Transformation of Tetrahymena to cycloheximide resistance with a ribosomal protein gene through sequence replacement.
Transformation of Tetrahymena to cycloheximide resistance with a ribosomal protein gene through sequence replacement.
复制标题
通过序列替换用核糖体蛋白基因将四膜虫转化为放线菌酮抗性。
DOI：
10.1073/pnas.88.21.9493
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发表时间：
1991
影响因子：
11.1
通讯作者：
Yao,CH
中科院分区：
文献类型：
--
作者：
Yao,MC;Yao,CH
早期已经建立了转化四膜虫的方法，但是由于缺少除了编码rRNA的DNA（rDNA）之外的选择标记，其应用受到限制。已知酵母核糖体蛋白L29基因（CYH2）中的突变赋予放线菌酮抗性。我们已经克隆并测序了该基因的同源物从野生型和抗环己酰亚胺（ChxA）四膜虫株。令人惊讶的是，比较显示ChxA突变不存在于CYH2同源物中。因此，我们通过定点诱变在四膜虫基因中产生酵母突变，并使用它们来转化四膜虫，无论是否连接到rDNA载体。当rDNA含有工程突变基因时，所有由rDNA载体转化的克隆也变得对放线菌酮具有抗性。在没有rDNA载体的情况下，突变基因转化了大约1%的注射细胞，使其对放线菌酮具有抗性。DNA分析表明，转化是通过宿主序列的置换而不是通过注射序列的随机整合发生的。在多倍体大核基因组中，该基因的一些但不是全部拷贝发生替换。因此，在四膜虫中的转化通过特定的序列置换发生，并且注入的突变基因可以作为该生物体中的显性选择转化标记。
A method for transforming Tetrahymena has been established earlier, but its application has been limited because of the lack of selectable markers other than the rRNA-encoding DNA (rDNA). Mutations in the yeast ribosomal protein L29 gene (CYH2) are known that confer cycloheximide resistance. We have cloned and sequenced the homologue of this gene from both a wild-type and a cycloheximide-resistant (ChxA) strain of Tetrahymena. Surprisingly, a comparison shows that the ChxA mutation is not present in the CYH2 homologue. We therefore created the yeast mutations in the Tetrahymena gene by site-directed mutagenesis and used them to transform Tetrahymena either with or without linking to an rDNA vector. All clones transformed by the rDNA vector also became resistant to cycloheximide when the rDNA contained the engineered mutant genes. Without the rDNA vector, the mutant genes transform approximately 1% of injected cells to become resistant to cycloheximide. DNA analysis indicates that transformation occurs by replacement of the host sequence and not by random integration of the injected sequence. The replacement occurs to some but not all copies of this gene in the polyploid macronuclear genome. Thus, transformation in Tetrahymena occurs by specific sequence replacement, and the injected mutant genes can serve as dominant selectable transformation markers in this organism.