Molecular cloning, sequencing, and expression of the 36 kDa protein present in pars planitis. Sequence homology with yeast nucleopore complex protein

PURPOSE: Patients with active pars planitis have increased levels of a 36 kDa protein (p-36) in their circulation. The current studies were undertaken to determine the primary structure of this protein. METHODS: A degenerate oligonucleotide probe based on the amino terminal sequence of p-36 was used to identify a clone from a human spleen cDNA library. The cDNA insert was subcloned into the EcoR1 site of pUC-19, and both strands were sequenced. Southern blot analysis was used to study the genomic hybridization pattern. p-36 cDNA was subcloned in a pSG5 expression vector, and the construct was used to transfect COS-7 cells. RESULTS: The cDNA sequence contained an open reading frame of 966 base pairs encoding a protein of 322 amino acids, an untranslated region of 322 base pairs, and 2693 base pairs at the 5' and 3' ends, respectively. The deduced amino acid sequence showed 96.8% identity with the carboxy-terminal region of a yeast nucleopore complex protein, nup 100. Southern blot analysis of human genomic DNA revealed a simple hybridization pattern. Transfection of p-36 cDNA in COS-7 cells resulted in the presence of p-36 mRNA and expression of protein. CONCLUSIONS: The 36 kDa protein (p-36) detected at increased levels in the blood of patients with active pars planitis was cloned from a human spleen cDNA library. Its deduced amino acid sequence is homologous with the carboxy-terminal region of a nucleopore complex protein. Thus, we refer to this protein as nup36.     

Complete sequence of rabbit kidney aminopeptidase N and mRNA localization in rabbit kidney by in situ hybridization

We have isolated and sequenced a full-length cDNA for rabbit kidney aminopeptidase N (APN). The 3-kilobase cDNA contains 12 nucleotides of the 5' noncoding region, a 2898 nucleotide long open reading frame, and 113 nucleotides of the 3' untranslated region. The open reading frame encodes a type II membrane protein of 966 amino acid residues composed of a 10 residue NH2-terminal cytosolic domain, a 23 residue transmembrane domain, and a large 933 residue ectodomain that contains the active site. Rabbit APN has eight potential N-glycosylation sites and seven cysteine residues, one of which is located in the transmembrane domain. Computer analysis showed that the enzymes from human, rat, and rabbit were highly conserved, except for the stalk region immediately downstream from the transmembrane domain. Using in situ hybridization techniques we showed that in rabbit kidney, APN mRNA is present in proximal tubules but not in glomeruli, which corresponds to the localization of the protein observed by immunohistochemistry. Taken together, our results strongly suggest that the expression of APN in kidney is modulated at mRNA levels and not at translational and (or) posttranslational levels.     

Somatostatin receptor subtypes sst1 and sst2 elicit opposite effects on the response to glutamate of mouse hypothalamic neurones: an electrophysiological and single cell RT-PCR study

To clone a new nuclear receptor, we screened a rabbit heart complementary DNA (cDNA) library with degenerate oligonucleotide probes corresponding to the DNA-binding domain of nuclear receptors, which is highly conserved among receptors. One of the cDNA clones, clone 23, encodes a novel protein of 596 amino acids, and predicted molecular mass is 66 kDa. Homology search analysis identified this protein as rabbit TR4 (TR4-0). We also cloned the cDNA encoding a rabbit TR4 isoform (TR4-1), which lacks the putative C-terminal ligand-binding domain (350 amino acids) caused by a 23-bp exon deletion, which probably occurred during messenger RNA (mRNA) splicing. Northern blot analysis showed that TR4s are expressed with two kinds of mRNAs (9.0 kb and 2.8 kb), both of which are relatively abundant in brain, testis, and bone. RT-PCR analysis, using pairs of primers specific for each TR4, showed that both types of receptor express in various tissues. Furthermore, both are present in primary osteoblasts and bone marrow cells, though the mRNA levels of TR4-0 were much higher than those of TR4-1. A functional study, using a transient transfection assay, showed that both receptors suppressed retinoid X receptor (RXR)-retinoid acid receptor, RXR-TR, and RXR-VDR-mediated transactivation significantly in COS-1 and osteosarcoma cells (UMR-106, ROS17/2.8) and that TR4-0 was much more effective than TR4-1. Unexpectedly, we found that the TR4s effectively suppressed estrogen receptor-mediated transactivation in bone cells, but neither in kidney (COS-1) nor breast cancer cells (MCF-7, one of the major target cells of the estrogen action). Thus, the present study shows a novel property of the TR4 orphan receptor, acting as a bone cell-specific repressor in the estrogen receptor-mediated signaling pathway.     

Molecular cloning, heterologous expression, and characterization of human glyoxalase II

A clone encoding glyoxalase II has been isolated from a human adult liver cDNA library. The sequence of 1011 base pairs consists of a full-length coding region of 780 base pairs, corresponding to a protein with a calculated molecular mass of 28,861 daltons. Identities (50-60%) were found to partial 5' and 3' cDNA sequences from Arabidopsis thaliana as well as within a limited region of glutathione transferase I cDNA from corn. A vector was constructed for heterologous expression of glyoxalase II in Escherichia coli. For optimal yield of enzyme, silent random mutations were introduced in the 5' coding region of the cDNA. A yield of 25 mg of glyoxalase II per liter of culture medium was obtained after affinity purification with immobilized glutathione. The recombinant enzyme had full catalytic activity and kinetic parameters indistinguishable from those of the native enzyme purified from human erythrocytes.     

Isolation and characterization of a 2.3-kilobase-pair cDNA fragment encoding the binding domain of the bovine leukemia virus cell receptor

An immunoscreening strategy was used to isolate a cDNA clone encoding the binding domain for the external glycoprotein gp51 of the bovine leukemia virus (BLV). Three recombinant phages demonstrating BLV binding activity and containing 2.3-kbp cDNA inserts with identical nucleotide sequences were isolated from a lambda gt11 cDNA library of bovine kidney cells (MDBK). One clone, BLVRcp1, hybridized with a 4.8-kb mRNA from cells of bovine origin and was also found to be conserved as a single-copy gene in murine, bovine, ovine, primate, canine, feline, and porcine DNAs. The same gene is amplified in caprine DNA isolated from a BLV-induced tumor. The longest open reading frame of BLVRcp1 encodes a protein fragment of 729 amino acids with a putative receptor structure. BLVRcp1 cDNA was cloned in the eucaryotic expression vector pXT-1 and transfected into murine NIH 3T3 and human HEp-2 cells. Cells expressing BLVRcp1 mRNA became susceptible to BLV infection. BLVRcp1 has no known physiological function and has no significant homology with sequences registered in the GenBank and EMBL data libraries (31 July 1992). Expression of deleted constructs of BLVRcp1 indicates that the BLV binding region is encoded at the 5' side of the receptor clone.     

cDNA sequence for rkST1, a novel member of the sodium ion-dependent glucose cotransporter family

A full length cDNA for rkST1, a novel member of the Na+/glucose cotransporter family, was cloned from rabbit kidney and sequenced. The coding sequence comprised 2022 base pairs and 674 amino acids. rkST1 beared 50-60% amino acid identity to the other cotransporters and was characteristic in respect of its expression in brain in addition to kidney among the cotransporters.     

The agonist selectivity of a mouse B1 bradykinin receptor differs from human and rabbit B1 receptors

A genomic clone encoding the mouse B1 receptor was isolated by homology to the human B1 receptor cDNA. The deduced amino acid sequence of the mouse B1 receptor is 72% identical to the human B1 receptor and 73% identical to the rabbit B1 receptor. Ligand binding studies of the mouse B1 receptor expressed in COS cells indicate that it has the pharmacological properties associated with the B1 receptor subtype. However the pharmacology of the mouse receptor is unique in that it possesses a 2-3-fold selectivity for the 'classical' B1 agonist des-Arg9BK over the agonist des-Arg10 kallidin. In contrast, the human and rabbit B1 receptors exhibit an approx. 2000- and 150-fold selectivity, respectively, for des-Arg10kallidin over des-Arg9BK. Thus relative to the human and rabbit B1 receptors the mouse B1 receptor has the opposite selectivity for kinin agonists. The DNA sequence of the region encoding bradykinin was determined for two different mouse kininogen cDNA clones, both encode the sequence Arg-BK. Antipeptide antibodies directed against a C-terminal peptide of the human B1 receptor were produced. Initial characterization of this antibody indicates that it detects specific bands by Western blot analyses that are present in membranes prepared from COS cells transfected with the human B1 receptor cDNA but not from mock transfected COS cells.     

Molecular cloning and characterization of rKlk10, a cDNA encoding T-kininogenase from rat submandibular gland and kidney

We have cloned and determined the nucleotide sequence of a novel kallikrein-like mRNA, designated rKlk10*, from rat submandibular gland and kidney with the aid of the polymerase chain reaction (PCR). This cDNA contains 737 base pairs comprising the sequence encoding a mature protein of 235 amino acid residues, partial zymogen peptide, and 3' noncoding sequence. Sequence comparisons showed that rKlk10 mRNA shares 87 and 88% sequence identity with rat tissue kallikrein at nucleic acid and amino acid levels, respectively. It encodes a 26,428-Da acidic protein whose derived amino acid sequence matches completely with the partial amino acid sequence of a kallikrein-like enzyme designated as T-kininogenase, K10 protein, or antigen-gamma purified from rat submandibular gland [Xiong et al. (1990) J. Biol. Chem. 265, 2822-2827; Gutman et al. (1991) Eur. J. Biochem. 784, 1-5; Berg et al. (1991) Biochem. J. 280, 19-25]. The protein encoded by rKlk10 retains the key amino acid residues determining kallikrein cleavage specificity. Northern blot analysis with an rKlk10-specific oligonucleotide probe showed that its mRNA level in the submandibular gland is decreased dramatically by administration of the beta agonist isoproterenol. Tissue-specific expression of rKlk10 was analyzed by Northern blotting and Southern blotting of PCR-amplified cDNA, which showed that rKlk10 is expressed at high levels in the submandibular gland and low levels in the kidney but not in seven other tissues including prostate, liver, heart, adrenal gland, testes, pituitary, and pancreas. rKlk10 cDNAs cloned from the kidney and submandibular gland show sequence identity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural requirements of the higher order RNA kissing element in the enteroviral 3'UTR

The origin of replication ( oriR ) involved in the initiation of (-) strand enterovirus RNA synthesis is a quasi-globular multi-domain RNA structure which is maintained by a tertiary kissing interaction. The kissing interaction is formed by base pairing of complementary sequences within the predominant hairpin-loop structures of the enteroviral 3' untranslated region. In this report, we have fully characterised the kissing interaction. Site-directed mutations which affected the different base pairs involved in the kissing interaction were generated in an infectious coxsackie B3 virus cDNA clone. The kissing interaction appeared to consist of 6 bp. Distortion of the interaction by mispairing of each of the base pairs involved in this higher order RNA structure resulted in either temperature sensitive or lethal phenotypes. The nucleotide constitution of the base which gaps the major groove of the kissing domain was not relevant for virus growth. The reciprocal exchange of the complete sequence involved in the kissing resulted in a mutant virus with wild type virus growth characteristics arguing that the base pair constitution is of less importance for the initiation of (-) strand RNA synthesis than the existence of the tertiary structure itself.     

Suppression of prostaglandin E receptor signaling by the variant form of EP1 subtype

A cDNA clone of prostaglandin (PG) E receptor EP1 subtype (rEP1) was isolated from a rat uterus cDNA library. It encodes 405 amino acid residues with seven transmembrane-spanning domains and couples to Ca2+ mobilization. In addition, three cDNA clones encoding a variant form of rEP1 were isolated. The open reading frame can code a 366-amino acid protein carrying a specific change of 49 amino acids from the middle of transmembrane segment VI to COOH terminus; it possesses a transmembrane segment VII-like structure lacking an intracellular COOH-terminal tail. Southern blot analysis of rat genomic DNA and genomic polymerase chain reaction demonstrated that these cDNAs were derived from a single copy gene. Northern blot analysis and ribonuclease protection assay revealed that both rEP1 and rEP1-variant receptor mRNAs were highly expressed in the kidney. Immunoblot with an antibody directed toward the specific region of rEP1-variant receptor showed that rEP1-variant receptor protein was expressed in the membrane of the kidney and Chinese hamster ovary (CHO) cells transfected with rEP1-variant cDNA. Thus, the rEP1-variant receptor is translated from mRNA which is not spliced at nucleotide position 952 in the segment VI transmembrane region. rEP1-variant receptor retained the ligand binding activity with affinity and specificity similar to rEP1 receptor, but lost the coupling of signal transduction systems by itself. However, when rEP1-variant receptor was stably co-expressed with rEP1 receptor in CHO cells, the Ca2+ mobilization mediated by EP1 receptor was significantly suppressed. Furthermore, when rEP1-variant receptor was expressed in CHO cells, cAMP formation by activation of endogenous EP4 receptor was strongly blocked. These results suggest that the rEP1-variant receptor may affect the efficiency of signal coupling of PGE receptors and attenuate the action of PGE2 on tissues.     

Rat 7,8-dihydro-8-oxoguanine DNA glycosylase: substrate specificity, kinetics and cleavagemechanism at an apurinic site

Reactive oxygen species produce different lesions in DNA. Among them, 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the major oxidative products implicated in mutagenesis. This lesion is removed from damaged DNA by base excision repair, and genes coding for 8-oxoG-DNA glycosylases have been isolated from bacteria, yeast and human cells. We have isolated and characterized the cDNA encoding the rat 8-oxoG-DNA glycosylase (rOGG1). Expression of the cDNA in the fgp mutY Escherichia coli double mutant allowed the purification of the untagged rOGG1 protein. It excises 8-oxoG from DNA with a strong preference for duplex DNA containing 8-oxoG:C base pairs. rOGG1 also acts on formamidopyrimidine (FaPy) residues, and the K m values on 8-oxoG and FaPy residues are 18.8 and 9.7 nM, respectively. When acting on an oligonucleotide containing an 8-oxoG residue, rOGG1 shows a beta-lyase activity that nicks DNA 3' to the lesion. However, rOGG1 acts on a substrate containing an apurinic site by a beta-delta elimination reaction and proceeds through a Schiff base intermediate. Expression of rOGG1 in E.coli fpg mutY suppresses its spontaneous mutator phenotype.