Sequence analysis of a cDNA encoding an isotocin precursor and localization of the corresponding mRNA in the brain of the cartilaginous fish Torpedo marmorata.

The nucleotide sequence of a cDNA encoding an isotocin hormone precursor has been elucidated by analyzing a lambda ZAPII library constructed using poly(A)+ RNA from the brain of the cartilaginous fish Torpedo marmorata. The sequence predicts a precursor of 126 amino acid residues that consists of a signal peptide, the isotocin moiety, and a neurophysin carrier protein. In contrast to other known fish isotocin precursor sequences, the Torpedo neurophysin moiety is not extended at its carboxy-terminus by a copeptin-like sequence. The T. marmorata isotocin precursor exhibits highest amino acid sequence identity (61%) to the toad mesotocin precursor. As demonstrated by in situ hybridization, the isotocin mRNA is present in neurons of the preoptic area of the Torpedo brain.

Molecular cloning of a bovine MSH receptor which is highly expressed in the testis.

A novel G protein-coupled receptor (BDF3) was isolated from bovine genomic DNA by a combined approach of polymerase chain reaction (PCR) and hybridization techniques. The predicted amino acid sequence is 317 amino acids in length and displays 80% homology to the human alpha-MSH receptor MC1. Stably transfected into CHO-K1 cells, BDF3 mediates an increase of intracellular cAMP-levels following incubation with NLE-alpha-MSH, a potent alpha-MSH analog. The stimulation with ACTH1-10 is only moderate and gamma-MSH is ineffective. Northern blot analysis of bovine tissues revealed that the BDF3 gene is highly expressed in the testis as a single 2.3 kb mRNA species, suggesting an involvement of the BDF3 receptor in spermatogenesis.

Sequence analysis of the promoter region of the rat somatostatin receptor subtype 1 gene.

Somatostatin receptor (SSTR) subtype genes are differentially expressed in brain and various peripheral tissues. RNA blotting and semiquantitative PCR analyses have revealed low levels of SSTR1 mRNA in the gastrointestinal tract and relatively high levels in GH3 anterior pituitary cells. As a first step in the investigation of the regulation of SSTR1 gene expression, we isolated a genomic fragment that contains the promoter region and determined the transcriptional initiation site. The SSTR1 gene lacks introns and TATA and CAAT motifs, but possesses several consensus recognition sequences for the transcription factors GCF and AP-2. The presence, also, of two Pit-1 binding sites could explain the high SSTR1 mRNA levels in GH3 cells.

Structure, function, and phylogeny of [Arg8]vasotocin receptors from teleost fish and toad.

[Arg8]Vasotocin (AVT) is considered to be the most primitive known vertebrate neurohypophyseal peptide of the vasopressin/oxytocin hormone family and may thus be ancestral to all the other vertebrate peptide hormones. The molecular evolution of the corresponding receptor family has now been studied by cloning an AVT receptor, consisting of 435 amino acid residues, from the teleost fish, the white sucker Catostomus commersoni. Frog oocytes injected with the AVT receptor-encoding cRNA respond to the application of AVT, but not to its structural and functional counterpart isotocin, by an induction of membrane chloride currents indicating the coupling of the AVT receptor to the inositol phosphate/calcium pathway. The pharmacological properties of the expressed AVT receptor show that it represents, or is closely related to, an ancestral nonapeptide receptor: oxytocin, aspargtocin, mesotocin, and vasopressin activated the receptor, but other members of the vasopressin/oxytocin family tested showed little or no potency; antagonists of the mammalian vasopressin V1 and oxytocin receptors blocked the AVT response. Comparison of AVT receptor sequences spanning transmembrane domains two to five, deduced by cloning cDNAs from the Pacific salmon Oncorhynchus kisutch, the cave-dwelling fish Astyanax fasciatus, and the anuran Xenopus laevis, with those of their mammalian counterparts emphasizes amino acid residues that are involved in hormone binding. The presence of a 5.0-kb transcript in various teleost tissues (pituitary, liver, gills, swim bladder, and lateral line) points to a physiological role for the fish AVT receptor in metabolic, osmoregulatory, and sensory processes.

Molecular cloning of a somatostatin-28 receptor and comparison of its expression pattern with that of a somatostatin-14 receptor in rat brain.

The tetradecapeptide somatotropin-release inhibiting factor somatostatin-14 regulates the release of peptide hormones and also functions as neurotransmitter. The octacosapeptide somatostatin-28, the N-terminally extended form of somatostatin-14, shows similar biological activities yet with different potencies. Both peptides most likely function through distinct receptors. Here we report on the molecular and functional characterization of a somatostatin-28 receptor (SSR-28) cloned from a rat brain cDNA library. The nucleotide sequence contains an open reading frame for a protein of 428 amino acid residues with a predicted molecular mass of 47 kDa. Binding assays using radiolabeled somatostatin-14 and membranes from COS cells transfected with the cloned cDNA show that this receptor, SSR-28, has a higher binding affinity for somatostatin-28 (IC50 = 0.24 nM) than for somatostatin-14 (IC50 = 0.89 nM). RNA blot analysis reveals a 4.4-kilobase mRNA in rat cerebellum and at significantly lower abundance in other brain regions. In situ hybridization indicates that SSR-28 mRNA is present in the granular and Purkinje cell layers of the cerebellum and in the large cells of the hypoglossal nucleus of the brain stem. Signals for SSR-28 mRNA do not overlap with those of a previously cloned rat receptor that preferentially binds somatostatin-14 (SSR-14). SSR-14 mRNA is found in the medial cerebellar nucleus, horizontal limb of the diagonal band, various hypothalamic nuclei, and in layers IV and V of the cortex. In the rat cerebellum, SSR-14 and SSR-28 mRNAs are developmentally regulated; the levels of the former are highest around birth and levels of the latter are highest at the adult stage.

Cloning of a cDNA encoding a novel putative G-protein-coupled receptor expressed in specific rat brain regions.

A cDNA clone encoding a novel putative G-protein-coupled receptor was isolated from a rat brain cDNA library using a PCR-amplified cDNA fragment as a hybridization probe. The 3,615-bp-long nucleotide sequence predicts a single open reading frame of 1,173 bp coding for 391 amino acids, giving a calculated molecular weight of 42.75 kD. The amino acid sequence shares features common to many other receptors, including the seven membrane-spanning hydrophobic regions and putative asparagine-linked glycosylation and phosphorylation sites. Northern blot analysis reveals that a corresponding approximately 3.7-kb mRNA is expressed in specific brain regions such as hypothalamus, cortex, hippocampus, and thalamus but not in other organs analyzed. Although the ligand for this receptor has not yet been identified, it shares some similarities with the vascular type-1 angiotensin II receptor, the vasoactive intestinal peptide (VIP) receptor, and the chemotactic receptors for human C5a anaphylatoxin and the formyl peptide fMet-Leu-Phe.

Corticotropin-releasing factor (CRF) gene family in the brain of the teleost fish Catostomus commersoni (white sucker): molecular analysis predicts distinct precursors for two CRFs and one urotensin I peptide.

Molecular cloning experiments indicate the presence of two distinct CRF genes in the sucker genome encoding independent 162-amino-acid precursors, which both consist of a signal sequence, succeeded by a cryptic peptide and subsequently by the hormone moiety. The two 41-amino-acid CRF peptides differ by an Ala-->Val substitution at amino acid position 28. CRF transcripts are primarily found in the sucker pre-optic nucleus (PON), to a much lesser extent in the lateral tuberal nucleus (LTN). In contrast, urotensin I (U I) encoding mRNA is equally present in both tissues. In urophysectomized fish, U I mRNA is elevated especially in LTN tissue, while CRF mRNA levels remain more or less constant in the PON and LTN regions.

Sodium and potassium ATPase of the teleost fish Catostomus commersoni. Sequence, protein structure and evolutionary conservation of the alpha-subunit.

The alpha-subunit of a Na+/K+ ATPase has been cloned by analysing a lambda gt11 library constructed from polyA+ RNA from the hypothalamic region of the teleost fish Catostomus commersoni (white sucker). The cDNA clone consists of 3853 bp and predicts a protein of 1027 amino-acid residues. Alignment of the sucker sequence with protein sequences previously published for alpha-subunits from various species reveals a high degree of homology throughout the entire sequence containing five potential sites for N-glycosylation, a phosphorylation site and a site for binding fluorescein 5'-isothiocyanate (FITC). A hydropathy profile predicts a secondary structure of the Na+/K+ ATPase alpha-subunit with at least eight membrane-spanning domains. Northern and southern blot analyses suggest the existence of two distinct Na+/K+ ATPase alpha-subunit genes in the sucker genome.

Vasotocin genes of the teleost fish Catostomus commersoni: gene structure, exon-intron boundary, and hormone precursor organization.

cDNA clones encoding two members of the vasotocin hormone precursor gene family have been isolated from the white sucker Catostomus commersoni. The hormone is encoded by at least two distinct genes, both of which are expressed, as indicated by Northern blot analysis. Genomic DNA amplified by the polymerase chain reaction has been used to define exon-intron boundaries. Both vasotocin genes contain introns in positions corresponding to those found in the gene of their mammalian counterpart vasopressin. The predicted vasotocin precursors show a surprising degree of sequence divergence, amounting to 45% at the amino acid level, of which only approximately half can be accounted for by conservative amino acid changes. The precursors include a hormone moiety followed by a putative neurophysin sequence that is longer at the C-terminus by a tract of some 30 amino acids by comparison to their mammalian counterpart. Each of these sequences contains a leucine-rich core segment resembling that found in copeptin, a glycopeptide moiety present in mammalian vasopressin precursors.