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.

An extracellular residue determines the agonist specificity of V2 vasopressin receptors.

The specific V2 agonist 1-deamino [8-D-arginine]-vasopressin (dDAVP), used for treatment of central diabetes insipidus, binds to vasopressin V2 receptors from human, bovine and rat kidney with an affinity that is similar to that of the natural hormone vasopressin. In contrast, the V1 receptors and the porcine V2 receptor do not tolerate a D-arginine in position 8 of vasopressin. By site directed mutagenesis of the cloned bovine and porcine V2 receptors we identified a residue (Asp-103) in the first extracellular loop of vasopressin receptors which is responsible for high affinity binding of dDAVP.

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.

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.

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.

[Biosynthesis and secretion of bovine growth hormone in Escherichia coli under the control of the secretory vector containing a promoter and signal region of the alkaline phosphatase gene]. / Biosintez i sekretsiia gormona rosta byka u Escherichia coli pod kontrolem sekretornogo vektora soderzhashchego promotor i signal'nuiu posledovatel'nost' gena shchelochnoi fosfatazy.

A recombinant plasmid carrying a bovine growth hormone gene fused with the regulatory and signal regions of the alkaline phosphatase gene of E. coli was constructed. The bovine growth hormone gene expression as well as protein partial processing and secretion into the periplasm have been shown to take place under phosphate starvation, i.e. conditions of alkaline phosphatase derepression.

[Genetic engineering of peptide hormones. III. Cloning of the swine growth hormone cDNA and construction of the gene for expression of the hormone in bacteria]. / Geneticheskaia inzheneriia peptidnykh gormonov. III. Klonirovanie kDNK gormona rosta svin'i i konstruirovanie gena dlia ékspressii gormona v bakteriiakh.

The clones containing cDNA of porcine growth hormone were obtained using poly(A)-RNA from porcine pituitary as a template for reverse transcriptase. The analysis of their nucleotide sequences revealed that these cDNAs have differences not only on the nucleotide level but also on the amino acid level, i. e. the polymorphism of mRNA and protein occurs in the case of porcine growth hormone. To create the construction for expression of porcine growth hormone in E. coli, the 5'-part of cDNA, coding the first 15 amino acids of the mature hormone, was substituted by the artificial sequence.

[Genetic engineering of peptide hormones. II. Possible polymorphism of bovine preprolactin. Molecular cloning data]. / Geneticheskaia inzheneriia peptidnykh gormonov. II. O vozmozhnom polimorfizme preprolaktina krupnogo rogatogo skota. Dannye molekuliarnogo klonirovaniia.

The primary structure of an insert from a clone isolated from the bovine pituitary cDNA library by hybridization with prolactin-specific probe has been determined. It was found that the rearrangement of cDNA took place in the process of cloning. The rearrangement includes the inversion of 5'-terminal and the deletion of the central part of cDNA. However from the structure of the insert we were able to deduce the sequences of 5'- and 3'-terminal regions of bovine preprolactin mRNA (257 and 551 bases long). The comparison of these sequences with those published earlier revealed several differences in the primary structure. The most essential of them is the additional triplet coding for alanine in position of -22 of the signal peptide. The heterogeneity of bovine preprolactin mRNA in the region coding for the signal peptide is considered to be a consequence of alternative splicing as it was shown for rat preprolactin mRNA.

[Genetic engineering of peptide hormones. I. Cloning and primary structure of cDNA of chicken growth hormone]. / Geneticheskaia inzheneriia peptidnykh gormonov. I. Klonirovanie i pervichnaia struktura kDNK gormona rosta kuritsy.

The cDNA coding for the chicken growth hormone was cloned and sequenced. The 795 base pairs long cDNA insert contains a 5'-untranslated region (35 b.p.), a sequence coding for precursor of growth hormone (648 b.p.), a 3'-untranslated region (96 b.p.) and a poly(A)-tail (16 b.p.). Comparison of the cDNA sequence cloned by us with that published earlier revealed several differences including the additional unique HinfI site at the position corresponding to codons for Leu-87 and Thr-88.

Stability of rubella virus after long-term persistence in human cell line.

Primary infection of HEp-2 cells with rubella virus resulted in non-cytophatic long-term persistent infection. During four years of persistence the virus was produced in sufficient quantities (up to 6 logs PFU/ml) and did not differ from the parental variant in its pathogenicity for BHK-21 or RK-13 cells, or hemagglutinating activity, but formed smaller plaques. Persistent virus preserved the original antigenicity as judged from reciprocal hemagglutination-inhibition or plaque reduction-neutralization tests with polyclonal antisera. Both original and persistent rubella viruses were thermoresistant (T 56 degrees C) and slightly temperature-sensitive. Clonal analysis revealed presence of ts-mutants among both original and persistent virus clones with different degrees of plating efficiency at 40 degrees/34 degrees C. RNA fingerprinting showed only minor changes in persistent rubella virus.

[Genetic engineering of peptide hormones]. / Geneticheskaia inzheneriia peptidnykh gormonov.

The application of different approaches for preparing DNAs coding for peptide hormones was demonstrated. The libraries of human, bovine and porcine pituitaries cDNA were obtained starting from their total mRNAs. Screening of these libraries revealed clones containing human, bovine and porcine growth hormone sequences, cDNAs for bovine ACTH-beta-lipotropin precursor and for bovine and porcine prolactin. The gene of human calcitonin was created by combination of chemical and enzymatic synthesis. This synthetic gene was further cloned in pBR322. The expression of cloned human growth hormone cDNA under control of different Escherichia coli promoters was studied and physico-chemical and biological properties of the growth hormone produced by E. coli were tested.

[Analysis of structural divergence of H1N1 serotype influenza virus individual genes]. / Analiz strukturnoi izmenchivosti individual'nykh genov épidemicheskikh shtammov virusa grippa serotipa H1N1.

A comparative structural analysis of H1N1 influenza virus isolated in 1977 (the A/USSR/90/77 strain) and H1N1 isolates of 1947 (the A/FM/1/47 strain) and 1950 (the A/FW/1/50 strain) have been carried out by oligonucleotide mapping of individual viral RNA segments. Seven of eight genes of A/USSR/90/77 strain have a high degree of homology with corresponding genes of A/FW/1/50 strain, especially the genes coding the NP and P2 proteins. At the same time the gene coding matrix (M) protein has higher structural similarity to the corresponding gene of A/FM/1/47 strain. Based on the results presented one may conclude that the A/USSR/90/77 epidemic strain is a recombinant virus.

On the origin of the H1N1 (A/USSR/90/77) influenza virus.

The influenza virus H1N1 (the A/USSR/90/77 strain) that reappeared in 1977 after the H1N1 influenza viruses had disappeared from the human population, is compared with the A/FM/1/47 and the A/FW/1/50 influenza viruses by the method of oligonucleotide mapping of individual segments of the viral RNAs. Seven genes of the A/USSR/90/77 virus appear to be very similar to the corresponding genes of the A/FW/1/50 virus, whereas the gene coding for the M protein displays considerable homology to the corresponding gene of the A/FM/1/47 virus. The data demonstrate that the A/USSR/90/77 strain is a recombinant virus.

Aminoacylation of barley stripe mosaic virus RNA: polyadenylate-containing RNA has a 3'-terminal tyrosine-accepting structure.

Barley stripe mosaic virus (BSMV) RNA which was previously reported to contain poly(A) sequences (Agranovsky et al., 1978) can be specifically esterified with tyrosine in vitro in the presence of an aminoacyl-tRNA synthetase fraction from wheat embryos. All the three RNA components of the BSMV strain with a three-component genome (Norwich) and both RNA components of a two-component strain (Russian) can be tyrosylated. The poly(A)-containing (bound to oligo(dT)-cellulose) and poly(A)-deficient (not bound to oligo(dT)-cellulose) fractions of BSMV RNA display a similar amino acid-accepting ability. The nucleotide sequence which accepts tyrosine is coupled with the intact genomic polyadenylated BSMV RNA. The viral RNA isolated after sucrose density gradient centrifugation under drastic denaturing conditions retains its aminoacylating activity, which suggests that this activity is not due to the presence in a BSMV RNA preparation of a tyrosine tRNA associated with BSMV RNA. Inhibition of aminoacylation of the 3'-oxidized (treated with sodium metaperiodate) BSMV RNA suggests that the tyrosine-accepting structure is localized at the 3' terminus of BSMV RNA molecules. It is shown that segments of different lengths obtained upon random fragmentation can be tyrosylated. The 3'-terminal (tyrosine-accepting) poly(A)+ segments can be isolated. The shortest segments of viral RNA capable of being aminoacylated [i.e., containing both tRNA-like structure and poly(A)] consists of approximately 150-200 nucleotides. The analysis of the oligonucleotides derived from individual BSMV RNA components labeled with (32)P at the 3' end revealed two types of 3'-terminal sequences different from poly(A). It is suggested that a poly(A) sequence is intercalated between a 3'-terminal tyrosine-accepting structure and the 5'-terminal portion of poly(A)+ BSMV RNA.

Primary structure of baker's yeast tRNAVal 2b.

The minor form of valine tRNA from baker's yeast-tRNAVal 2b--purified by column chromatography was completely digested with guanylo-RNase and pancreatic RNase. The products of these digestions were separated by a combination of thin-layer chromatography on cellulose and high voltage electrophoresis on DEAE-paper and then identified. The halves of tRNA Val 2b were prepared by partial digestion with pancreatic RNase, and their complete guanylo-RNase and pancreatic RNase digests were analysed. Basing on the obtained data the primary structure of baker's yeast tRNA Val 2b was reconstructed.

The separation of oligonucleotides of baker's-yeast value transfer ribonucleic acid 2b by high-voltage electrophoresis on DEAE-paper and by thin-layer chromatography.

A modified procedure for the separation of oligoribonucleotides is described that is based on the combination of t.l.c. on cellulose and electrophoresis on DEAE-paper at 4000 V on a cooling plate. The technique is relatively rapid and allows the analysis of larger quantities than is possible by electrophoresis on cellulose acetate.