DNA-mediated transformation in Dictyostelium discoideum: regulated expression of an actin gene fusion
DNA-mediated transformation in Dictyostelium discoideum: regulated expression of an actin gene fusion
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盘基网柄菌中 DNA 介导的转化：肌动蛋白基因融合的调节表达
DOI：
10.1128/mcb.4.12.2890-2898.1984
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发表时间：
1984
影响因子：
5.3
通讯作者：
Colleen Silan
中科院分区：
文献类型：
--
作者：
W. Nellen;Colleen Silan
我们构建了一种新的转化载体，该载体将盘状盘孢肌动蛋白6启动子基因和5'侧翼区与细菌Tn5 NeoR （KanR）基因融合，可以赋予对氨基糖苷G418的抗性。该载体可用于转化盘状天竺鼠细胞。每10(7)个细胞获得约200至2000个转化子。转化后的细胞群体携带载体DNA的平均拷贝数约为每个细胞5个，在没有选择的情况下，DNA可以稳定地保存40代以上。我们已经证明转化的细胞合成功能性的卡那霉素磷酸转移酶，并且肌动蛋白6- neor基因融合的转录起始发生在肌动蛋白6帽位点。此外，对转化细胞和未转化细胞在营养生长和发育过程中分离的RNA分析表明，肌动蛋白6- neor基因融合与内源性肌动蛋白6基因平行调节，表明肌动蛋白6的上游侧翼区域含有足以在盘状天牛发育过程中对该基因进行差异调节的顺式调控序列。这些结果表明，该系统可用于检测盘状田鼠发育过程中基因表达的控制。
We have constructed a new vector for transformation that carries a fusion of the Dictyostelium discoideum actin 6 promoter gene and 5' flanking region with the bacterial Tn5 NeoR (KanR) gene which can confer resistance to the aminoglycoside G418. This vector can be used to transform D. discoideum cells. Approximately 200 to 2,000 transformants were obtained per 10(7) cells. Transformed cell populations carried vector DNA at an average copy number of ca. 5 per cell, and the DNA was stable for more than 40 generations in the absence of selection. We have shown that transformed cells synthesize functional kanamycin phosphotransferase and that initiation of transcription of the actin 6-NeoR gene fusion occurs at the actin 6 cap site. Moreover, analysis of RNA isolated from transformed and untransformed cells during vegetative growth and during development indicated that the actin 6-NeoR gene fusion was regulated in parallel with the endogenous actin 6 gene, suggesting that the upstream flanking regions of actin 6 contain the cis-acting regulatory sequences sufficient for differential regulation of this gene during D. discoideum development. These results indicate that this system can be used to examine control of gene expression during D. discoideum development.
DNA 介导的盘基网柄菌转化系统。
DOI：
10.1073/pnas.79.23.7356
发表时间：
1982
影响因子：
11.1
作者：
Hirth,KP;Edwards,CA;Firtel,RA
通讯作者：
Firtel,RA