Deletion analysis of the mouse m1 muscarinic acetylcholine receptor: effects on phosphoinositide metabolism and down-regulation.
Deletion analysis of the mouse m1 muscarinic acetylcholine receptor: effects on phosphoinositide metabolism and down-regulation.
复制标题
小鼠 m1 毒蕈碱乙酰胆碱受体的删除分析：对磷酸肌醇代谢和下调的影响。
DOI：
10.1021/bi00448a039
复制
发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Nathanson,NM
中科院分区：
文献类型：
--
作者：
Shapiro,RA;Nathanson,NM
缺失突变的材料和构建方法。一个1.8 kb的包含小鼠ml mAChR整个编码区的Kpn-BamHl片段被亚克隆到pGEM3中（Shapiro et al., 1988）。4个mAChR缺失突变（AvaA、NcoA、BclA和Bel-Ava A）构建如下图（图1）。AvaA：用Aval消化1.8-kb ml/pGEM，用Klenow填充，并连接BamHI连接器，用BamHI切割，并进行宗教处理。由此产生的亚克隆（ml S'-Ava/Bam）含有ml受体的氨基末端，氨基酸含量高达220。然后将ml 5'-Ava/Bam片段与BamHI线性化，并连接到带有BamHI连接物的550 bp Aval/ small片段上。由此产生的结构体删除了氨基酸221-343，并用三个氨基酸取而代之：Arg-Ile-Arg。NcoA: ml S'-Ava/Bam与BamHI线性化，并连接到一个3'800 bp的Ncol/ small片段（用Klenow填充并连接BamHI连接物）。由此产生的构建体删除了氨基酸221-284，并将其替换为氨基酸Arg-Ile-Arg。BclA: ml S ' -Ava/Bam与BamHI线性化，并连接到y-Bcl\/BamHI片段（750 bp）。由此产生的结构删除了氨基酸221-301，并用氨基酸Arg代替它们。Bel/AvaA：用Bel I和BamHI酶切1.8 kb/pGEM，分离出载体序列和5'ml序列。然后将该片段连接到用于构建AvaA的相同y-Aval/ small ml片段。由此产生的结构删除了303-343氨基酸，并用三个氨基酸取而代之：Arg-Ile-Arg。转染和细胞培养。然后用合适的限制性内切酶对所有缺失构建物进行酶切，去除mpgem中的编码区，用Klenow填充，并用琼脂糖凝胶纯化。然后将钝端片段连接到表达载体ZEM 228的BamHI填充位点（E. Mulvihill, Zymogenetics）。然后转染小鼠Y1肾上腺癌细胞，并在基因素（G418）存在下分离稳定的转染物，如前所述（Shapiro等人，1988）。
Materials and MethodsConstruction of Deletion Mutations. A 1.8-kb Kpn-BamHl fragment containing the entire coding region of the mouse ml mAChR was subcloned into pGEM3 (Shapiro et al., 1988). Four mAChR deletion mutations {AvaA, NcoA, BclA, and Bel-Ava A) were constructed as described below (Figure 1). AvaA: The 1.8-kb ml/pGEM was digested with Aval, filled in with Klenow and a BamHI linker attached, cut with Bam-HI, and religated. Theresulting subclone (ml S'-Ava/Bam) contains the amino-terminal end of the ml receptor up to amino acid 220. The ml 5'-Ava/Bam fragment was then linearized withBamHI and ligated to a 550 bp Aval/Smal fragment with BamHI linkers attached. The resulting con-struct deletes amino acids 221-343 and replaces them with three amino acids: Arg-Ile-Arg. NcoA: ml S'-Ava/Bam was linearized with BamHI and ligated to a 3'800 bp Ncol/Sall fragment (which was filled in with Klenow and had BamHI linkers attached). The resulting construct deletes amino acids 221-284 andreplaces them with the amino acids Arg-Ile-Arg. BclA: ml S’-Ava/Bam was linearized with BamHI and ligated to a y-Bcl\/BamHI fragment (750 bp). The resulting con-struct deletes amino acids 221-301 and replaces them with the amino acid Arg. Bel/AvaA: The 1.8-kb/pGEM was digested with Bel I and BamHI and the vector sequence and5'ml sequence isolated. This fragment was then ligated to the same y-Aval/Smal ml fragment used in the construction of the AvaA. The resulting construct deletes amino acids 303-343 and replaces them with three amino acids: Arg-Ile-Arg.Transfection and Cell Culture. All deletion constructs were then digested with the appropriate restriction enzymes to remove the coding region frompGEM, filled in with Klenow, and purified on agarose gels. The blunt-ended fragments were then ligated into the filled-in BamHI site of the expression vector ZEM 228 (E. Mulvihill, Zymogenetics). Mouse Y1 adrenal carcinoma cells were then transfected and stable transfectants isolated in thepresence of Geneticin (G418) as described (Shapiro et al., 1988).