Isolation of a clone coding for the alpha-subunit of a mouse acetylcholine receptor.
The mouse cell line BC3H-I synthesizes an acetylcholine receptor (AChR) with the pharmacological properties of a muscle nicotinic cholinergic receptor. We have purified mRNA from this cell line and used the size-fractionated poly(A)+RNA to produce a cDNA library of approximately 50,000 clones. The library was screened with a subclone containing genomic sequences coding for the putative acetylcholine-binding site of the alpha-subunit of chicken AChR. We obtained a plasmid, pMAR alpha 15, with a 1,717-base pair insert. The insert cDNA has 26 nucleotides at the 5'-end which code for a portion of the signal peptide followed by a single open reading frame of 1,311 nucleotides which code for a protein of 49,896 daltons. The insert has 377 bases of 3'-untranslated sequence with 3 polyadenylation sites. Radiolabeled plasmid DNA has been used to identify homologous RNA species of about 2 kilobases in Northern blot analyses of poly(A)+ selected RNA from BC3H-I cells. A similar size mRNA is seen in innervated mouse diaphragm and leg muscle, and both mouse and rat brain. Comparisons of the deduced amino acid sequence of the mouse AChR alpha-subunit with Torpedo marmorata, T. californica, chicken, human, and calf sequences show overall homologies of 80%, 80%, 86%, 96%, and 95%, respectively. More detailed analyses reveal a non-random distribution of amino acid substitutions in several structural domains. Based on the absolute conservation of cysteine residues, a new model for the arrangement of the disulfide bonds in the extracellular portion of the alpha-subunit is proposed.
Boulter
J
J
Luyten
W
W
Evans
K
K
Mason
P
P
Ballivet
M
M
Goldman
D
D
Stengelin
S
S
Martin
G
G
Heinemann
S
S
Patrick
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D004251:DNA Transposable Elements; D004270:DNA, Circular / Q000235:genetics; D004220:Disulfides / Q000378:metabolism; D051379:Mice / Q000235:genetics*; D008957:Models, Genetic; D011950:Receptors, Cholinergic / Q000235:genetics* / Q000378:metabolism
D004251:DNA Transposable Elements; D004270:DNA, Circular; D004220:Disulfides; D011950:Receptors, Cholinergic