-2 is 1,000-fold lower than that of fibulin-1, the data indicate that these two isoforms are not always coordinately expressed. This is also suggested by Northern blots of tissue mRNAs and by immunofluorescence localizations using mouse tissues. The latter studies also demonstrated an extracellular localization for fibulin-2 in basement membranes and other connective tissue compartments.     

Molecular cloning and expression of a mouse muscle cDNA encoding adenylosuccinate synthetase

Adenylosuccinate synthetase (EC 6.3.4.4) catalyzes the first step in formation of AMP from IMP. At least two isozymes exist in vertebrate tissue. An acidic form, present in most tissues, has been suggested to be involved in de novo biosynthesis while a basic isozyme, which predominates in muscle, appears to function in the purine nucleotide cycle. Antibodies specific for the basic isozyme detect a single protein in mouse tissues with highest levels in skeletal muscle, tongue, esophagus, and heart tissue consistent with a role for the enzyme in muscle metabolism. A series of degenerate oligonucleotides were constructed based on peptide sequences from purified rat muscle enzyme and then used to clone a mouse muscle cDNA encoding the basic isozyme. The clone contains a open reading frame of 1356 bases with 452 amino acids. Northern analysis of RNA from mouse tissues showed a tissue distribution similar to that of the protein, indicating a high level of gene expression in muscle. Transfection of COS cells with the mouse muscle cDNA allows expression of a functional protein with a molecular mass of approximately 50 kDa, consistent with the open reading frame and the size of the isolated rat enzyme. The deduced amino acid sequence of the mouse synthetase is 47 and 37% identical to the synthetase sequences from Dictyostelium discoideum and Escherichia coli, respectively. The availability of antibodies and cDNA clones specific for the basic isozyme of adenylosuccinate synthetase from muscle will facilitate future genetic and biochemical analysis of this protein and its role in muscle physiology.     

A protein with homology to the C-terminal repeat sequence of Octopus rhodopsin and synaptophysin is a member of a multigene family in Dictyostelium discoideum

Monoclonal antibodies were raised against a protein with a molecular mass of 24 kDa that has been described as a membrane-associated, actin binding protein from Dictyostelium discoideum [( 1985) J. Cell Biol. 100, 727-735]. Using these monoclonal antibodies we isolated from a lambda gt11 expression library cDNA clones coding for this protein. The cDNA deduced amino acid sequence revealed the presence of an unusual carboxy-terminus which has homologies to the C-termini of Octopus rhodopsin and synaptophysin. This part of the protein sequence contains 5 direct repeats with the motif GYP (P)Q(P). Southern and Northern blots showed that this sequence is present in a series of Dictyostelium genes transcribed in all stages of development.     

Cloning and sequencing of the 23 kDa mouse photoreceptor cell-specific protein.

The 23 kDa protein was localized by immunocytochemistry to photoreceptor cells of the mouse retina, and bovine and mouse cDNA clones were isolated and sequenced. The deduced amino acid sequences showed that the mouse 23 kDa protein is 91% identical to the bovine protein, and is the same as S-modulin, the CAR (cancer-associated retinopathy) protein and recoverin, the Ca(2+)-dependent activator of photoreceptor guanylate cyclase. The amino acid sequence reveals two Ca2+ binding sites, no internal repeats, 59% homology to the chicken visinin protein and 40% homology to calmodulin while Northern analysis demonstrated a single 1.0 kb mRNA species in bovine and mouse retina.     

Mouse and human GTPBP2, newly identified members of the GP-1 family of GTPase

We earlier identified the GTPBP1 gene which encodes a putative GTPase structurally related to peptidyl elongation factors. This finding was the result of a search for genes, the expression of which is induced by interferon-gamma in a macrophage cell line, THP-1. In the current study, we probed the expressed sequence tag database with the deduced amino acid sequence of GTPBP1 to search for partial cDNA clones homologous to GTPBP1. We used one of the partial cDNA clones to screen a mouse brain cDNA library and identified a novel gene, mouse GTPBP2, encoding a protein consisting of 582 amino acids and carrying GTP-binding motifs. The deduced amino acid sequence of mouse GTPBP2 revealed 44.2% similarity to mouse GTPBP1. We also cloned a human homologue of this gene from a cDNA library of the human T cell line, Jurkat. GTPBP2 protein was found highly conserved between human and mouse (over 99% identical), thereby suggesting a fundamental role of this molecule across species. On Northern blot analysis of various mouse tissues, GTPBP2 mRNA was detected in brain, thymus, kidney and skeletal muscle, but was scarce in liver. Level of expression of GTPBP2 mRNA was enhanced by interferon-gamma in THP-1 cells, HeLa cells, and thioglycollate-elicited mouse peritoneal macrophages. In addition, we determined the chromosomal localization of GTPBP1 and GTPBP2 genes in human and mouse. The GTPBP1 gene was mapped to mouse chromosome 15, region E3, and human chromosome 22q12-13.1, while the GTPBP2 gene is located in mouse chromosome 17, region C-D, and human chromosome 6p21-12.     

Isolation and sequence analysis of cDNA for rat carboxypeptidase E [EC 3.4.17.10], a neuropeptide processing enzyme

Carboxypeptidase E (CPE) is the carboxypeptidase B-like enzyme associated with the biosynthesis of numerous peptide hormones and neurotransmitters. This enzyme has been previously purified to homogeneity from bovine tissues, and cDNA clones (non-full length) isolated from a bovine pituitary cDNA library. In the present study, cDNA encoding full-length rat CPE has been isolated and sequenced. Both the nucleotide and amino acid sequences of rat CPE show substantial homology with the bovine sequences. The bovine and rat nucleotide sequences are homologous within the entire coding region, as well as within several portions of the 3'-untranslated region. The predicted amino acid sequence of rat CPE is greater than 90% homologous with the bovine enzyme. Northern blot analyses indicate a single species of CPE mRNA approximately 2100 nucleotides in length to be present in many neural and endocrine tissues. High levels of CPE mRNA are present in rat hypothalamus, hippocampus, midbrain, striatum, and cerebral cortex; and moderate levels are present in the brain stem, cerebellum, heart, adrenal, and eye. Low levels are detected in testis and duodenum, but not in liver or thymus. This tissue-specific expression of CPE mRNA is consistent with the proposed role for this enzyme in the production of numerous peptide hormones and neurotransmitters.     

cDNA of a Novel mRNA Expressed Predominantly in Mouse Kidney

We examined embryonic carcinoma (EC) cells for a potential prototype molecule of C3, the third component of complement. PCR primers, corresponding to the base sequence derived from the C3 cDNA of several species, were used for PCR amplification of the EC cell cDNA. All the PCR products obtained had the same sequence and showed no sequence homology to C3. Subsequently, cDNA clones were isolated from a mouse liver cDNA library using the PCR product as a probe. Unexpectedly, neither the base sequence of the cDNA clones nor the amino acid sequence deduced from the cDNA showed homology to C3, although partial homology was observed to a number of sequences from EST databases. We designated this new clone NCU-G1. Northern hybridization experiments revealed that NCU-G1 is expressed constitutively not only in the mouse fetus but also in various mouse tissues, and is most abundant in the kidney cortex.     

cDNA clone and expression analysis of rodent fast and slow skeletal muscle troponin I mRNAs

We have characterized the structure and expression of rodent mRNAs encoding the fast and slow skeletal muscle isoforms of the contractile regulatory protein, troponin I (TnIfast and TnIslow). TnIfast and TnIslow cDNA clones were isolated from mouse and rat muscle cDNA clone libraries and were used as isoform-specific probes in Northern blot and in situ hybridization studies. These studies showed that the TnIfast and TnIslow mRNAs are expressed in skeletal muscle, but not cardiac muscle or other tissues, and that they are differentially expressed in individual muscle fibers. Fiber typing on the basis of in situ hybridization analysis of TnI isoform mRNA content showed an excellent correlation with fiber type as assessed by myosin ATPase histochemistry. These results directly demonstrate that the differential expression of skeletal muscle TnI isoforms in the various classes of vertebrate striated muscle cells is based on gene regulatory mechanisms which control the abundances of specific TnI mRNAs in individual muscle cells. Both TnIfast and TnIslow mRNAs are expressed, at comparable levels, in differentiated cultures of rat L6 and mouse C2 muscle cell lines. Thus, although neuronal input has been shown to be an important factor in determining fast versus slow isoform-specific expression in skeletal muscle, both TnIfast and TnIslow genes can be expressed in muscle cells in the absence of nerve. Comparison of the deduced rodent TnI amino acid sequences with previously determined rabbit protein sequences showed that residues with potential fast/slow isoform-specific function are present in several discrete clusters, two of which are located near previously identified actin and troponin C binding sites.     

Prediction of the coding sequences of mouse homologues of KIAA gene: III. the complete nucleotide sequences of 500 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have conducted a human cDNA project to predict protein-coding sequences (CDSs) in large cDNAs (> 4 kb) since 1994, and the number of newly identified genes, known as KIAA genes, already exceeds 2000. The ultimate goal of this project is to clarify the physiological functions of the proteins encoded by KIAA genes. To this end, the project has recently been expanded to include isolation and characterization of mouse KIAA-counterpart genes. We herein present the entire sequences and the chromosome loci of 500 mKIAA cDNA clones and 13 novel cDNA clones that were incidentally identified during this project. The average size of the 513 cDNA sequences reached 4.3 kb and that of the deduced amino acid sequences from these cDNAs was 816 amino acid residues. By comparison of the predicted CDSs between mouse and human KIAAs, 12 mKIAA cDNA clones were assumed to be differently spliced isoforms of the human cDNA clones. The comparison of mouse and human sequences also revealed that four pairs of human KIAA cDNAs are derived from single genes. Notably, a homology search against the public database indicated that 4 out of 13 novel cDNA clones were homologous to the disease-related genes.     

Identification of human CPI-17, an inhibitory phosphoprotein for myosin phosphatase

CPI-17 is a phosphorylation-dependent inhibitor of myosin phosphatase. cDNA clones encoding CPI-17 were isolated from a human aorta library. Overlapping clones indicated two isoforms: CPI-17alpha was 147 residues and mass of 16.7 kDa; CPI-17beta (120 residues, mass 13.5 kDa) resulted from a deletion in the alpha-isoform of 27 residues, sequence 68-94. N-terminal 67 residues of all CPI-17 isoforms (human, porcine, rat and mouse) were highly conserved (for the human and porcine isoforms the identity was 91%). The presence of the two human isoforms was detected from cDNA sequences amplified by RT-PCR and by Western blots on human aorta. The cloned human CPI-17 gene indicated 4 coding exons and CPI-17beta was an alternative splice variant due to deletion of the second exon. FISH analysis located the human CPI-17 gene on chromosome 19q13.1.