Cloned Drosophila alcohol dehydrogenase genes are correctly expressed after transfection into Drosophila cells in culture.
Cloned Drosophila alcohol dehydrogenase genes are correctly expressed after transfection into Drosophila cells in culture.
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克隆的果蝇乙醇脱氢酶基因转染至培养的果蝇细胞后正确表达。
DOI：
10.1073/pnas.81.6.1701
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发表时间：
1984
影响因子：
11.1
通讯作者：
Dray,JF
中科院分区：
文献类型：
--
作者：
Benyajati,C;Dray,JF
通过 DNA 介导的基因转移到培养的果蝇细胞中，我们获得了克隆的黑腹果蝇乙醇脱氢酶 (Adh) 基因的正确转录。通过磷酸钙-DNA 转染技术将超螺旋质粒导入 Schneider 2 系 (SL2) 细胞，每个超螺旋质粒均含有克隆于 pBR327 中的 Adh 基因的不同区域。尽管这些细胞通常不表达其内源性 Adh 基因，但它们确实表达外源基因，如转染后 48 小时分离的 RNA 的引物延伸和核酸酶 S1 分析所示。所得的乙醇脱氢酶 (ADH) 转录物（无论是幼虫还是成虫）都具有正确的 5' 末端并正确剪接。转染的细胞还获得了 ADH 酶活性。转染不同Adh质粒的细胞中酶活性和ADH蛋白交叉反应物质的水平与ADH转录物的水平直接相关。当从 ADH RNA 剪接缺陷的 ADH 阴性突变果蝇克隆的突变 Adh 基因转染到 Schneider line 2 细胞中时，所得 ADH RNA 无法正确剪接，无法合成 ADH；因此，将突变基因转染到细胞培养物中模拟了在突变果蝇中观察到的突变表型。
We have obtained correct transcription of the cloned alcohol dehydrogenase (Adh) gene of Drosophila melanogaster after DNA-mediated gene transfer into Drosophila cells in culture. Supercoiled plasmids, each containing various regions of the Adh gene cloned in pBR327, were introduced into Schneider line 2 (SL2) cells by the calcium phosphate-DNA transfection technique. Although these cells do not normally express their endogenous Adh genes, they do express the exogenous genes as shown by primer extension and nuclease S1 analyses of RNA isolated 48 hr after transfection. The resulting alcohol dehydrogenase (ADH) transcripts, both the larval and adult types, have the correct 5' ends and are properly spliced. The transfected cells have also acquired ADH enzyme activity. The levels of enzyme activity and of ADH protein crossreacting material in cells transfected with different Adh plasmids correlate directly with the level of ADH transcripts. When a mutant Adh gene cloned from an ADH-negative mutant fly with a defect in the splicing of ADH RNA is transfected into the Schneider line 2 cells, the resulting ADH RNA is not spliced properly and there is no synthesis of ADH; thus, the mutant gene transfection into cell culture mimics the mutant phenotypes observed in the mutant fly.