Cloning and sequence analysis of DNA complementary to porcine cerebral messenger RNA encoding the muscarinic acetylcholine receptor predict the complete amino-acid sequence of this protein. Expression of the complementary DNA produced functional muscarinic receptor in Xenopus oocytes. The muscarinic receptor is homologous with the beta-adrenergic receptor and rhodopsin in both amino-acid sequence and suggested transmembrane topography.
We report a robust display technology for the screening of disulfide-rich peptides, based on cDNA–protein fusions, by developing a novel and versatile puromycin-linker DNA. This linker comprises four major portions: a ‘ligation site’ for T4 RNA ligase, a ‘biotin site’ for solid-phase handling, a ‘reverse transcription primer site’ for the efficient and rapid conversion from an unstable mRNA–protein fusion (mRNA display) to a stable mRNA/cDNA–protein fusion (cDNA display) whose cDNA is covalently linked to its encoded protein and a ‘restriction enzyme site’ for the release of a complex from the solid support. This enables not only stabilizing mRNA–protein fusions but also promoting both protein folding and disulfide shuffling reactions. We evaluated the performance of cDNA display in different model systems and demonstrated an enrichment efficiency of 20-fold per selection round. Selection of a 32-residue random library against interleukin-6 receptor generated novel peptides containing multiple disulfide bonds with a unique linkage for its function. The peptides were found to bind with the target in the low nanomolar range. These results show the suitability of our method for in vitro selections of disulfide-rich proteins and other potential applications.
The primary structures of two muscarinic acetylcholine receptor (mAChR) species, designated as mAChR I and mAChR II, have been elucidated by cloning and sequence analysis of DNAs complementary to the porcine cerebral and cardiac messenger RNAs, respectively. mAChR I and mAChR II expressed in Xenopus oocytes differ from each other both in acetylcholine-induced response and in antagonist binding properties. These results, together with the differential tissue location of the two mAChR mRNAs, have indicated that pharmacologically distinguishable subtypes of the mAChR represent distinct gene products. The primary structures of two additional mammalian mAChR species, designated as mAChR III and mAChR IV, have subsequently been deduced from the nucleotide sequences of the cloned cDNAs or genomic DNAs. We report here that mAChR I and mAChR III expressed in NG108-15 neuroblastoma-glioma hybrid cells, but not mAChR II and mAChR IV, efficiently mediate phosphoinositide hydrolysis, activation of a Ca2+-dependent K+ current and inhibition of the M-current, a voltage-dependent K+ current sensitive to muscarinic agonists.
The primary structure of porcine preproenkephalin B has been elucidated by cloning and sequencing cDNA: it contains neoendorphin, dynorphin and leumorphin (containing rimorphin as its amino-terminus). These opioid peptides, each having a leucine-enkephalin structure, act on the kappa-receptor. We have now cloned a human genomic DNA segment containing the preproenkephalin B gene. The structural organization of this gene resembles those of the genes encoding the other opioid peptide precursors, that is, preproenkephalin A and the corticotropin-beta-lipotropin precursor (ACTH-beta-LPH precursor). The primary structure of human preproenkephalin B has been deduced from the gene sequence. The amino acid sequence homology observed between preproenkephalin B and preproenkephalin A, together with the similarity between their gene organizations, suggests that the two genes have been generated from a common ancestor by gene duplication.
In the marine mollusk Aplysia, the CCAAT/enhancer-binding protein, ApC/EBP, serves as an immediate early gene in the consolidation of long-term facilitation in the synaptic connection between the sensory and motor neurons of the gill-withdrawal reflex. To further examine the role of ApC/EBP as a molecular switch of a stable form of long-term memory, we cloned the full-length coding regions of two alternatively spliced forms, the short and long form of ApC/EBP. Overexpression of each isoform by DNA microinjection resulted in a l6-fold increase in the expression of the coinjected luciferase reporter gene driven by an ERE promoter. In addition, when we overexpressed ApC/EBP in Aplysia sensory neurons, we found that the application of a single pulse of 5-HT that normally induced only short-term facilitation now induced long-term facilitation. Conversely, when we attempted to block the synthesis of native ApC/EBP by microinjecting double-strand RNA or antisense RNA, we blocked long-term facilitation in a sequence-specific manner. These data support the idea that ApC/EBP is both necessary and sufficient to consolidate short-term memory into long-term memory. Furthermore, our results suggest that this double-strand RNA interference provides a powerful tool in the study of the genes functioning in learning and memory in Aplysia by specifically inhibiting both the constitutive and induced expression of the genes.
The complete amino acid sequence of the porcine cardiac muscarinic acetylcholine receptor has been deduced by cloning and sequencing the cDNA. The tissue location of the RNA hybridizing with the cDNA suggests that this muscarinic receptor species represents the M2 subtype.
The tissue distribution of the mRNAs encoding muscarinic acetylcholine receptors (mAChRs) I, II, III and IV has been investigated by blot hybridization analysis with specific probes. This study indicates that exocrine glands contain both mAChR I and III mRNAs, whereas smooth muscles contain both mAChR II and III mRNAs. All four mAChR mRNAs are present in cerebrum, whereas only mAChR II MRNA is found in heart.
The muscarinic acetylcholine receptor (mAChR) mediates various cellular responses, including inhibition of adenylate cyclase, breakdown of phosphoinositides and modulation of potassium channels, through the action of guanine nucleotide-binding regulatory proteins. Pharmacologically distinguishable forms of the mAChR occur in different tissues and have provisionally been classified into M1 (I), M2 cardiac (II) and M2 glandular (III) subtypes on the basis of their difference in apparent affinity for antagonists. In an attempt to elucidate the molecular basis of the functional heterogeneity of the mAChR, we have cloned and sequenced DNAs complementary to porcine cerebral and cardiac messenger RNAs encoding mAChRs and have thereby deduced the primary structures of the receptor proteins. We report here that the messenger RNA generated by transcription of the cardiac complementary DNA directs the formation of a functional mAChR in Xenopus oocytes and that this mAChR differs from the mAChR formed by expression of the cerebral cDNA both in acetylcholine (ACh)-induced response and in antagonist binding properties. Our results provide evidence indicating that the mAChR encoded by the cerebral cDNA (designated as mAChR I) and the mAChR encoded by the cardiac cDNA (mAChR II) are of the M1 (I) and the M2 cardiac (II) subtype, respectively.
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