J. Craig Venter

Cloning, localization, and permanent expression of a Drosophila octopamine receptor

A cDNA for a member of the G protein-coupled receptor family was isolated from Drosophila using a probe derived from a human beta 2-adrenergic receptor cDNA. This Drosophila receptor gene is localized at 99A10-B1 on the right arm of chromosome 3 and is preferentially expressed in Drosophila heads. The insect octopamine receptor has been permanently expressed in mammalian cells, where it mediates the attenuation of adenylate cyclase activity and exhibits a pharmacological profile consistent with an octopamine type 1 receptor. Sequence and pharmacological comparisons indicate that the octopamine receptor is unique but closely related to mammalian adrenergic receptors, perhaps as an evolutionary precursor.

Muscarinic cholinergic receptor structure: molecular biological support for subtypes

Abstract Pharmacological evidence has indicated the existence of multiple subtypes of muscarinic receptors, while biochemical and immunological data have uncovered the highly conserved nature of muscarinic receptor structure. Molecular biological data now appear to have confirmed the notion of subtypes. Tony Kerlavage, Claire Fraser and Craig Venter discuss recent cloning and sequence analyses of the genes encoding muscarinic receptors in brain and heart which have revealed the existence of four distinct but highly homologous gene products.

Cloning and sequence analysis of the human brain β-adrenergic receptor: Evolutionary relationship to rodent and avian β-receptors and porcine muscarinic receptors

Two cDNA clones, λ‐CLFV‐108 and λ‐CLFV‐119, encoding for the β‐adrenergic receptor, have been isolated from a human brain stem cDNA library. One human genomic clone, LCV‐517 (20 kb), was characterized by restriction mapping and partial sequencing. The human brain β‐receptor consists of 413 amino acids with a calculated M r of 46 480. The gene contains three potential glucocorticoid receptor‐binding sites. The β‐receptor expressed in human brain was homology with rodent (88%) and avian (52%) β‐receptors and with porcine muscarinic cholinergic receptors (31%), supporting our proposal [(1984) Proc. Natl. Acad. Sci USA 81, 272‐276] that adrenergic and muscarinic cholinergic receptors are structurally related. This represents the first cloning of a neurotransmitter receptor gene from human brain.Two cDNA clones, lambda-CLFV-108 and lambda-CLFV-119, encoding for the beta-adrenergic receptor, have been isolated from a human brain stem cDNA library. One human genomic clone, LCV-517 (20 kb), was characterized by restriction mapping and partial sequencing. The human brain beta-receptor consists of 413 amino acids with a calculated Mr of 46480. The gene contains three potential glucocorticoid receptor-binding sites. The beta-receptor expressed in human brain was homology with rodent (88%) and avian (52%) beta-receptors and with porcine muscarinic cholinergic receptors (31%), supporting our proposal [(1984) Proc. Natl. Acad. Sci. USA 81, 272 276] that adrenergic and muscarinic cholinergic receptors are structurally related. This represents the first cloning of a neurotransmitter receptor gene from human brain.

Cloning, sequence analysis and chromosome localization of a Drosophila muscarinic acetylcholine receptor

Two cDNA clones (3.7 kb and 4.8 kb) encoding a Drosophila muscarinic acetylcholine receptor were isolated from a Drosophila head cDNA library and characterized by automated DNA sequence analysis. The Drosophila muscarinic receptor contains 788 amino acids with a calculated M r of 84 807 and displays greater than 60% homology with mammalian muscarinic receptors. The muscarinic receptor maps to the tip of the right arm of the second chromosome of the Drosophila genome.

Chromosomal assignment of 46 brain cDNAs

Expressed sequence tags (ESTs) have been obtained from several hundred brain cDNAs as an initial effort to characterize expressed brain genes. These ESTs will become tools for human genome mapping and they will also provide candidate causative genes for inherited disorders affecting the central nervous system. We have developed a procedure for the rapid chromosomal assignment of these ESTs: cDNA sequences are first analyzed by a computer program to determine regions likely not to be interrupted by introns in the genomic DNA. A pair of oligonucleotide primers is then designed to amplify this region by the polymerase chain reaction using DNA template from human-rodent somatic cell hybrid chromosomal panels. The chromosomal assignment of the cDNA is determined by studying the segregation of the amplified products in these panels. In this paper we describe the mapping of 46 brain ESTs, as well as observations on the amplification of rodent sequences.

The cloned murine M1 muscarinic receptor is associated with the hydrolysis of phosphatidylinositols in transfected murine B82 cells

A rat genomic DNA clone was isolated by its homology with a conserved primary sequence among the mammalian and avian beta adrenergic and porcine muscarinic receptors. A gene identified in this clone was highly homologous to the rat M1 muscarinic receptor. Stable expression of this gene was achieved in an established murine fibroblast cell line, B82. The gene product exhibits M1 type muscarinic receptor characteristics, as it has high affinity for PZ but low affinity for AF-DX 116. Carbachol stimulated the hydrolysis of phosphatidylinositols in the transfected cells. Pirenzepine had a more potent inhibitory effect on this response than AF-DX 116 since their functional inhibition constants were 13 nM and 480 nM, respectively, which is consistent with an M1 pharmacological profile. These data suggest that the M1 muscarinic receptor encoded by the gene is coupled to the hydrolysis of phosphatidylinositols after transfecting this gene into the B82 cells.

Genome sequence analysis : scientific objectives and practical strategies

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The differential loss of [3H]pirenzepine vs [3H](−)Quinuclidinylbenzilate binding to soluble rat brain muscarinic receptors indicates that pirenzepine binds to an allosteric state of the muscarinic receptor

[3H]Pirenzepine [( 3H]PZ) and [3H] (-)Quinuclidinylbenzilate [( 3H] (-)QNB) specific binding to soluble rat brain muscarinic cholinergic receptors was assessed as a function of time subsequent to receptor solubilization. The soluble brain muscarinic receptor is stable at 4 degrees C when assayed by [3H] (-)QNB binding (t 1/2 = 80 hrs). In contrast the pirenzepine state of the receptor decays rapidly (t 1/2 = 3.0 hrs). Prior occupation of the receptor with [3H] (-)QNB or [3H]PZ increases the receptor stability by two to five fold (t 1/2 QNB greater than 1,000 hrs; t 1/2 PZ = 6.5 hrs). These data indicate that pirenzepine binds to an allosteric state of the muscarinic receptor and that caution should be employed in the assignment of receptor subtypes based solely upon the binding of ligands which recognize unique conformational states.

Size of Neurotransmitter Receptors as Determined by Radiation Inactivation–Target Size Analysis

Publisher Summary The principal receptors of the autonomic nervous system are the adrenergic and cholinergic receptors. Adrenergic and cholinergic responses have been the most extensively studied because of their overall physiological importance. β-Adrenergic receptors, which modulate a diverse array of cellular and organ functions including heart rate and vascular and airway diameter, have received extensive attention because of their demonstrated role in the activation of adenylate cyclase activity. Nicotinic acetylcholine receptors, which control skeletal muscle contraction and certain aspects of ganglionic function, are the most extensively studied receptors and consequently the receptor about which the most is known. The genes for nicotinic acetylcholine receptors have been cloned and the complete amino acid sequence has been determined. The purification and structural elucidation of the nicotinic acetylcholine receptor has been aided tremendously, particularly in the early stages, by having a rich source of the receptor (electric eels) and specific receptor probes (cobra toxins). The resolution of the structure of adrenergic (α and β), muscarinic cholinergic, and dopaminergic receptors is clearly underway in a variety of laboratories throughout the world. Structural determinations have generally depended on the application of new technologies and the development of affinity ligands. The chapter discusses the application of target size analysis, a reemerging technique, for obtaining structural information for a number of receptors.

Cloning and Expression of Adrenergic and Muscarinic Cholinergic Receptor Genes

We have extensively characterized the structure and evolution of the adrenergic and muscarinic cholinergic receptors. This analysis has included studies with monoclonal antibodies, protein purification, target size analysis, gene cloning and sequencing and gene expression (1–7). Based upon the results of biochemical and immunological studies, we proposed that adrenergic and muscarinic cholinergic receptors are highly conserved proteins. This hypothesis has been confirmed by gene cloning experiments which have provided the primary structures of a number of adrenergic and muscarinic cholinergic receptors.