Cloning and characterization of a cDNA encoding a novel subtype of rat thyrotropin-releasing hormone receptor

A cDNA encoding a thyrotropin-releasing hormone (TRH) receptor expressed in the pituitary was previously cloned (De La Pena, P., Delgado, L. M., Del Camino, D., and Barros, F. (1992) Biochem. J. 284, 891-899; De La Pena, P., Delgado, L. M., Del Camino, D., and Barros, F. (1992) J. Biol. Chem. 267, 25703-25708; Duthie, S. M., Taylor, P. L., Anderson, J., Cook, J., and Eidne, K. A. (1993) Mol. Cell Endocrinol. 95, R11-R15). We now describe the isolation of a rat cDNA encoding a novel subtype of TRH receptor (termed TRHR2) displaying an overall homology of 50% to the pituitary TRH receptor. Introduction of TRHR2 cDNA in HEK-293 cells resulted in expression of high affinity TRH binding with a different pharmacological profile than the pituitary TRH receptor. De novo expressed receptors were functional and resulted in stimulation of calcium transient as assessed by fluorometric imaging plate reader analysis. The message for TRHR2 was exclusive to central nervous system tissues as judged by Northern blot analysis. Studies of the expression of TRHR-2 message by in situ hybridization revealed a pattern of expression remarkably distinct (present in spinothalamic tract, spinal cord dorsal horn) from that of the pituitary TRH receptor (present in hypothalamus, and ventral horn of the spinal cord, anterior pituitary). Therefore, we have identified a novel, pharmacologically distinct receptor for thyrotropin-releasing hormone that appears to be more restricted to the central nervous system particularly to the sensory neurons of spinothalamic tract and spinal cord dorsal horn, which may account for the sensory antinociceptive actions of TRH.     

Cloning and characterization of a cDNA encoding a novel heterogeneous nuclear ribonucleoprotein-like protein and its expression in myeloid leukemia cells

Breast cancer is the second leading cause of cancer-related deaths among women in the United States. Approximately 180,000 new cases of breast cancer are diagnosed each year and a quarter of these are fatal. Early detection is a key to survival of these patients. Unfortunately, no definitive markers are available to diagnose breast cancer at early stages. Identification of such early markers, therefore, is an important priority in breast cancer research. In order to identify early markers, we have focussed on understanding the molecular mechanisms that can lead to conversion of the normal mammary epithelial cells into precancerous immortal cells. Over last several years, we have developed in vitro models of human mammary epithelial cell immortalization which have allowed us to invoke the critical roles of the known tumor suppressor pathways in the maintenance of the untransformed state of mammary epithelial cells. These models are now being used to identify novel genes whose expression is important for normal mammary epithelial cell growth and whose altered expression contributes to breast cell transformation. Characterization of the molecular machinery whose alterations result in early preneoplastic transformation should help identify candidate genes for evaluation as potential early diagnostic markers.     

Cloning, sequencing and expression of a cDNA encoding mammalian valyl-tRNA synthetase

The incus of the right ear from 4 growing mongrel dogs was surgically disarticulated and left in the middle ear space. The external auditory canal was then filled with teflon paste and sutured. After 6 weeks (D-6 group) and 13 weeks (D-13 group) the animals were sacrificed. The right experimental incus and the left control one were embedded in methyl methacrylate and sectioned in single 50-microns-thick sections according to the principal axis of the two processes. On the microradiographs of each section we evaluated the thickness of the body and of both processes and the percentage area of the primary channels of the secondary osteons and that of the appositional bone tissue. The thickness of the body and of the two processes was more pronounced in all the experimental incuses, in which 6% (in D-6) and 8% (in D-13) of the total area were occupied by new appositional woven bone. In the body of the D-13 group, 9% of the pre-existing bone was substituted by secondary osteons. The results indicate that the incus react to the variations of mechanical stimuli.     

Cloning and expression of a cDNA encoding a bovine brain brefeldin A-sensitive guanine nucleotide-exchange protein for ADP-ribosylation factor

A 200-kDa guanine nucleotide-exchange protein (p200 or GEP) for ADP-ribosylation factors 1 and 3 (ARF1 and ARF3) that was inhibited by brefeldin A (BFA) was purified earlier from cytosol of bovine brain cortex. Amino acid sequences of four tryptic peptides were 47% identical to that of Sec7 from Saccharomyces cerevisiae, which is involved in vesicular trafficking in the Golgi. By using a PCR-based procedure with two degenerate primers representing sequences of these peptides, a product similar in size to Sec7 that contained the peptide sequences was generated. Two oligonucleotides based on this product were used to screen a bovine brain library, which yielded one clone that was a partial cDNA for p200. The remainder of the cDNA was obtained by 5' and 3' rapid amplification of cDNA ends (RACE). The ORF of the cDNA encodes a protein of 1,849 amino acids (approximately 208 kDa) that is 33% identical to yeast Sec7 and 50% identical in the Sec7 domain region. On Northern blot analysis of bovine tissues, a approximately 7.4-kb mRNA was identified that hybridized with a p200 probe; it was abundant in kidney, somewhat less abundant in lung, spleen, and brain, and still less abundant in heart. A six-His-tagged fusion protein synthesized in baculovirus-infected Sf9 cells demonstrated BFA-inhibited GEP activity, confirming that BFA sensitivity is an intrinsic property of this ARF GEP and not conferred by another protein component of the complex from which p200 was originally purified.     

Cloning and sequence analysis of cDNA encoding rat carboxypeptidase D

Carboxypeptidase D (CPD) is a recently described 180-kD enzyme with carboxypeptidase E-like enzymatic properties. CPD has been proposed to be present in the secretory pathway and to contribute to peptide hormone processing in the Cpe(fat)/Cpe(fat) mouse, which lacks functional CPE. Sequence analysis of cDNA clones encoding rat CPD show the protein to contain an amino-terminal signal peptide, three carboxypeptidase-like domains, a putative transmembrane domain, and a 60-amino-acid cytoplasmic tail. Whereas active site, substrate-binding, and metal-binding residues of other metallocarboxypeptidases are conserved in the first two domains of CPD, several of the critical residues are not conserved in the third domain; this third domain is not predicted to form an active carboxypeptidase. The overall homology between rat CPD and the duck homolog gp180 is high, with 75% amino acid identity. The three carboxypeptidase domains show 66%, 83%, and 82% amino acid identity between rat CPD and duck gp180. Homology is also high in the transmembrane domain (86%) and in the cytoplasmic tail (97%). The mouse Cpd gene maps to the medial portion of chromosome 11, approximately 45.5 cM distal to the centromere. Northern blot analysis of CPD mRNA shows major bands of approximately 8 and 4 kb in many rat tissues, and additional species ranging from 1.4 to 5 kb that are expressed in some tissues or cell lines. CPD mRNA is detectable in most tissues examined, and is most abundant in hippocampus, spinal cord, atrium of the heart, colon, testis, and ovaries. In situ hybridization of CPD mRNA shows a distribution in many cells in rat brain and other tissues, with high levels in hippocampus, olfactory bulb, and the intermediate pituitary. The broad distribution is consistent with a role for CPD in the processing of many peptides and proteins that transit the secretory pathway.     

Cloning, expression, and characterization of a cDNA encoding a novel human growth factor for primitive hematopoietic progenitor cells

Multiple growth factors synergistically stimulate proliferation of primitive hematopoietic progenitor cells. A human myeloid cell line, KPB-M15, constitutively produces a novel hematopoietic cytokine, termed stem cell growth factor (SCGF), possessing species-specific proliferative activities. Here we report the molecular cloning, expression, and characterization of a cDNA encoding human SCGF using a newly developed lambdaSHDM vector that is more efficient for differential and expression cloning. cDNA for SCGF encodes a 29-kDa polypeptide without N-linked glycosylation. SCGF transiently produced by COS-1 cells supports growth of hematopoietic progenitor cells through a short-term liquid culture of bone marrow cells and exhibits promoting activities on erythroid and granulocyte/macrophage progenitor cells in primary semisolid culture with erythropoietin and granulocyte/macrophage colony-stimulating factor, respectively. Expression of SCGF mRNA is restricted to myeloid cells and fibroblasts, suggesting that SCGF is a growth factor functioning within the hematopoietic microenvironment. SCGF could disclose some human-specific mechanisms as yet unidentified from studies on the murine hematopoietic system.     

Isolation of a cDNA encoding Fasciola hepatica cathepsin L2 and functional expression in Saccharomyces cerevisiae

Cathepsin L2 is a major cysteine proteinase secreted by adult Fasciola hepatica. The enzyme differs from other reported cathepsin Ls in that it can cleave peptide substrates that contain proline in the P2 position. A cDNA was isolated from an expression library by immunoscreening with antiserum prepared against purified native cathepsin L2. This cDNA was sequenced and shown to encode a complete preprocathepsin L proteinase. Functionally active recombinant cathepsin L proteinase was expressed and secreted by Saccharomyces cerevisiae transformed with the cDNA. The recombinant enzyme was purified from large-scale fermentation broths using ultrafiltration and gel filtration chromatography on Sephacryl S200 HR columns. NH2-terminal amino acid sequencing showed that the cleavage point for activation of the recombinant pro-enzyme is identical to that of the F. hepatica-produced cathepsin L2. The mature active recombinant proteinase behaved similarly to the native enzyme when analysed by SDS-PAGE, immunoblotting and zymography and also cleaved peptides containing proline in the P2 position. Finally, the recombinant cathepsin L2 cleaved fibrinogen to form a fibrin clot, a property we described for F. hepatica cathepsin L2.     

Isolation and expression of a rat brain cDNA encoding glutamate carboxypeptidase II

N-acetylated alpha-linked acidic dipeptidase (NAALADase) hydrolyzes acidic peptides, such as the abundant neuropeptide N-acetyl-alpha-L-aspartyl-L-glutamate (NAAG), thereby generating glutamate. Previous cDNA cloning efforts have identified a candidate rat brain NAALADase partial cDNA, and Northern analyses have identified a family of related RNA species that are found only in brain and other NAALADase-expressing cells. In this report, we describe the cloning of a set of rat brain cDNAs that describe a full-length NAALADase mRNA. Transient transfection of a full-length cDNA into the PC3 cell line confers NAAG-hydrolyzing activity that is sensitive to the NAALADase inhibitors quisqualic acid and 2-(phosphonomethyl)glutaric acid. Northern hybridization detects the expression of three similar brain RNAs approximately 3,900, 3,000, and 2,800 nucleotides in length. In situ hybridization histochemistry shows that NAALADase-related mRNAs have an uneven regional distribution in rat brain and are expressed predominantly by astrocytes as demonstrated by their colocalization with the astrocyte-specific marker glial fibrillary acidic protein.     

Identification of a novel cDNA clone encoding protein tyrosine kinase in murine skin

Tyrosine phosphorylation is widely recognized as playing important roles in cell differentiation, proliferation, and carcinogenesis. We have used the polymerase chain reaction (PCR) method to identify protein tyrosine kinases that are expressed in the skin. Mixed oligonucleotide probes were used to amplify and screen a neonatal murine skin cDNA pool for clones encoding amino acid contiguities whose conservation is characteristic of the protein tyrosine kinase family. When the PCR products were sequenced, 13 distinct clones were found, of which one is novel to date and has provisionally been named tks (for tyrosine kinase identified from skin). Sequence homology comparison showed that the tks gene is homologous to the src and fes/fps families. Northern blotting using PCR products of tks as a probe revealed that the mRNA of tks is detected ubiquitously and weakly in other tissues such as brain, lung, liver, thymus and kidney. This fact suggests that the tks gene is expressed in widely distributed cell types.     

Cloning of a cDNA encoding SjIrV1, a Schistosoma japonicum calcium-binding protein similar to calnexin, and expression of the recombinant protein in Escherichia coli

Membrane-associated proteins were isolated from adult Philippine strain Schistosoma japonicum by partitioning into the detergent phase of Triton X-114. A rabbit polyclonal antiserum raised against these proteins was used to screen an S. japonicum expression cDNA library. Positive clones were identified which encoded the species orthologue of SmIrV1, a Schistosoma mansoni protein which was initially identified by screening with sera from mice protectively vaccinated with irradiated cercariae [Hawn et al., J. Biol. Chem. 268 (1993) 7692-7698]. The S. japonicum molecule, which we term SjIrV1, is 83% identical to SmIrV1 at the predicted amino acid level and is a member of the calreticulin family of non-EF-hand, calcium-binding proteins. The Chinese strain S. japonicum orthologue of SjIrV1 was obtained by screening with the radiolabelled insert of the Philippine strain clone. Northern blot analysis revealed a single message of around 2.4 kb and gave no indication of alternative splicing. Southern blot analysis gave a simple pattern, indicating a single-copy gene, and showed a single restriction fragment length polymorphism between the genomes of Chinese and Philippine strains of S. japonicum. Recombinant, full-length SjIrV1 was expressed with a hexahistidine tag in Escherichia coli and the recombinant protein isolated by nickel-chelate chromatography. Recombinant SjIrV1 was shown to exhibit calcium-dependent, differential electrophoretic migration and to bind ruthenium red in the absence but not in the presence of calcium ions. The presence of conserved Ca(2+)-binding motifs predicted from the primary sequence, together with the Ca(2+)-dependent electrophoretic mobility of recombinant SjIrV1, confirmed that SjIrV1 was a functional calcium-binding protein.     

Cloning, sequencing, and heterologous expression of rat methionine synthase cDNA

Methionine synthase catalyzes cobalamin-dependent methyl transfer reaction from 5-methyltetrahydrofolate to homocysteine, forming methionine. Rat methonine synthase cDNA was cloned and analyzed by RT-PCR, 3'- and 5'-RACE techniques. The cDNA consists of a 0.3-kb upstream untranslated region, a 3.8-kb coding region, and a 0.4-kb downstream untranslated region. The open reading frame encoded a polypeptide of 1,253 amino acid residues with a calculated molecular weight of 139,162. This molecular weight was in good agreement with the observed one (143,000) of the purified rat liver enzyme. The deduced amino acid sequence was 53, 92, and 64% identical with those of the Escherichia coli, human, and presumptive Caenorhabditis elegans enzymes, respectively. All the fingerprint sequences, forming parts of the cobalamin- and S-adenosylmethionine-binding sites, were completely conserved in the rat methionine synthase. A high-level expression of catalytically active enzyme in insect cells was done by infection with a baculovirus containing the rat methionine synthase cDNA.     

Cloning and functional expression of the cDNA encoding a novel ATP-sensitive potassium channel subunit expressed in pancreatic beta-cells, brain, heart and skeletal muscle

A cDNA clone encoding an inwardly-rectifying potassium channel subunit (Kir6.2) was isolated from an insulinoma cDNA library. The mRNA is strongly expressed in brain, skeletal muscle, cardiac muscle and in insulinoma cells, weakly expressed in lung and kidney and not detectable in spleen, liver or testis. Heterologous expression of Kir6.2 in HEK293 cells was only observed when the cDNA was cotransfected with that of the sulphonylurea receptor (SUR). Whole-cell Kir6.2/SUR currents were K(+)-selective, time-independent and showed weak inward rectification. They were blocked by external barium (5 mM), tolbutamide (Kd = 4.5 microM) or quinine (20 microM) and by 5 mM intracellular ATP. The single-channel conductance was 73 pS. Single-channel activity was voltage-independent and was blocked by 1 mM intracellular ATP or 0.5 mM tolbutamide. We conclude that the Kir6.2/SUR channel complex comprises the ATP-sensitive K-channel.     

Cloning and analysis of a cDNA encoding the BALB/c murine erythrocyte band 7 integral membrane protein

Phospholipid metabolism abnormalities have been suggested by a number of postmortem brain and red blood cell studies in schizophrenia. 31P magnetic resonance spectroscopy enables the examination of phospholipid metabolism in living patients. These in vivo studies have demonstrated that schizophrenic patients have lower prefrontal levels of phosphomonoesters and higher levels of phosphodiesters compared to matched controls. Patients with psychotic depression also seem to show lower levels of phosphomonoesters compared to controls. This suggests that membrane phospholipid differences may not be specific to schizophrenia. Preliminary 31P magnetic resonance spectroscopy studies at high field strength on postmortem temporal lobe samples show no differences between treated schizophrenic patients and controls for phosphoethanolamine and phosphocholine which are the main constituents of the phosphomonoester peak. Further studies are underway in the prefrontal region. While 31P magnetic resonance spectroscopy studies have demonstrated membrane phospholipid abnormalities in schizophrenia, it is not clear whether these findings are specific to schizophrenia or part of a generalized membrane phospholipid abnormality.