In vitro translation of a 2.3-kb splicing variant of the hamster pgp1 gene whose presence in transfectants is associated with decreased drug resistance.
In vitro translation of a 2.3-kb splicing variant of the hamster pgp1 gene whose presence in transfectants is associated with decreased drug resistance.
复制标题
仓鼠 pgp1 基因 2.3-kb 剪接变体的体外翻译，其在转染子中的存在与耐药性降低相关。
DOI：
10.1007/s002800050858
复制
发表时间：
1999
影响因子：
3
通讯作者：
Melera,PW
中科院分区：
文献类型：
--
作者：
Ma,JF;Grant,G;Staelens,B;Howard,DL;Melera,PW
目的：P-糖蛋白（P-gp），中国仓鼠pgp 1基因的产物，赋予哺乳动物细胞的多药耐药性，其中它是通过转染或作为药物选择的结果过表达。它由4.3 kb mRNA编码，其非糖基化形式的预测分子量约为141 kDa。当含有该序列的cDNA转染到药物敏感的中国仓鼠肺细胞中并在β-肌动蛋白启动子的控制下表达时，产生全长4.3-kb mRNA和2.3-kb转录物。后者由剪接事件产生，该剪接事件利用了存在于全长mRNA中的近共有5′和3′剪接信号，并且还发现其存在于表达天然基因的细胞系中。因此，它本身是pgp 1的剪接产物。方法：体外转录和翻译实验表明，该2.3kb转录本的开放阅读框编码一个新的57 kDa蛋白（p57 pgp 1），该蛋白含有跨膜结构域9-12和P-gp的C端ATP结合折叠。为了阐明p57 pgp 1的功能，构建了含有代表（1）2.3-kb转录本，（2）全长4.3-kb mRNA和（3）剪接失活的4.3-kb转录本（其中2.3-kb转录本的产生通过3′剪接位点的框内突变消除）的cDNA的表达载体，并转染到DC-3F细胞中。另外的表达载体，其中p57 pgp 1代表的N-末端的一个绿色荧光蛋白融合constructs. Results的瞬时表达studies.Results：过表达的2.3 kb的转录单独没有赋予多药耐药。与不表达2.3kb转录物但仅表达4.3kb mRNA的水平相同的转染子相比，存在4.3kb转录物和2.3kb转录物的转染子在交叉抗性模式上几乎没有变化。然而，后者细胞的总体抗性水平增加了约两倍。因此，2.3 kb转录本的存在与耐药性的降低有关。结论：p57 pgp 1的剪接变异体含有一个开放的阅读框架，能够在体外和体内进行翻译，提示剪接的改变可能直接或间接地参与pgp 1介导的CHL细胞多药耐药的机制。
Purpose: P-glycoprotein (P-gp), a product of the Chinese hamster pgp1 gene, confers multidrug resistance to mammalian cells in which it is overexpressed either by transfection or as a result of drug selection. It is encoded by a 4.3-kb mRNA and in its unglycosylated form has a predicted molecular weight of approximately 141 kDa. When a cDNA containing this sequence is transfected into drug-sensitive Chinese hamster lung cells and is expressed under the control of the β-actin promoter, both the full-length 4.3-kb mRNA and a 2.3-kb transcript are produced. The latter results from a splicing event that utilizes near consensus 5′ and 3′ splicing signals resident in the full-length mRNA, and it has also been found to be present in cell lines that express the native gene. Therefore, it is a splicing product of pgp1 per se. This report is concerned with the biological relevance of this transcript.Methods: In vitro transcription and translation experiments were used to show that the putative open reading frame of the 2.3-kb transcript encodes a novel 57-kDa protein (p57pgp1) that contains transmembrane domains 9–12 and the C-terminal ATP binding fold of P-gp. To elucidate the function of p57pgp1, expression vectors containing cDNAs representing (1) the 2.3-kb transcript, (2) the full-length 4.3-kb mRNA, and (3) a splice-disabled 4.3-kb transcript in which production of the 2.3-kb transcript is eliminated by an in-frame mutation at the 3′ splice site, were constructed and transfected into DC-3F cells. Additional expression vectors in which p57pgp1represented the N-terminus of a green fluorescent protein fusion construct were also prepared and used for transient expression studies.Results: Overexpression of the 2.3-kb transcript alone did not confer multidrug resistance. Transfectants in which both the 4.3-kb transcript and the 2.3-kb transcript were present, compared with transfectants in which no 2.3-kb transcript was expressed, but in which the level of expression of the 4.3-kb mRNA alone was the same, showed little change in cross-resistance pattern. However, the overall level of resistance in the latter cells was increased by approximately twofold. Hence the presence of the 2.3-kb transcript was associated with a decrease in drug resistance. In vitro transcription and translation experiments and transient expression studies indicate that p57pgp1can be expressed both in vitro and in vivo.Conclusion: These results demonstrate that a splicing variant of pgpl contains an open reading frame capable of translation in vitro and in vivo and suggest that alterations in splicing may contribute both directly and indirectly to the overall mechanism of pgp1-mediated multidrug resistance in CHL cells.