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.     

Molecular cloning, structural characterization and functional expression of the human substance P receptor.

A cDNA encoding the human substance P receptor (SPR) was isolated and the primary structure of the protein was deduced by nucleotide sequence analysis. This SPR consists of 407 residues and is a member of the G-protein coupled receptor superfamily. Comparison of rat and human SPR sequences demonstrated a 94.5% identity. The receptor was expressed in a COS-7 cell line and displayed a Kd for Tyr-1-SP binding of 0.24 nM. Ligand displacement by naturally occurring tachykinin peptides was SP much greater than neurokinin A greater than neurokinin B. SP stimulation of transfected cells resulted in a rapid and transient inositol 1,4,5-trisphosphate response. RNA blot hybridization and solution hybridization demonstrated that SPR mRNA was about 4.5 Kb in size, and was expressed in IM-9 lymphoblast and U373-MG astrocytoma cells, as well as in spinal cord and lung but not in liver.     

Molecular cloning and expression of the murine interleukin-5 receptor

Murine interleukin-5 (IL-5) is known to play an essential role in Ig production of B cells and proliferation and differentiation of eosinophils. Here, we have isolated cDNA clones encoding a murine IL-5 receptor by expression screening of a library prepared from a murine IL-5 dependent early B cell line. A cDNA library was expressed in COS7 cells and screened by panning with the use of anti-IL-5 receptor monoclonal antibodies. The deduced amino acid sequence analysis demonstrates that the receptor is a glycoprotein of 415 amino acids (Mr 45,284), including an N-terminal hydrophobic region (17 amino acids), a glycosylated extracellular domain (322 amino acids), a single transmembrane segment (22 amino acids) and a cytoplasmic tail (54 amino acids). COS7 cells transfected with the cDNA expressed a 60 kd protein that bound IL-5 with a single class of affinity (KD = 2-10 nM). FDC-P1 cells transfected with the cDNA for murine IL-5 receptor showed the expression of IL-5 binding sites with both low (KD = 6 nM) and high affinity (KD = 30 pM) and acquired responsiveness to IL-5 for proliferation, although parental FDC-P1 cells did not show any detectable IL-5 binding. In addition, several cDNA clones encoding soluble forms of the IL-5 receptor were isolated. Northern blot analysis showed that two species of mRNAs (5.0 kb and 5.8 kb) were detected in cell lines that display binding sites for murine IL-5. Homology search for the amino acid sequence of the IL-5 receptor reveals that the IL-5 receptor contains a common motif of a cytokine receptor family that is recently identified.     

Molecular cloning and expression of a murine homolog of the human poliovirus receptor gene.

The poliovirus receptor (Pvr) is a member of the immunoglobulin superfamily of proteins, but its function in the cell is not known. Southern blot hybridization analysis indicated that the murine genome contains a sequence homolog of pvr. As a first step toward using the murine pvr homolog (mph) to study the function of Pvr, murine genomic and cDNA clones encoding mph were isolated. mph encodes a polypeptide with extensive sequence similarity to the extracellular domains of the human PVR. mph mRNAs of 2.0 and 3.0 kb are transcribed in the adult mouse brain, the spinal cord, the spleen, the kidney, the heart, and the liver. The Mph protein does not function as a receptor for poliovirus. However, substitution of domain 1 of the Mph protein with the corresponding sequence from pvr produced a chimeric receptor that could bind poliovirus and lead to productive infection. By constructing pvr-mph chimeras, it will be possible to identify the contact points of poliovirus within domain 1 of Pvr. Identification of the ligand and the cellular function of the Mph protein may help us understand the role of Pvr in the cell.     

Molecular cloning of interferon-gamma inducible genes from a murine pre-B cell leukemia.

In the present study, a pre-B cell leukemia L1210-C7, representing a very early stage of the B lineage, was used to characterize the molecular mechanisms exploited by IFN-gamma to modulate B cell activity. A cDNA library was prepared with poly (A)+ RNA from cells stimulated with IFN-gamma and three cDNAs clones complementary to IFN-gamma inducible mRNAs were isolated by differential screening. Of these, the 9.5 cDNA hybridized to a 2.4 kb mRNA not homologous with previously cloned IFN-gamma inducible mRNAs. Furthermore, when compared with RNAs obtained from cells of different origins (fibroblasts and T cells) the 9.5 mRNA appeared to be increased only in cells belonging to the B lineage. Taken as a whole, these results demonstrate that in leukemic pre-B cells IFN-gamma induces the expression of a gene that could be employed as specific cell activation marker.     

Molecular characterization of rat substance K receptor and its mRNAs

The nucleotide sequence and the amino acid sequence for rat substance K receptor were deduced by molecular cloning and sequence analysis of its cDNAs. The rat substance K receptor consists of 390 amino acid residues and belongs to the family of G protein-coupled receptors. The comparison of the amino acid sequences of the rat and bovine substance K receptors indicated that they are highly homologous in the regions covering seven putative transmembrane domains, and this similarity is particularly remarkable in the transmembrane segments III and VII and their surrounding regions. RNA blot hybridization analysis showed that the rat substance K receptor is encoded by two species of mRNAs which differ in the lengths of the extreme 5' sequence of the 5'-untranslated regions. This analysis also indicated that the substance K receptor mRNAs are expressed in the gastrointestinal tract. Interestingly, no appreciable substance K receptor mRNAs were detected in poly(A)+ RNAs isolated from the brain and spinal cord, even though these tissues are known to not only contain substance K but also express the mRNA encoding the substance K precursor.     

Molecular cloning of menaquinone biosynthetic genes of Escherichia coli K12

Summary A tranducing phage carrying some of the genes (men) defining the early stages of menaquinone biosynthesis was isolated from a pool of recombinant lambda phages that had been constructed from R.HindIII digests of E. coli DNA and the corresponding insertion vector. The lesions of menB and menC mutants were complemented by the phage but menD mutants were transduced either at low frequencies or not at all. This indicates that the transducing phage contains functional menB and menC genes but that only part of the menD gene had been cloned. The phage (G68) was accordingly disignated menCB(D). Studies with the transducing phage enabled earlier mapping data (Guest 1979) to be reinterpreted in favour of the gene order nalA.... menC..menB..menD.... purF. Restriction analyses established the presence of a bacterial DNA fragment (11.5 kb) linked by a R.HindIII target to the right arm of the genome but fused to the left arm of the vector. Hybridization studies confirmed that the cloned DNA was derived from a larger R.HindIII fragment (21 kb). A physical map of the men region was constructed and some flanking and overlapping fragments were identified.     

Molecular cloning of pertussis toxin genes.

We have cloned a 4.5 kb EcoRI/BamHI DNA fragment from Bordetella pertussis which contains at least two genes responsible for expression of pertussis toxin. The S4 subunit of the toxin was isolated by high pressure liquid chromatography and the NH2-terminal amino acid sequence determined. Using a mixed synthetic oligonucleotide probe designed by reverse translation of a portion of the protein sequence, a cloned DNA fragment was identified which contains the coding information for at least the S4 structural subunit of the toxin. Sequence analyses indicate that the mature protein is derived by proteolytic cleavage of a precursor molecule. Southern blot analyses of Tn5-induced B. pertussis toxin-deficient mutants show that the Tn5 DNA is inserted 1.3 kb downstream from the S4 subunit gene. This second gene could code for another subunit required for assembly of the mature toxin or a non-structural transport protein, possibly in the same polycistronic operon. The molecular cloning of pertussis toxin genes provides the basis for development of a safer recombinant "new generation" vaccine for whooping cough.     

Molecular cloning of the murine cMOAT ATPase

cMOAT encodes an ATPase within the family of cMOAT/MRP ATPases that functions as an ATP dependent, multispecific anion transporter within the canalicular surface of hepatocytes that has pharmacologic significance. We describe here the cloning of a murine cMOAT cDNA isolated from mouse liver. The open reading frame of this cDNA incorporates 4627 nucleotides encoding 1309 amino acids with 77.5% and 86.7% identity with the human and rat encoded amino acids, respectively. Northern blotting showed that the expression of cMOAT mRNA occurs primarily in mouse liver in the form of two variants with approximately 5.6 and 7.8 kb of sequence each. cMOAT mRNA was also detected in mouse kidney at a low level but was not detected in other mouse organs or tumors except the Hep 1-6 murine hepatoma where expression was also in the form of the same two mRNA variants.     

Photoaffinity labeling the substance P receptor using a derivative of substance P containing p-benzoylphenylalanine.

A novel photoreactive substance P (SP) analogue has been synthesized by solid-phase peptide synthesis methodology to incorporate the amino acid p-benzoyl-L-phenylalanine [L-Phe(pBz)] in place of the Phe8 residue of SP. [Phe8(pBz)]SP was equipotent with SP in competing for SP binding sites on rat submaxillary gland membranes and had potent sialagogic activity in vivo. In the absence of light, the 125I-labeled Bolton-Hunter conjugate of [Phe8(pBz)]SP bound in a saturable and reversible manner to an apparently homogeneous class of binding sites (Bmax = 0.2 pmol/mg of membrane protein) with an affinity KD = 0.4 nM. The binding of 125I-[Phe8(pBz)]SP was inhibited competitively by various tachykinin peptides and analogues with the appropriate specificity for SP/NK-1 receptors. Upon photolysis, up to 70% of the specifically bound 125I-[Phe8(pBz)]SP underwent covalent linkage to two polypeptides of Mr = 53,000 and 46,000, identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Quantitative analysis of the inhibitory effects of SP and related peptides on 125I-[Phe8(pBz)]SP photoincorporation indicated that the binding sites of the two photolabeled polypeptides have the same peptide specificity, namely, that typical of NK-1-type SP receptors. In addition, the labeling of the two polypeptides was equally sensitive to inhibition by guanyl-5'-yl imidodiphosphate, a nonhydrolyzable analogue of GTP. Further information on the relationship between the two labeled SP binding sites was provided by enzymatic digestion studies: the Mr = 46,000 polypeptide contains N-linked carbohydrates and is derived most likely from the higher molecular weight species by proteolytic nicking.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning and physical mapping of murine cytomegalovirus DNA.

Murine cytomegalovirus (MCMV) Smith strain DNA is cleaved by restriction endonuclease HindIII into 16 fragments, ranging in size from 0.64 to 22.25 megadaltons. Of the 16 HindIII fragments, 15 were cloned in plasmid pACYC177 in Escherichia coli HB101 (recA). The recombinant plasmid clones were characterized by cleavage with the enzymes XbaI and EcoRI. In addition, fragments generated by double digestion of cloned fragments with HindIII and XbaI were inserted into the plasmid vector pACYC184. The results obtained after hybridization of 32P-labeled cloned fragments to Southern blots of MCMV DNA cleaved with HindIII, XbaI, EcoRI, BamHI, ApaI, ClaI, EcoRV, or KpnI allowed us to construct complete physical maps of the viral DNA for the restriction endonucleases HindIII, XbaI, and EcoRI. On the basis of the cloning and mapping experiments, it was calculated that the MCMV genome spans about 235 kilobase pairs, corresponding to a molecular weight of 155,000,000. All fragments were found to be present in equimolar concentrations, and no cross-hybridization between any of the fragments was seen. We conclude that the MCMV DNA molecule consists of a long unique sequence without large terminal or internal repeat regions. Thus, the structural organization of the MCMV genome is fundamentally different from that of the human cytomegalovirus or herpes simplex virus genome.     

Expression cloning of the murine erythropoietin receptor

Two independent cDNA clones encoding the erythropoietin receptor (EPO-R) were isolated from a pXM expression library made from uninduced murine erythroleukemia (MEL) cells. The clones were identified by screening COS cell transfectants for binding and uptake of radioiodinated recombinant human erythropoietin. As inferred from the cDNA sequence, the murine erythropoietin receptor is a 507 amino acid polypeptide with a single membrane-spanning domain. It shows no similarities to known proteins or nucleic acid sequences in the data bases. Although the MEL cell EPO-R has a single affinity with a dissociation constant of approximately 240 pM, the EPO-R cDNA, expressed in COS cells, generates both a high-affinity (30 pM) and a low-affinity (210 pM) receptor.     

Molecular characterization of a functional cDNA for rat substance P receptor

This paper describes the amino acid sequence of the rat substance P receptor and its comparison with that of the rat substance K receptor on the basis of molecular cloning and sequence analysis. From a rat brain cDNA library constructed with an RNA expression vector, we identified a cDNA mixture containing a functional substance P receptor cDNA by examining electrophysiologically a receptor expression following injection of the mRNAs synthesized in vitro into Xenopus oocytes. A receptor cDNA clone was then isolated by cross-hybridization with the bovine substance K receptor cDNA. The clone was confirmed by selective binding of substance P to the cloned receptor expressed in mammalian COS cells. The deduced amino acid sequence (407 amino acid residues) possesses seven putative membrane spanning domains and shows a sequence similarity to the members of G-protein-coupled receptors. The rat substance P and substance K receptors are very similar in both size and amino acid sequences, particularly in the putative transmembrane regions and the first and second cytoplasmic loops. This similarity is in marked contrast to the sequence divergence in the amino- and carboxyl-terminal regions and the third cytoplasmic loop. The observed sequence similarity and divergence would thus contribute to the expression of similar but pharmacologically distinguishable activities of the two tachykinin receptors.     

IL-4 and IFN-gamma up-regulate substance P receptor expression in murine peritoneal macrophages

While the ability of macrophages to express authentic substance P receptors (i.e., NK-1 receptors) has been inferred from radioreceptor binding assays and functional assays and, most recently, by identification of NK-1 receptor mRNA expression, we know little about NK-1 expression at the protein level or what host factors might up-regulate expression of this receptor. In the present study we demonstrate that the cytokines IL-4 and IFN-gamma can increase the expression of NK-1 receptors on murine peritoneal macrophages. Specifically, we show that IL-4 and IFN-gamma can elicit increases in the level of mRNA encoding the NK-1 receptor by up to 12- and 13-fold, respectively. Furthermore, these cytokines can significantly increase the expression of the NK-1 receptor protein as measured by Western blot and FACS analysis using specific Abs developed in our laboratory. In addition, we have demonstrated the ability of both IL-4 and IFN-gamma to enhance the ability of macrophages to bind substance P as measured by radiolabeled binding assay. The observation that the level of expression of this receptor protein can be enhanced by cytokines that promote either cell-mediated (Th1) or humoral (Th2) immune responses supports the idea that this receptor can be induced during either type of immune response. As such, these results may point to a more ubiquitous role for substance P in the generation of optimal immune responses than previously appreciated.     

中华蜜蜂酪胺受体基因克隆及表达分析

酪胺(tyramine)属于生物多聚胺类,是昆虫中枢神经系统内重要的神经递质、神经调质和神经激素,参与调控昆虫的多种行为和生理过程,如酪胺受体基因参与调控动物的学习与记忆。本研究首次克隆获得中华蜜蜂(Apis cerana cerana)酪胺受体基因Actyr1和Actyr2的全长cDNA序列,利用qRT-PCR方法鉴定了Actyr1和Actyr2在中华蜜蜂不同组织器官中的表达谱,采用地高辛原位杂交技术对Actyr1和Actyr2在大脑中的表达进行了定位。中华蜜蜂Actyr1、Actyr2的cDNA全长序列分别为1241 bp(GenBank登录号：KC814693)和1270 bp(GenBank登录号：KC814694),分别编码297、399个氨基酸残基。qRT-PCR分析结果表明,Actyr1和Actyr2在不同组织中的表达量为头部最高,其次是腹部表皮,触角和胸部肌肉的表达量最低,并且头部的表达量显著高于其他组织的表达量;原位杂交结果显示,Acytr1和Actyr2在中华蜜蜂大脑蘑菇体的凯尼恩细胞、触角叶周围的细胞处均有较强阳性着色。这些研究表明,Acytr1和Actyr2基因可能参与了蜜蜂的学习记忆,并且在相同的细胞中互相作用,共同调控蜜蜂的生物学功能。     

Molecular cloning of the fMet-Leu-Phe receptor from neutrophils.

The bacterial chemotactic peptide fMet-Leu-Phe (fMLP) activates neutrophils upon binding to surface receptors. In a previous communication we reported the functional reconstitution of the fMLP receptor in Xenopus laevis oocytes (Coats, W. D., and Navarro, J. (1990) J. Biol. Chem. 265, 5964-5966). In this work we report the isolation of the cDNA encoding the fMLP receptor from neutrophils. A rabbit neutrophil cDNA library was screened with an oligonucleotide probe deduced from the nucleotide sequence of G-protein-coupled receptors, and a cDNA encoding the fMLP receptor was isolated. This cDNA was characterized according to the following criteria: 1) Analysis of the deduced amino acid sequence revealed that the clone belongs to a G-protein-coupled receptor. 2) Tissue distribution analysis of the mRNA indicated that the message is only found in neutrophils. 3) In vitro translation of the message revealed a protein corresponding in size to the deglycosylated fMLP receptor. 4) X. laevis oocytes injected with the fMLP receptor message exhibited fMLP-dependent calcium mobilization and specific binding to the fMLP analog 125I-labeled fNle-Leu-Phe-Nle-Tyr-Lys (where Nle is norleucine and fNle is formylnorleucine). The molecular cloning of the fMLP receptor should provide the framework to analyze the relationship between structure, function, and regulation of this receptor.