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.

Identification of bacterial clones encoding bovine caseins by direct immunological screening of the cDNA library.

A sensitive immunoassay was used to identify recombinant plasmids carrying cDNA fragments of bovine caseins in the cDNA library from bovine mammary gland mRNA. Colonies grown on nitrocellulose filters were lysed in situ and proteins from the lysates were blotted onto CNBr-activated cellulose filter paper. Antigens covalently bound to CNBr-activated paper or bound to nitrocellulose filters were detected by reaction with antiserum to caseins, followed by 125I-labelled Staphylococcus aureus protein A and autoradiography. Six clones were found positive among 5400 of the cDNA library: 3-A1, 3-B2, 3-B5, 3-H7, 2-A5 and 2-C9. The molecular weights of chimeric pre-beta-lactamase: casein proteins synthesized in Escherichia coli were estimated by immunoblotting. Colony hybridization and nucleotide sequence analysis showed that clone 3-B5 contained a cDNA fragment of bovine chi-casein, clone 3-H7 contained a cDNA fragment of beta-casein, while clones 2-A5 and 2-C9 carried cDNA fragments of alpha s1-casein.

Organization and chromosomal localization of the gene for the human bombesin receptor subtype expressed in pregnant uterus.

The gene encoding the human homologue of the guinea pig uterine bombesin receptor [(1992) Eur. J. Biochem. 208, 405] was isolated from a genomic lambda library by the PCR/homology screening approach. The gene spans more than 4 kb and consists of 3 exons and 2 introns. The deduced amino acid sequence shows about 86% identity to that of guinea pig bombesin receptor. This subtype of bombesin receptor is expressed in the pregnant uterus and in two human tumour cell lines, T47D (ductal breast carcinoma) and A431 (epidermal carcinoma). PCR analysis of genomic DNA from human-mouse cell hybrids allows the cloned gene to be localized to the region q26-q28 on chromosome X.

[Identification of bacterial clones that encode cow's caseins by direct immunological screening of the cDNA library]. / Identifikatsiia bakterial'nykh klonov, kodiruiushchikh kazeiny korovy, priamym immunologicheskim skriningom banka kDNK.

A sensitive immunoassay was used to identify recombinant DNA plasmids carrying cDNA fragments of bovine caseins in the cDNA library from rRNA of bovine mammary glands. Colonies grown on nitrocellulose filters were lysed in situ and proteins from the lysates were blotted onto CNBr-activated filter paper. Antigens covalently bound to the CNBr-activated paper or bound to the nitrocellulose filters were detected by reaction with antiserum to caseins, followed by 125I-labeled protein A from Staphylococcus aureus and autoradiography. Four clones were positive among 5400 bacterial clones of the cDNA library--al, b2, b5, h7. Molecular weights of chimeric proteins pre-beta-lactamase:casein synthesized in Escherichia coli were determined by immunoblotting. Colony hybridization and DNA sequence analysis showed that clone b5 contained cDNA fragment of bovine kappa-caseins and clone h7 cDNA fragment of beta-casein. The last clone was designated pKcas beta-7.

Molecular cloning and functional characterization of V2 [8-lysine] vasopressin and oxytocin receptors from a pig kidney cell line.

[Arg8]vasopressin and oxytocin are the two main members of the neurohypophysial hormone family found to be present in nearly all mammals. [Lys8]vasopressin ([Lys8]VP) has been identified as the antidiuretic hormone in pig and some marsupial families. The porcine-derived kidney epithelial cell line, LLC-PK1, expresses both [Lys8]VP receptors coupled to the activation of adenylate cyclase (V2 receptors) and oxytocin receptors. Here we report the molecular cloning of the V2 [Lys8]VP receptor and the oxytocin receptor from LLC-PK1 cells. The cloned V2 [Lys8]VP receptor differs from human and rat V2 [Arg8] receptors mainly in its N-terminal region, in residues located in the extracellular loops and in intracellular phosphorylation sites. When expressed in COS7 cells, the V2 [Lys8]VP receptor exhibits the relative order of ligand affinity [Lys8]VP = [Arg8]VP >> 1-deamino[D-Arg8]VP > or = oxytocin and adenylate-cyclase stimulation, expected for the porcine V2 [Lys8]VP receptor but different from V2 [Arg8]VP receptors. Adenylate-cyclase activation by [Lys8]VP was inhibited in COS7 cells by a V2 antagonist. The cloned oxytocin receptor exhibits in COS7 cells a ligand specificity typical of mammalian oxytocin receptors. mRNA-distribution analysis revealed a single 5.5-kb transcript in the uterus from pregnant guinea pig.

Molecular cloning of a new bombesin receptor subtype expressed in uterus during pregnancy.

The homology screening approach has been used to clone a new member of the guanine-nucleotide-binding-protein-coupled receptor superfamily from guinea pig uterus. The cloned cDNA encodes a 399-amino-acid protein and shows the highest amino acid similarity to members of the bombesin receptor family; 52% and 47% similarity to the gastrin-releasing-peptide (GRP) receptor and the neuromedin-B receptor, respectively. Binding experiments with the stably transfected LLC-PK1 cell line expressing the new receptor protein confirmed the bombesin-like nature of the cloned receptor. The relative order of ligand affinity, GRP = neuromedin C much greater than neuromedin B, suggests that the cloned cDNA represents the GRP subtype rather than the neuromedin-B subtype of bombesin receptors. Northern-blot analysis of mRNA species from several guinea-pig tissues showed that the mRNA for the new bombesin receptor subtype is expressed mainly in uteri of pregnant animals.

[In vitro RNA-synthesis on immobilized DNA-templates]. / Sintez RNK in vitro na immobilizovannykh DNK-matritsakh.

The possibility of RNA-synthesis by E. coli RNA-polymerase using denatured DNA-templates from mouse liver, immobilized on nitrocellulose filters was shown. The size of RNA molecules, synthesized on immobilized templates was estimated by electrophoresis in polyacrylamide gel. The length of the RNA molecules was found to be about 30 nucleotides. Data from alkaline hydrolisys and sedimentation in sucrose density gradient suggest that there is no connection between the DNA-primer and the RNA-product, therefore the DNA-primer is not necessary for the initiation of RNA-synthesis.

[mRNA localization and content in bovine kininogen]. / Lokalizatsiia mesta sinteza i soderzhanie mRNK kininogena byka.

The bradykinin (BK) gene chemically synthesized and cloned into pBR 322 was used for the study of tissue localization and quantitation of mRNA coding for the BK precursor, kininogen. Poly(A+)-mRNAs from bovine liver, spleen, kidney, mammary gland and pancreas were used for dot-hybridization to [32P]DNA of the BK gene or to the plasmid-containing BK gene. The experimental results demonstrate that [32P]DNA of BK is hybridized only to the liver poly(A+)-RNA, which proves the liver to be the main kininogen mRNA-producing tissue. In other tissues, the kininogen mRNA synthesis is either altogether absent or its level is two orders of magnitude less as compared to the liver. Several approaches for the quantitation of the kininogen mRNA were developed. The amount of this mRNA was shown to be about 0.6% of the total cellular poly(A+)-RNA. Poly(A+)-RNA which is bound to BK DNA-cellulose and is enriched with BK-coding sequences was used for the study of the hybridization kinetics and translation in a cell-free system from rabbit reticulocytes. The polypeptides synthesized contain BK as was shown by the use of a bio-test in rat uterus.

Cloning and functional characterization of the amphibian mesotocin receptor, a member of the oxytocin/vasopressin receptor superfamily.

Mesotocin is the oxytocin-like hormone found in most terrestrial vertebrates from lungfishes to marsupials, which includes all non-mammalian tetrapods (amphibians, reptiles, and birds). It has the largest distribution in vertebrates after vasotocin found in all non-mammalian vertebrates and isotocin identified in bony fishes. In this study, we report the cloning and functional characterization of the cDNA for the mesotocin receptor (MTR) from the urinary bladder of the toad Bufo marinus. The cloned cDNA encodes a polypeptide of 389 amino acids that shows the greatest similarity to the teleost fish isotocin receptor and to mammalian oxytocin receptors with mutations in extracellular loops which are involved in ligand binding. When expressed in COSM6 cells, MTR exhibits the following relative order of ligand affinity: mesotocin > vasotocin = oxytocin > vasopressin > hydrin 1, isotocin, hydrin 2. Injection of MTR cRNA into Xenopus laevis oocytes induces membrane chloride currents in response to mesotocin, which indicates the coupling of the mesotocin receptor to the inositol phosphate/calcium pathway. This response is inhibited by an oxytocin antagonist, but not by a vasopressin antagonist specific for V2 vasopressin receptors. MTR mRNA is not only found in toad urinary bladder, but also in kidney, muscle, and brain tissue of the toad as revealed by northern blot analysis and reverse-transcriptase PCR. The results suggest a variety of function for mesotocin and its receptor including, in particular, an involvement in the regulation of water and salt transport.