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来自小鼠肌肉细胞系BC3H-1的乙酰胆碱受体α亚基的cDNA克隆。

cDNA clone for the alpha subunit of the acetylcholine receptor from the mouse muscle cell line BC3H-1.

作者信息

Merlie J P, Sebbane R, Gardner S, Lindstrom J

出版信息

Proc Natl Acad Sci U S A. 1983 Jun;80(12):3845-9. doi: 10.1073/pnas.80.12.3845.

DOI:10.1073/pnas.80.12.3845
PMID:6344089
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC394149/
Abstract

Sequences from a gene coding for mouse acetylcholine receptor alpha subunit have been inserted into a recombinant plasmid and cloned in Escherichia coli. mRNAs for acetylcholine receptors occur in low abundance in vertebrate muscle. To clone the mouse alpha-subunit cDNA, we made use of (i) a cell line, BC3H-1, that overproduces the alpha-subunit mRNA and (ii) a polysome fractionation procedure that results in enrichment of alpha-subunit mRNA. Polyadenylylated RNA was used to construct a cDNA library of 750 recombinant clones. Acetylcholine receptor-specific sequences were identified by hybrid-selected translation, followed by monoclonal antibody precipitation and peptide mapping of the translation product. One clone (pA59) that fit these criteria was found in the first 80 isolates. It had a 700-base-pair insert that was excised with Pst I. Blot-hybridization experiments with nick-translated pA59 DNA showed that BC3H-1 cells contain 100-1,000 times more alpha-subunit mRNA than does newborn or adult mouse muscle. Blot hybridization of restriction digests of mouse liver DNA revealed that pA59 is homologous to a very small number (probably one) of genomic sequences.

摘要

编码小鼠乙酰胆碱受体α亚基的基因序列已被插入到重组质粒中，并在大肠杆菌中进行克隆。乙酰胆碱受体的mRNA在脊椎动物肌肉中的丰度较低。为了克隆小鼠α亚基cDNA，我们利用了（i）一种过量产生α亚基mRNA的细胞系BC3H-1，以及（ii）一种导致α亚基mRNA富集的多核糖体分级分离程序。使用聚腺苷酸化RNA构建了一个包含750个重组克隆的cDNA文库。通过杂交选择翻译，随后进行单克隆抗体沉淀和翻译产物的肽图谱分析，鉴定出乙酰胆碱受体特异性序列。在前80个分离株中发现了一个符合这些标准的克隆（pA59）。它有一个700个碱基对的插入片段，用Pst I酶切下来。用缺口平移的pA59 DNA进行印迹杂交实验表明，BC3H-1细胞中α亚基mRNA的含量比新生或成年小鼠肌肉中的多100 - 1000倍。对小鼠肝脏DNA限制性消化产物进行印迹杂交显示，pA59与极少数（可能一个）基因组序列同源。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b119/394149/c5b1d3bd5840/pnas00638-0315-c.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b119/394149/92d0bb6984cb/pnas00638-0314-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b119/394149/4241f9d2c06c/pnas00638-0314-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b119/394149/d94d0165c70a/pnas00638-0314-c.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b119/394149/2d86d865e51a/pnas00638-0315-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b119/394149/ec71cdbe7617/pnas00638-0315-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b119/394149/c5b1d3bd5840/pnas00638-0315-c.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b119/394149/92d0bb6984cb/pnas00638-0314-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b119/394149/4241f9d2c06c/pnas00638-0314-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b119/394149/d94d0165c70a/pnas00638-0314-c.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b119/394149/2d86d865e51a/pnas00638-0315-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b119/394149/ec71cdbe7617/pnas00638-0315-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b119/394149/c5b1d3bd5840/pnas00638-0315-c.jpg

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本文引用的文献

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Acetylcholine receptor: complex of homologous subunits.乙酰胆碱受体：同源亚基复合体。
Science. 1980 Jun 27;208(4451):1454-6. doi: 10.1126/science.7384786.
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Acetylcholine receptor synthesis from membrane polysomes.来自膜多核糖体的乙酰胆碱受体合成
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Acetylcholine receptor subunits transit a precursor pool before acquiring alpha-bungarotoxin binding activity.乙酰胆碱受体亚基在获得α-银环蛇毒素结合活性之前会经过一个前体池。
骨骼肌去神经超敏反应：用克隆的cDNA探针进行分析
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Isolation and characterization of a cDNA clone for the complete protein coding region of the delta subunit of the mouse acetylcholine receptor.小鼠乙酰胆碱受体δ亚基完整蛋白质编码区cDNA克隆的分离与鉴定。
Proc Natl Acad Sci U S A. 1984 Dec;81(24):7970-4. doi: 10.1073/pnas.81.24.7970.
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Immunochemical demonstration that amino acids 360-377 of the acetylcholine receptor gamma-subunit are cytoplasmic.免疫化学证明乙酰胆碱受体γ亚基的360 - 377位氨基酸位于细胞质中。
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An acetylcholine receptor precursor alpha subunit that binds alpha-bungarotoxin but not d-tubocurare.一种能结合α-银环蛇毒素但不结合d-筒箭毒碱的乙酰胆碱受体前体α亚基。
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Regulation of nicotinic acetylcholine receptors by protein phosphorylation.蛋白质磷酸化对烟碱型乙酰胆碱受体的调控
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Molecular studies of the neuronal nicotinic acetylcholine receptor family.神经元烟碱型乙酰胆碱受体家族的分子研究
Mol Neurobiol. 1987 Winter;1(4):281-337. doi: 10.1007/BF02935740.
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Mouse muscle nicotinic acetylcholine receptor gamma subunit: cDNA sequence and gene expression.小鼠肌肉烟碱型乙酰胆碱受体γ亚基：cDNA序列与基因表达
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Activity regulates the levels of acetylcholine receptor alpha-subunit mRNA in cultured chicken myotubes.活性调节培养的鸡肌管中乙酰胆碱受体α亚基mRNA的水平。
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J Biol Chem. 1981 Apr 25;256(8):3605-8.
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Mammalian muscle acetylcholine receptor purification and characterization.哺乳动物肌肉乙酰胆碱受体的纯化与特性分析。
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Amino acid sequence homology between "alpha" subunits from Torpedo and Electrophorus acetylcholine receptor.电鳐和电鳗乙酰胆碱受体“α”亚基之间的氨基酸序列同源性。
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Inhibition of glycosylation with tunicamycin blocks assembly of newly synthesized acetylcholine receptor subunits in muscle cells.用衣霉素抑制糖基化可阻断肌肉细胞中新合成的乙酰胆碱受体亚基的组装。
J Biol Chem. 1982 Mar 10;257(5):2694-701.
8
The nicotinic cholinergic receptor: correlation of molecular structure with functional properties.烟碱型胆碱能受体：分子结构与功能特性的相关性
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Turnover of acetylcholine receptors in skeletal muscle.骨骼肌中乙酰胆碱受体的周转
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