ELECTROPORATION-MEDIATED REPLACEMENT OF A POSITIVELY AND NEGATIVELY SELECTABLE BETA-TUBULIN GENE IN TETRAHYMENA-THERMOPHILA
ELECTROPORATION-MEDIATED REPLACEMENT OF A POSITIVELY AND NEGATIVELY SELECTABLE BETA-TUBULIN GENE IN TETRAHYMENA-THERMOPHILA
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DOI：
10.1073/pnas.91.10.4549
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发表时间：
1994-05-10
影响因子：
11.1
通讯作者：
GOROVSKY, MA
中科院分区：
文献类型：
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作者：
GAERTIG, J;THATCHER, TH;GOROVSKY, MA
衣原体β-微管蛋白基因中赖氨酸-350被甲硫氨酸取代后，对微管解聚药物产生耐药性，对微管稳定药物紫杉醇的敏感性增加。这种突变是在克隆的BTU 1中产生的，BTU 1是嗜热四膜虫的两个共表达的β-微管蛋白基因之一。当通过电穿孔引入时，突变的基因通过在同源位点的基因置换专门转化四膜虫。紫杉醇敏感的转化体可以用野生型基因再转化并选择紫杉醇抗性。转化细胞中的表型分类和mRNA的分析表明，可以获得在多倍体大核中BTU 1基因的完全替换。这些研究证明了该标记物用于研究微管蛋白基因功能的实用性，并表明电穿孔允许在四膜虫中进行简单的基因置换。
Replacement of lysine-350 by methionine in the beta-tubulin gene of Chlamydomonas confers resistance to microtubule-depolymerizing drugs and increased sensitivity to the microtubule-stabilizing drug taxol. This mutation was created in cloned BTU1, one of two coexpressed beta-tubulin genes of Tetrahymena thermophila. When introduced by electroporation, the mutated gene transformed Tetrahymena exclusively by gene replacement at the homologous locus. Taxol-sensitive transformants could be retransformed with a wildtype gene and selection for taxol resistance. Analyses of phenotypic assortment and of the mRNA in transformed cells suggest that complete replacement of the BTU1 gene in the polyploid macronucleus can be obtained. These studies demonstrate the utility of this marker for studying tubulin gene function and show that electroporation allows facile gene replacement in Tetrahymena.