Cloning and localization of rgpr85 encoding rat G-protein-coupled receptor

In an attempt to isolate genes involved in the brain development using ordered differential display PCR, we cloned rgpr85 which encodes rat G-protein-coupled receptor with high degree of identity to the amine-like neurotransmitter receptors. This gene was found to be localized at rat chromosome 4q21. In situ hybridization demonstrated that rgpr85 was predominantly expressed in the developing brain and spinal cord. Hybridization signal was especially abundant within the embryonic cortical plates where postmitotic cortical neurons are localized. In the cerebral cortex, the expression of rgpr85 was gradually decreased postnatally and became undetectable by P18. However, weak but significant expression of rgpr85 was maintained in the adult hippocampal formation, olfactory bulb, and cerebellum. Interestingly, rgpr85 expression was transiently induced in the adult hippocampal formation, piriform cortex, and amygdaloid complex by kainic acid (KA) treatment. Thus, dynamic regulation of rgpr85 expression suggests an importance of rgpr85-mediated signaling in the development of cerebral cortex and in the KA-induced responses in the adult brain.     

Cloning and chromosomal mapping of four putative novel human G-protein-coupled receptor genes

We report the discovery of four novel human putative G-protein-coupled receptor (GPCR) genes. Gene GPR20 was isolated by amplifying genomic DNA with oligos based on the opioid and somatostatin related receptor genes and subsequent screening of a genomic library. Also, using our customized search procedure of a database of expressed sequence tags (dbEST), cDNA sequences that partially encoded novel GPCRs were identified. These cDNA fragments were obtained and used to screen a genomic library to isolate the full-length coding region of the genes. This resulted in the isolation of genes GPR21, GPR22 and GPR23. The four encoded receptors share significant identity to each other and to other members of the receptor family. Northern blot analysis revealed expression of GPR20 and GPR22 in several human brain regions while GPR20 expression was detected also in liver. Fluorescence in situ hybridization (FISH) was used to map GPR20 to chromosome 8q, region 24.3–24.2, GPR21 to chromosome 9, region q33, GPR22 to chromosome 7, region q22–q31.1, and GPR23 to chromosome X, region q13–q21.1.© 1997 Elsevier Science B.V. All rights reserved.     

Cloning and functional analysis of a gene encoding a novel muscarinic acetylcholine receptor expressed in chick heart and brain

Previous studies have demonstrated that muscarinic acetylcholine receptors (mAChR) expressed in chick heart are pharmacologically, immunologically, and biochemically distinct from mAChR expressed in mammalian heart. A chicken genomic clone encoding a mAChR whose deduced amino acid sequence is most homologous to the mammalian m4 receptor has been isolated. Northern blot analysis demonstrated that this gene is expressed in both chick heart and brain. The receptor encoded by this gene was expressed in stably transfected Chinese hamster ovary (CHO) and Y1 adrenal carcinoma cells in order to examine its ligand binding and functional properties. The receptor expressed in CHO and Y1 cells exhibits high affinity binding for the muscarinic antagonists quinuclidinyl benzilate and atropine, as well as the M1-selective antagonist pirenzepine and the M2-selective antagonist AF-DX 116. Therefore, when expressed in two heterologous cell lines, the cloned chick m4 receptor exhibits pharmacological properties similar to those previously reported for the chick cardiac receptor. This m4 receptor was able to mediate both agonist-dependent inhibition of forskolin-stimulated cAMP accumulation and agonist-dependent stimulation of phosphoinositide metabolism when expressed in CHO cells. In contrast, when expressed in Y1 cells, the chick m4 receptor mediated agonist-dependent inhibition of forskolin-stimulated cAMP accumulation, but not stimulation of phosphoinositide metabolism. Thus, as with the mammalian cardiac (m2) receptor, the functional specificity of the chick cardiac receptor appears to be dependent on the cell type in which it is expressed.     

Cloning of cDNA encoding rat TCP-1.

We have isolated and sequenced a cDNA encoding a rat homolog of the mouse t-complex polypeptide 1 (TCP-1). Its deduced gene product is a polypeptide of 556 amino acids, with a predicted Mr of 60,341. The similarity between mouse Tcp-1 and the rat homolog is about 94.0% at the nucleotide level and 97.1% at the amino acid level showing the evolutionary conservation of this protein. The similarity of the amino acid sequence of the rat TCP-1 is not significantly biased to any of those from wild (TCP-1B) or from t-haplotype mice (TCP-1A). From a comparison of deduced amino acid sequences of eukaryotic TCP-1 proteins, we found highly conserved domains. Southern blot analysis revealed that there are at least two similar sequences to Tcp-1 in the rat, one is a structural gene and the other seems to be a processed pseudogene.     

Molecular cloning of a novel putative G-protein coupled receptor expressed during rat spermiogenesis.

A cDNA clone encoding a novel putative G-protein coupled receptor has been isolated from a rat testis cDNA library using a PCR-amplified cDNA fragment as a hybridization probe. Northern blot analysis reveals that a corresponding 1.5 kb mRNA is exclusively expressed in testis. By in situ hybridization experiments this mRNA has been localized in spermatocytes and spermatids but not in spermatogonia, Leydig or sertoli cells. Ontogenic studies show that expression of the receptor-encoding mRNA and sexual maturation are correlated reaching highest levels during the second and third months. Although the ligand for this receptor has not yet been identified, this receptor may play a role during reproduction.     

A cDNA encoding a putative lipid transfer protein expressed in sunflower seeds

Based on the N-terminal sequence of a sunflower antifungal protein, a full length cDNA (Ha-LTP5) encoding a putative lipid transfer protein from sunflower seeds was cloned using a RT-PCR based strategy. However, the sequence of the deduced protein is not identical to that of the antifungal protein previously isolated. The nucleotide sequence presents an ORF of 116 amino acids with a putative signal peptide, thus encoding a mature protein of 90 amino acids that is basic and hydrophobic. In contrast to the pattern of expression described for most LTP-like genes from dicots, Northern blot analyses detected constitutive expression of Ha-LTP5 in seeds, but not in aerial parts of sunflower plants.     

Cloning of a glycine receptor subtype expressed in rat brain and spinal cord during a specific period of neuronal development.

Complementary (c) DNAs encoding a glycine receptor (GlyR) isomer were cloned from libraries constructed in lambda ZAPII with poly (A)+ RNA of neonatal rat spinal cord. Northern blot analysis revealed that RNA hybridized to the cloned cDNA is detectable only for a period of late embryonic/early postnatal stage of the spinal cord. Moreover, other central nervous tissues, such as hippocampus and cerebral cortex, in the infant rats are also rich in this message. The 'neonatal (N) GlyR' has 71% homology to that of another GlyR isoform in which adult rad cord is rich (AGlyR). Injection of a single RNA transcribed from the NGlyr-cDNA into Xenopus oocyte induced functional formation of glycine-gated Cl- channels, however, its pharmacological property differed from that of AGlyR.     

Cloning and characterization of a cDNA encoding a novel fibroblast growth factor preferentially expressed in human heart

A novel human fibroblast growth factor (hFGF), which shows 75% sequence homology with fibroblast growth factor-9, was isolated in random sequencing of a human heart cDNA library. The full-length sequence is 928 bp, the encoded protein is composed of 168 amino acid residues, and its pI value and molecular weight were estimated to be 8.13 and 19.1 kDa, respectively. RT-PCR using Marathon human heart cDNA shows that the coding region is approximately 507 bp. Southern hybridization showed a single band which indicates that this is a single copy gene. Northern hybridization done on a human multiple tissues blot showed that the gene is preferentially expressed in human heart, very weakly detectable in human brain and not detectable in 18 other different human tissues.     

Cloning of a cDNA encoding porcine brain natriuretic peptide

Complimentary DNA (cDNA) clones encoding porcine brain natriuretic peptide (BNP) were isolated from a porcine atrial cDNA library. The longest of the cDNA clones (1507 nucleotides) apparently originated from an unprocessed messenger RNA, since the nucleotide sequence encoding BNP-26 was interrupted by an intron of 554 nucleotides. A partial cDNA clone representing processed BNP mRNA was prepared by polymerase chain reaction. A comparison of the sequence of these two cDNAs reveals the presence of an additional intron within the sequence encoding the BNP precursor. The identification of these introns suggests that the BNP gene structure differs from the atrial natriuretic peptide gene in the location of intron 2. BNP mRNA encodes a propeptide of 131 amino acids, including a signal peptide domain (25 amino acids) and a prohormone domain (106 amino acids). Like atrial natriuretic peptide, the bioactive BNP sequence is localized at the carboxyl terminus of the prohormone. Although the carboxyl-terminal peptide sequences of porcine atrial natriuretic peptide and BNP are well conserved, there is relatively little homology within their propeptide regions.     

Cloning and sequence analysis of cDNA encoding a precursor for rat brain natriuretic peptide

Brain natriuretic peptide (BNP) is a new type of natriuretic peptide, which has so far been identified only in porcine brain and atrium. Immunological observations suggest that rat and porcine BNP may have structural difference according to species. To identify rat BNP, we constructed a rat atrial cDNA library, and screened for clones encoding rat BNP-precursor by using part of porcine BNP cDNA as a probe. By sequencing a cloned cDNA, the amino acid sequence of rat BNP-precursor comprising 121 residues was deduced as carrying a 26-residue putative signal peptide at the N-terminus and a region homologous to porcine BNP-32 at the C-terminus. In addition, remarkably high homology between rat and porcine BNP-precursors was observed in the 3'-untranslated AT-rich region. Comparing sequences of precursors of ANP and BNP thus far identified, structural and processing features characteristic of the BNP family were discussed.     

Cloning and characterization of osteoactivin, a novel cDNA expressed in osteoblasts.

Osteoblast development is a complex process involving the expression of specific growth factors and regulatory proteins that control cell proliferation, differentiation, and maturation. In this study, we used the rat mutation, osteopetrosis (op), to examine differences in skeletal gene expression between mutant op and normal littermates. Total RNA isolated from long bone and calvaria was used as a template for mRNA differential display. One of many cDNAs that were selectively expressed in either normal or mutant bone was cloned and sequenced and found to share some homology to the human nmb and Pmel 17 genes. This novel cDNA was named osteoactivin. Osteoactivin has an open reading frame of 1716 bp that encodes a protein of 572 amino acids with a predicted molecular weight of 63.8 kD. Protein sequence analysis revealed the presence of a signal peptide and a cleavage site at position 23. The protein also has thirteen predicted N-linked glycosylation sites and a potential RGD integrin recognition site at position 556. Northern blot analysis confirmed that osteoactivin was 3- to 4-fold overexpressed in op versus normal bone. RT-PCR analysis showed that osteoactivin is most highly expressed in bone compared with any of the other non-osseous tissues examined. In situ hybridization analysis of osteoactivin in normal bone revealed that it is primarily expressed in osteoblasts actively engaged in bone matrix production and mineralization. In primary rat osteoblast cultures, osteoactivin showed a temporal pattern of expression being expressed at highest levels during the later stages of matrix maturation and mineralization and correlated with the expression of alkaline phosphatase and osteocalcin. Our findings show that osteoactivin expression in bone is osteoblast-specific and suggest that it may play an important role in osteoblast differentiation and matrix mineralization. Furthermore, osteoactivin overexpression in op mutant bone may be secondary to the uncoupling of bone resorption and formation resulting in abnormalities in osteoblast gene expression and function.     

Molecular cloning and characterization of a rat brain cDNA encoding a 5-hydroxytryptamine1B receptor.

To date, there have been at least eight different receptors for the neurotransmitter serotonin (5-HT) identified in the central nervous system. These receptors fall into four pharmacological classes: 5-HT1, 5-HT2, 5-HT3 and 5-HT4. The 5-HT1 class has been shown to contain at least four pharmacologically distinct subtypes, 5-HT1A-D. Of these, cDNAs encoding the 5-HT1A and 5-HT1C receptors have been previously characterized. We now report the cloning and expression of a rat brain cDNA encoding another member of the 5-HT1 receptor family. Transient expression of this clone demonstrated high-affinity binding of [3H]5-HT with a pharmacological profile corresponding to that of the 5-HT1B subtype: 5-CT, 5-HT greater than propranolol greater than methysergide greater than rauwolscine greater than 8-OH-DPAT. In situ hybridization revealed expression of cognate mRNA within cells of the dorsal and median raphe nuclei, consistent with previous reports that the 5-HT1B receptor acts as an autoreceptor on 5-HT terminals in this species. mRNA expression was also detected in cells within the CA1 region of hippocampus, striatum, layer 4 of cortex and in the cerebellum, suggesting a previously unrecognized post-synaptic role for the 5-HT1B receptor.