cDNA cloning, sequence analysis and tissue distribution of rat preproendothelin-1 mRNA.

We report the cloning of a full-length cDNA encoding rat preproendothelin-1 (preproET-1). The predicted rat preproET-1 consists of 202 amino acid residues and highly similar to human, porcine and bovine preproET-1, respectively. The deduced 21-residue sequence of mature rat ET-1 is identical to human, porcine, canine and bovine ET-1. As in other mammalian species, the mature ET-1 is predicted to be produced from a 39-residue big ET-1 in the rat. Northern blot analysis showed that a single 2.3-kb preproET-1 mRNA is expressed not only in vascular endothelial cells but also in other rat tissues, including the lung, brain, uterus, stomach, heart, adrenal gland and kidney. These findings suggest that ET-1 may play roles as a local mediator in multiple organs both within and outside the cardiovascular system in the rat.     

Three types of mouse peptidylarginine deiminase: characterization and tissue distribution.

Three types of mouse peptidylarginine deiminase were separated by DEAE-Sephacel ion-exchange column chromatography, and we propose designating them peptidylarginine deiminase type I, II, and III according to the order of elution. The type II enzyme was widely distributed in various tissues including the skeletal muscle, whereas the type I enzyme was localized in the epidermis and uterus, and the type III enzyme was detected in the epidermis and hair follicles. These enzymes were distinguished by their molecular weights and substrate specificity. The molecular weights were estimated to be approximately 54,000 (type I) and 100,000 (type II and III) by Sephacryl S-200 gel filtration column chromatography. On SDS-PAGE the type II and III enzymes gave Mr = 81,000 and Mr = 76,000, respectively. Among the substrates tested, the type I enzyme showed highest activity toward BZ-L-Arg-NH2, type II toward BZ-L-Arg-O-Et, and type III toward protamine. Western blot analysis showed that antibodies against the type II enzyme were immuno-crossreactive to the type III enzyme.     

Bovine tryptases. cDNA cloning, tissue specific expression and characterization of the lung isoform.

A complementary DNA encoding a new bovine tryptase isoform (here named BLT) was cloned and sequenced from lung tissue. Analysis of sequence indicates the presence of a 26-amino acid prepro-sequence and a 245 amino acid catalytic domain. It contains six different residues when compared with the previously characterized tryptase from bovine liver capsule (BLCT), with the most significant difference residing at the primary specificity S1 pocket. In BLT, the canonical residues Asp-Ser are present at positions 188-189, while in BLCT these positions are occupied by residues Asn-Phe. This finding was confirmed by mass fingerprinting of the peptide mixture obtained upon in-gel tryptic digestion of BLT. Analysis by gel filtration of the purified protein shows that BLT is probably tetrameric, similar to the previously identified tryptases from other species, with monomer migrating as 35-40 kDa multiple bands in SDS/PAGE. As expected, the catalytic abilities of the two bovine tryptases are different. The specificity constant values (kcat/Km) assayed with model substrates are 10- to 60-fold higher in the case of BLT. The tissue-specific expression of the two tryptases was evaluated at the RNA level by analysis of their different restriction patterns. In lung, only BLT was found to be expressed, while in liver capsule only BLCT is present. Both isoforms are distributed in similar amounts in heart and spleen. Analysis of the two gene sequences reveals the presence of several recognition sequences in the promoter regions and suggest a role for hormones in governing the mechanism of tissue expression of bovine tryptases.     

cDNA cloning and sequence of rat ribonuclease inhibitor, and tissue distribution of the mRNA.

A cDNA clone encoding ribonuclease inhibitor was isolated from a rat lung cDNA library. The deduced amino acid sequence revealed a high degree of conservation of a repeated structure. The mRNA was detected in all seven tissues of rat examined, the amount being highest in the lung and lowest in the heart.     

cDNA cloning and tissue distribution of a rat ubiquitin carboxyl-terminal hydrolase PGP9.5.

We have isolated a cDNA clone encoding ubiquitin carboxyl-terminal hydrolase PGP9.5 from a rat brain cDNA library and examined the tissue distribution. The primary structure of the cDNA consists of 856 nucleotides including the entire coding region for 223 amino acids, and the calculated molecular mass is 24,782 Da. The rat PGP9.5 is strikingly homologous to the human PGP9.5, 75.2% of nucleic acids and 95.1% of amino acids being identical. The mRNA of PGP9.5 is most abundant in the rat brain and to a lesser degree in the testis. In other peripheral tissues we tested, the mRNA was undetectable. Western blotting using an anti-rat PGP9.5 antibody revealed the parallel distribution of mRNA and protein in various brain regions and testis. The availability of the rat PGP9.5 clone provides a new approach to examine the function of PGP9.5 and the role that it plays in the pathology of neurodegenerative diseases.     

A specific human lysophospholipase: cDNA cloning, tissue distribution and kinetic characterization

Lysophospholipases are critical enzymes that act on biological membranes to regulate the multifunctional lysophospholipids; increased levels of lysophospholipids are associated with a host of diseases. Herein we report the cDNA cloning of a human brain 25 kDa lysophospholipid-specific lysophospholipase (hLysoPLA). The enzyme (at both mRNA and protein levels) is widely distributed in tissues, but with quite different abundances. The hLysoPLA hydrolyzes lysophosphatidylcholine in both monomeric and micellar forms, and exhibits apparent cooperativity and surface dilution kinetics, but not interfacial activation. Detailed kinetic analysis indicates that the hLysoPLA binds first to the micellar surface and then to the substrate presented on the surface. The kinetic parameters associated with this surface dilution kinetic model are reported, and it is concluded that hLysoPLA has a single substrate binding site and a surface recognition site. The apparent cooperativity observed is likely due to the change of substrate presentation. In contrast to many non-specific lipolytic enzymes that exhibit lysophospholipase activity, hLysoPLA hydrolyzes only lysophospholipids and has no other significant enzymatic activity. Of special interest, hLysoPLA does not act on plasmenylcholine. Of the several inhibitors tested, only methyl arachidonyl fluorophosphonate (MAFP) potently and irreversibly inhibits the enzymatic activity. The inhibition by MAFP is consistent with the catalytic mechanism proposed for the enzyme - a serine hydrolase with a catalytic triad composed of Ser-119, Asp-174 and His-208.     

Porcine lung surfactant protein D: complementary DNA cloning, chromosomal localization, and tissue distribution

Porcine organs and lung surfactant have medically important applications in both xenotransplantation and therapy. We have started to characterize porcine lung surfactant by cloning the cDNA of porcine surfactant protein D (SP-D). SP-D and SP-A are important mediators in innate immune defense for the lung and possibly other mucosal surfaces. Porcine SP-D will also be an important reagent for use in existing porcine animal models for human lung infections. The complete cDNA sequence of porcine SP-D, including the 5' and 3' untranslated regions, was determined from two overlapping bacteriophage clones and by PCR cloning. Three unique features were revealed from the porcine sequence in comparison to SP-D from other previously characterized species, making porcine SP-D an intriguing species addition to the SP-D/collectin family. The collagen region contains an extra cysteine residue, which may have important structural consequences. The other two differences, a potential glycosylation site and an insertion of three amino acids, lie in the loop regions of the carbohydrate recognition domain, close to the carbohydrate binding region and thus may have functional implications. These variations were ruled out as polymorphisms or mutations by confirming the sequence at the genomic level in four different pig breeds. Porcine SP-D was shown to localize primarily to the lung and with less abundance to the duodenum, jejunum, and ileum. The genes for SP-D and SP-A were also shown to colocalize to a region of porcine chromosome 14 that is syntenic with the human and murine collectin loci.     

Molecular cloning of substance P receptor cDNA from guinea-pig uterus.

A cDNA encoding guinea-pig uterine substance P (SP) receptor has been isolated using the homology screening approach. Northern blot analysis reveals that the corresponding mRNA, of approx. 4.8 kb, is expressed in all tissues tested, but predominantly in the uteri of non-pregnant animals; during pregnancy its expression is reduced. The guinea-pig SP receptor was expressed in COS-7 cells and demonstrated relative ligand affinity in the order: SP much greater than neurokinin A greater than neurokinin B.     

Partial molecular cloning and tissue distribution of hexokinase I cDNA in common carp

The full C-terminal half of HK-I cDNA was cloned from common carp Cyprinus carpio . This HK-I cDNA is homologous to mammalian HK-I, in its sequence and tissue distribution and probably derives from duplication and fusion of an ancestral gene.     

Selective tissue distribution of G protein gamma subunits, including a new form of the gamma subunits identified by cDNA cloning.

The GTP-binding regulatory proteins (G proteins) that transduce signals from receptors to effectors are composed of alpha, beta, and gamma subunits. Whereas the role of alpha subunits in directly regulating effector activity is widely accepted, it has recently been demonstrated that beta gamma subunits may also directly regulate effector activity. This has made clear the importance of identifying and characterizing beta and gamma subunits. We have isolated a cDNA clone encoding a new gamma subunit, referred to here as the gamma 7 subunit, using probes based on peptide sequences of a gamma subunit previously purified from bovine brain. The clone contains a 1.47-kilobase cDNA insert, which includes an open reading frame of 204 base pairs that predicts a 68-amino acid polypeptide with a calculated M(r) of 7553. The predicted protein shares amino acid identities with the other known gamma subunits, ranging from 38 to 68%. Also characteristic of gamma subunits is a carboxyl-terminal CAAX motif. The expression of the gamma 7 subunit as well as the gamma 2, gamma 3, and gamma 5 subunits was examined in several bovine tissues at both the mRNA and protein levels. Whereas the gamma 2 and gamma 3 subunits were selectively expressed in brain, the gamma 5 and gamma 7 subunits were expressed in a variety of tissues. Thus, the gamma 5 and gamma 7 subunits are the first G protein gamma subunits known that could participate in the regulation of widely distributed signal transduction pathways.     

Concurrent up-regulation of guanine-nucleotide-binding proteins Gi1 alpha, Gi2 alpha and Gi3 alpha in adipocytes of hypothyroid rats.

Rat white adipocytes express three distinct 'Gi-like' guanine-nucleotide-binding proteins (G-proteins) [Mitchell, Griffiths, Saggerson, Houslay, Knowler & Milligan (1989) Biochem. J. 262, 403-408]. We have previously noted elevated levels of Gi in membranes of adipocytes from hypothyroid rats [Milligan, Spiegel, Unson & Saggerson (1987) Biochem. J. 247, 223-227]. Using a series of anti-peptide antisera able to discriminate between the individual gene products we have examined levels of each Gi-like G-protein in adipocyte membranes of hypothyroid rats compared with euthyroid controls. We demonstrate that up-regulation of Gi in adipocytes of hypothyroid rats is not restricted to a single subtype of Gi but that each of Gi1 alpha, Gi2 alpha and Gi3 alpha is present at markedly higher levels compared with euthyroid animals. In contrast, levels of both the 45 and 42 kDa forms of Gs alpha were not altered substantially in the hypothyroid state.     

A low molecular weight insulin-like growth factor binding protein from rat: cDNA cloning and tissue distribution of its messenger RNA

Rat serum contains two major forms of insulin-like growth factor (IGF) binding proteins (BPs) that have apparent mol wts of about 35,000 and 150,000. We have isolated a cDNA clone encoding an IGF-BP whose N-terminal sequence is completely homologous to the NH2-terminal of the Buffalo rat liver cells-3A BP. The 270 amino acid mature protein has a predicted mol wt of 29,500. It contains a cysteine rich domain at each end of the molecule and an Arg-Gly-Asp (RGD) tripeptide motif near its C-terminus which suggests that this BP might associate with integrin cell surface receptors. The mature protein shares only partial homology with two published human IGF-BPs. Northern blot analysis shows that its mRNA is abundant in several fetal tissues, in adult brain, testes, ovaries, and kidney. Expression in the liver is high in fetal life but decreases to a barely detectable level in adulthood. However, upon hypophysectomy, the mRNA level increases at least 20-fold which suggests a hormonal regulation for the hepatic production of this small IGF-BP.     

Human insulin-like growth-factor-binding protein. Low-molecular-mass form: protein sequence and cDNA cloning

Two different insulin-like growth-factor (IGF)-binding proteins have been found in human blood, one of high molecular mass and dependent on growth hormone for synthesis, the other of low molecular mass and independent of growth hormone. The small IGF-binding protein is abundant in human amniotic fluid. Its amino acid sequence has now been determined by direct analysis of the protein and its proteolytic fragments. Also, by immunoscreening a partial cDNA clone was isolated from a human hepatoma cell line. The mature protein consists of 234 amino acids and is coded for by an mRNA of approximately 1700 nucleotides in length. The primary structure of the protein reveals 18 Cys residues in N-terminal and C-terminal clusters and an Arg-Gly-Asp peptide sequence, common to extracellular proteins binding to receptors of the integrin family. A protein-sequence polymorphism was detected at position Ile/Met-228, indicating possible allelic variation. The 3'-untranslated mRNA sequence has a high A + T content and shows five copies of an ATTTA sequence, which has been shown to be involved in the regulation of the stability of certain mRNAs coding for growth-regulating proteins.     

cDNA cloning, sequence analysis and tissue distribution of a precursor for vasoactive intestinal contractor (VIC)

A full-length cDNA encoding preprovasoactive intestinal contractor (PPVIC) has been cloned. From the deduced 160 amino acid PPVIC, the mature VIC is predicted to be produced via a 37 residue intermediate, big VIC. The PPVIC also contains a VIC-like peptide of 16 amino acids structurally related to to the amino-terminal residues of VIC and flanked by pairs of dibasic amino acids, putative processing sites. RNA blot hybridization with PPVIC cDNA confirmed the PPVIC gene to be expressed in the small and large intestinal tract in a tissue specific manner.     

Vitamin-D dependent 9 kDa calcium-binding protein gene: cDNA cloning, mRNA distribution and regulation

Cholecalciferol (calcitriol) the active hormonal form of vitamin D induces the synthesis of at least two intracellular calcium-binding proteins (Ka = 10(6) M-1), the cholecalcins (CaBP) in mammals. We used the synthesis of these proteins to study the genomic steroid-like action of vitamin D. The 9 kDa CaBP is mainly concentrated in the duodenum while 28 kDa CaBP is located in the kidney and cerebellum. Complementary DNA copies of rat intestinal 9 kDa CaBP mRNA were cloned in E. coli. The deduced amino acid sequence for 9 kDa CaBP contains two 'EF hand' domains corresponding to calcium-binding sites I and II. The homology observed suggests, after comparison with the structures of other intracellular CaBPs, that rat 9 kDa CaBP mRNA contains the remains of an untranslated calcium-binding site III-like structure seen in 28 kDa CaBP from kidney and cerebellum of rat. Northern blots showed that the cDNA sequence hybridizes to a homogeneous 500-600 nucleotide mRNA species from rat duodenum. Larger mRNA species encoding 28 kDa CaBP were undetectable in rat kidney and cerebellum even under low stringency conditions. These findings demonstrate that there is no cross-hybridization between 9 kDa and 28 kDa CaBP mRNAs, and Southern analysis indicates that there are distinct genes coding for each rat cholecalcin. The cDNA probe was used to analyze the specific 9 kDa CaBP gene expression along the intestine of growing rats and during gestation and fetal development.(ABSTRACT TRUNCATED AT 250 WORDS)     

cDNA cloning and characterization of a novel nucleolar protein.

In an initial study of anti-nuclear antibodies in the chronic inflammatory bladder disease interstitial cystitis, we reported that 7% of interstitial cystitis patients studied had autoantibodies to the nucleolus. We now report that, using an autoimmune serum from a patient with interstitial cystitis, we have identified and partially characterized a novel protein with an M(r) of approximately 55 kDa (hereafter referred to as No55) localized to the granular component of the nucleolus. No55 was initially characterized by diffuse nucleolar immunofluorescence staining in interphase cells and by Western blotting as a 55-kDa doublet on whole-cell extracts. During mitosis, No55 was associated with chromosomes and appeared in prenucleolar bodies during telophase, but it did not colocalize with p80-coilin in coiled bodies. Immunoelectron microscopy revealed that No55 was localized uniformly throughout the granular component of the nucleolus compared with a more peripheral localization of nucleolar granular component protein B23. On segregation of the nucleolus with actinomycin D, No55 remained with the granular component of the segregated nucleolus, whereas protein B23 was found predominantly in the nucleoplasm. Finally, a cDNA expression library was screened with the human autoantibody against No55, and a 2.4-kb insert was isolated, subcloned to homogeneity, and then sequenced. Analysis of this sequence showed an open reading frame of approximately 1.3 kb coding for 437 amino acids with a predicted molecular weight of 50 kDa. A search of the gene sequence database indicated homology with SC65, a rat synaptonemal complex protein. Therefore, on the basis of molecular weight, nucleolar sublocalization, response to actinomycin D, and cDNA sequence determination, No55 is a novel protein of the interphase nucleolus.     

Purification and characterization of Go alpha and three types of Gi alpha after expression in Escherichia coli

Complementary DNAs for the G protein alpha subunits Gi alpha 1, Gi alpha 2, Gi alpha 3, and Go alpha were expressed in Escherichia coli, and the four proteins were purified to homogeneity. The recombinant proteins exchange and hydrolyze guanine nucleotide, are ADP-ribosylated by pertussis toxin, and interact with beta gamma subunits. The rates of dissociation of GDP from Gi alpha 1 and Gi alpha 3 (0.03 min-1) are an order of magnitude slower than that from rGo alpha; release of GDP from Gi alpha 2 is also relatively slow (0.07 min-1). However, the values of kcat for the hydrolysis of GTP by rGo alpha and the three rGi alpha proteins are approximately the same, about 2 min-1 at 20 degrees C. The recombinant proteins restore inhibition of Ca2+ currents in pertussis toxin-treated dorsal root ganglion neurons in response to neuropeptide Y and bradykinin, indicating that the proteins can interact functionally with all necessary components of at least one signal transduction system. The two different receptors function with different arrays of G proteins to mediate their responses, since all four G proteins restored responses to bradykinin, while Gi alpha 2 was inactive with neuropeptide Y. Despite these results, high concentrations of activated Gi alpha proteins are without effect on adenylyl cyclase activity, either in the presence or absence of forskolin or Gs alpha, the G protein that activates adenylyl cyclase. These results are consistent with the hypothesis that G protein beta gamma subunits are primarily responsible for inhibition of adenylyl cyclase activity.     

革胡子鲶生长激素cDNA克隆与蛋白质结构分析

从革胡子鲶(Clarias lazera(Burchell))的脑垂体组织中提取总RNA, 应用RT-PCR方法, 扩增得到了革胡子鲶生长激素(Growth hormone, GH)基因cDNA的开放阅读框(Open reading frame, ORF)序列。ORF全长为603 nt, 编码由22个信号肽氨基酸和178个成熟肽氨基酸共同组成的生长激素前体蛋白。序列同源比较结果表明, 研究中得到的革胡子鲶生长激素氨基酸序列与GenBank中已报道的其他6种鲶形目鱼类的氨基酸序列同源性高达95.8%。二级结构预测分析结果表明, 革胡子鲶生长激素蛋白中含有a 螺旋、b 折叠和b 转角以及无规卷曲等二级结构, 以a 螺旋为主, 是典型的a 型结构蛋白质。此外, 抗原性分析表明, 在氨基酸序列中的4个区域均可形成优势抗原表位, 其结构特点非常适合改造成为重组生长激素疫苗或单克隆抗体制剂加以开发利用。