Cloning and characterization of a mouse brain calcitonin receptor complementary deoxyribonucleic acid and mapping of the calcitonin receptor gene

We have identified and characterized a mouse brain calcitonin receptor (CTR) complementary DNA (cDNA). This cDNA encodes a receptor protein that, after expression, has high affinity binding for salmon calcitonin (Kd approximately, 12.5 nM) and is coupled to adenylate cyclase. The binding affinity of this expressed receptor for salmon calcitonin is lower than that described for the previously cloned porcine renal and human ovarian CTRs, but is similar to that of the recently described rat brain CTR, designated the C1b form of the receptor. Analysis of the deduced structure of the mouse brain CTR reveals that it is highly related to the other CTR cDNAs that belong to a distinct family of G-protein-coupled receptors with seven transmembrane-spanning domains. The major structural feature that distinguishes the mouse cDNA clone from the other CTRs is the presence of a consecutive 111-basepair nucleotide sequence that encodes a 37-amino acid sequence which is predicted to localize to the first extracellular loop between the second and third transmembrane-spanning domains. We have mapped the CTR gene in the mouse to the proximal region of chromosome 6, which is homologous to the 7q region of human chromosome 7; only a single CTR gene was identified. Preliminary analysis of the mouse CTR gene reveals that it is complex, consisting of multiple exons separated by lengthy introns that would allow for splice variants consistent with the existence of multiple CTR isoforms predicted from the CTR cDNA clones. The differential cellular and tissue distribution of these functionally distinct CTR isoforms provides the molecular basis for the previously reported widespread distribution and functional heterogeneity of the CTR.     

Cloning of the cDNA encoding the urotensin II precursor in frog and human reveals intense expression of the urotensin II gene in motoneurons of the spinal cord

Urotensin II (UII) is a cyclic peptide initially isolated from the caudal neurosecretory system of teleost fish. Subsequently, UII has been characterized from a frog brain extract, indicating that a gene encoding a UII precursor is also present in the genome of a tetrapod. Here, we report the characterization of the cDNAs encoding frog and human UII precursors and the localization of the corresponding mRNAs. In both frog and human, the UII sequence is located at the C-terminal position of the precursor. Human UII is composed of only 11 amino acid residues, while fish and frog UII possess 12 and 13 amino acid residues, respectively. The cyclic region of UII, which is responsible for the biological activity of the peptide, has been fully conserved from fish to human. Northern blot and dot blot analysis revealed that UII precursor mRNAs are found predominantly in the frog and human spinal cord. In situ hybridization studies showed that the UII precursor gene is actively expressed in motoneurons. The present study demonstrates that UII, which has long been regarded as a peptide exclusively produced by the urophysis of teleost fish, is actually present in the brain of amphibians and mammals. The fact that evolutionary pressure has acted to conserve fully the biologically active sequence of UII suggests that the peptide may exert important physiological functions in humans.     

Processing of human prosomatostatin in AtT-20 cells: S-28 and S-14 are generated in different secretory pathways

Somatostatin-14 (S-14) and somatostatin-28 (S-28) are generated by differential processing of a single precursor at a dibasic (R-K) or monobasic (R) proteolytic cleavage site, respectively. To study the pathways of processing of prosomatostatin, we have expressed in AtT20 cells cDNA encoding human prosomatostatin and prosomatostatin mutated in one or the other processing site. Analysis of the peptides present in cell extracts or culture media before and after stimulation of the cells with 8-BrcAMP indicated that prosomatostatin can enter three distinct secretory pathways where it is differently processed: 1) prosomatostatin was secreted through the constitutive pathway; 2) the regulated secretory pathway generated S-14 which was released upon stimulation of the cells with 8-BrcAMP; 3) an alternative pathway, insensitive to 8-BrcAMP produced S-28 and S-14. Moreover, our results suggest that the R-K processing site used to produce S-14 is an important structural feature for targeting the precursor to the regulated secretory pathway.     

Expression and localization of the Th2-type cytokine interleukin-13 and its receptor in the placenta during human pregnancy

The gene encoding the mouse somatostatin receptor subtype 5 has been isolated from a genomic library and the mRNA start point mapped to position -95 relative to the translational start codon. The promoter region is devoid of TATA and CAAT boxes but contains putative binding sites for AP-1, AP-2 and SP1 and response elements for glucocorticoids (GRE) and phorbol esters (TRE). The encoded receptor protein with a predicted molecular weight of 42.5 kDa is comprised of 385 amino acids and thus contains 22 and 21 amino acids more than rat and human counterparts. The extra amino acids are caused by another translational initiation codon located further upstream. In the region of overlap the mouse somatostatin receptor subtype 5 displays 96.7% sequence identity to the rat and 81.7% to the human homologue. Application of somatostatin-14 and -28 to human embryonic kidney cells expressing the recombinant receptor resulted in the inhibition of forskolin-stimulated adenylyl cyclase with comparable EC50 values. Consistent with the observed sequence relationship, the mouse somatostatin receptor subtype 5 displays a pharmacological profile that resembles the rat homologue more closely than the human counterpart. mRNA for the mouse somatostatin type 5 receptor has been detected in pituitary, kidney, spleen and ovary and, to a lesser extent, in brain, stomach, intestine and thymus but was not observed in heart, pancreas and liver.     

Cloning, cDNA sequence analysis and patch clamp studies of a toxin from the venom of the armed spider (Phoneutria nigriventer)

The cDNAs (Tx3-2 and Pn3A) encoding precursor of toxin Tx3-2 and an isoform called Pn3A have been isolated from a library constructed from stimulated venom glands of the spider Phoneutria nigriventer. The cDNA of Tx3-2 reveals the presence of a signal peptide of 21 amino acids and of an intervening propeptide (with 16 amino acids) preceding the toxin sequence, which was followed by additional amino acid residues at the C-terminus (C-terminal peptide), implying post-translational modifications of the synthesised peptide. The deduced amino acid sequence for the mature toxin confirms the previous sequence published. In addition, by using the whole-cell patch clamp technique, we have determined that purified Tx3-2 decreases L-type currents present in GH3 cells. Finally, the presence of the cDNA Pn3A, with high sequence identity with Tx3-2, reveals the existence of a putative new toxin showing, at the cDNA level, 85.4% identity in its whole segment.     

Sex determination of infant and juvenile skeletons: I. Morphognostic features

The human neutrophil-derived cationic peptide HP-4 exhibits corticostatic activity on adrenal cells and is an L-type calcium channel agonist at nanomolar concentrations. Complementary DNA clones encoding the HP-4 precursor have been isolated from a human bone marrow cDNA library by screening with oligonucleotide probes. The nucleotide sequence shares about 72% identity with the cDNA encoding defensin HP-1, but differs from it, and from other genes of this family characterized to date, by an extra 83-base segment. This extra segment is not adjacent to an intron and is apparently the result of a recent duplication within the coding region corresponding to most of the mature HP-4 peptide. The predicted amino acid sequence shows the HP-4 precursor structure to be typical of this family of molecules. By analysis of DNA from a pannel of hamster/human hybrid cell lines, the HP-4 gene was found to be on chromosome 8, as is the gene for human peptide HP-1. Comparison with the few sequences of other corticostatin/defensin genes available does not indicate distinct lineages of corticostatic and noncorticostatic peptides, since HP-1 and HP-4 cDNA sequences share more identity with each other than either shares with cDNAs encoding rabbit MCP-1 or MCP-2, or guinea pig GNCP-1.     

Molecular cloning of a murine N-acetylgalactosamine transferase cDNA that determines expression of the T lymphocyte-specific CT oligosaccharide differentiation antigen

Two monoclonal antibodies termed CT1 and CT2 define a cell surface oligosaccharide molecule expressed on restricted populations of murine lymphocytes. This oligosaccharide structure is largely associated with the extracellular domain of the CD45 family of tyrosine phosphatases, molecules required for lymphocytes to proliferate in response to antigen stimulation. Previous work has shown that the oligosaccharide structure recognized by the CT antibodies is identical, at least in part, to that of the human Sda blood group, a structure formed through enzymatic addition of N-acetylgalactosamine in beta 1,4-linkage to a sub-terminal galactose substituted with an alpha 2,3-linked N-acetylneuraminic acid residue. We have used a mammalian transient expression cloning approach to isolate a murine cDNA that determines expression of an oligosaccharide structure recognized by the CT antibodies as well as human anti-Sda serum. The nucleotide sequence predicts a 510-amino acid type II transmembrane protein characteristic of other mammalian glycosyltransferases. Enzymatic characterization of the protein expressed by this cDNA demonstrates that it encodes a beta 1,4-N-acetylgalactosamine transferase activity that can add to the low molecular weight acceptor 3'-sialyl-N-acetyllactosamine, to form the nonreducing terminal tetrasaccharide Sda blood group structure. This cDNA shares 51% nucleotide sequence identity with a cDNA encoding the human GM2/GD2 synthase, particularly throughout the regions encoding their putative catalytic domains. Southern blot analysis demonstrates that these two cDNA's represent distinct loci in the murine genome. The CT-GalNAc transferase cDNA isolated here represents a tool with which to define the role(s) of lymphocyte cell surface CT determinants, and may facilitate the isolation of the human Sda blood group locus through cross-hybridization approaches.     

Cloning and characterization of mycovirus double-stranded RNA from the plant pathogenic fungus, Fusarium solani f. sp. robiniae

A mycovirus (named FusoV) from the phytopathogenic fungus, Fusarium solani f. sp. robiniae SUF704, has two kinds of double-stranded (ds) RNA genomes, designated M1 and M2. The cDNAs were constructed from FusoV genomic dsRNAs. The sequences of M1 and M2 cDNAs comprised 1645 and 1445bp, respectively. Sequence analysis showed that each dsRNA had a single long open reading frame (ORF) on only one of the strands. M1 ORF encodes a 519-amino acid residue polypeptide with a predicted molecular mass of 60 kDa. RNA-dependent RNA polymerase-conserved motifs were identified in the predicted amino acid sequence, and the polymerase synthesized dsRNA in vitro. The M2 ORF encodes a polypeptide of 413 amino acid residues with a predicted molecular mass of 44 kDa. The predicted amino acid sequence contained the sequence corresponding to those found in the purified 44-kDa capsid protein of FusoV.     

Diversity of the troponin C genes during chordate evolution

To elucidate the diversity of troponin C (TnC) during chordate evolution, we determined the organization of TnCs from the amphioxus, the lamprey, and the frog. Like the ascidian, the amphioxus possesses a single gene of TnC, and the fundamental gene structure is identical with the ascidian TnC. However, because alternative splicing does not occur in amphioxus, the potential for generation of TnC isoforms through this event arises only in the ascidian lineage. From the frog Xenopus laevis, two distinct cDNAs encoding fTnC isoforms and a single s/cTnC cDNA were determined. The duplication of the fTnC gene may be a character of only Xenopus or closely related species. The lamprey possesses two cDNAs each encoding fTnC and s/cTnC. The lamprey is the earliest diverged species among vertebrates, and thus it is supposed that the presence of both fTnC and s/cTnC is universal among vertebrate species, and that the gene duplication might have occurred at a vertebrate ancestor after the protochordate/vertebrate divergence. The position of the 4th intron is 3.24/0 in protochordate TnC genes, but at 3. 11/2 in vertebrate fTnCs and s/cTnCs. It is suggested that the 4th intron sliding might have occurred prior to the gene duplication.     

Nature and regulation of pistil-expressed genes in tomato

The specialized reproductive functions of angiosperm pistils are dependent in part upon the regulated activation of numerous genes expressed predominantly in this organ system. To better understand the nature of these pistil-predominant gene products we have analyzed seven cDNA clones isolated from tomato pistils through differential hybridization screening. Six of the seven cDNAs represent sequences previously undescribed in tomato, each having a unique pistil- and/or floral-predominant expression pattern. The putative protein products encoded by six of the cDNAs have been identified by their similarity to sequences in the database of previously sequenced genes, with a seventh sequence having no significant similarity with any previously reported sequence. Three of the putative proteins appear to be targeted to the endomembrane system and include an endo-beta-1,4-glucanase which is expressed exclusively in pistils at early stages of development, and proteins similar in sequence to gamma-thionin and miraculin which are expressed in immature pistils and stamens, and in either sepals or petals, respectively. Two other clones, similar in sequence to each other, were expressed primarily in immature pistils and stamens and encode distinct proteins with similarity to leucine aminopeptidases. An additional clone, which encodes a protein similar in sequence to the enzyme hyoscyamine 6-beta-hydroxylase and to other members of the family of Fe2+/ascorbate-dependent oxidases, was expressed at high levels in pistils, stamens and sepals, and at detectable levels in some vegetative organs. Together, these observations provide new insight into the nature and possible functional roles of genes expressed during reproductive development.     

des-AA-1,2,5[D-Trp8, IAmp9]somatostatin-14 allows the identification of native rat somatostatin sst1 receptor subtype

Somatostatin exerts multiple activities by interacting with at least five different receptor subtypes (sst[1-5]). The affinity of des-AA(1,2,5)-[D-Trp8, IAmp9]somatostatin-14 (CH-275) was studied by competition experiments using the non-selective radioligand [125I][Leu8, D-Trp22, Tyr25]somatostatin-28 in areas of the rat brain and pituitary known to express identified receptor subtypes. In the cerebellar nuclei and cerebral cortex, which possess the somatostatin sst1 receptor subtype, CH-275 exhibited a moderate affinity (IC50: 10-50 nM). Conversely, in the hippocampus, immature cerebellum and pituitary which contain different subsets of receptors mRNAs (sst[2-5]), the IC50 values were > 1 microM. These data indicate that CH-275 is an appropriate ligand for the identification of native rat somatostatin sst1 receptor subtype.     

Amino acid sequence of a putative sodium channel expressed in the giant axon of the squid Loligo opalescens

A full-length cDNA encoding a putative Na+ channel (GFLN1) has been cloned from a library prepared from the stellate ganglion of Loligo opalescens. The cDNA encodes a predicted protein of 1784 amino acids. Regions of the GFLN1 protein with defined functional importance (membrane span S4, the SS1 and SS2 segments, and interdomain III-IV) are highly conserved among all vertebrate Na+ channel alpha-subunit structures. Northern blot hybridization and RNase protection assays verify that mRNA corresponding to GFLN1 is expressed in neurons of the giant fiber lobe that form the giant axon. We propose that GFLN1 encodes the Na+ channel that has been extensively studied in the squid axon.