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首页> 外文期刊>Molecular and Cellular Biology >Direct selection for mutations affecting specific splice sites in a hamster dihydrofolate reductase minigene.
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Direct selection for mutations affecting specific splice sites in a hamster dihydrofolate reductase minigene.

机译：直接选择影响仓鼠二氢叶酸还原酶微型基因中特定剪接位点的突变。

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摘要

A Chinese hamster cell line containing an extra exon 2 (50 bp) inserted into a single intron of a dihydrofolate reductase (dhfr) minigene was constructed. The extra exon 2 was efficiently spliced into the RNA, resulting in an mRNA that is incapable of coding for the DHFR enzyme. Mutations that decreased splicing of this extra exon 2 caused it to be skipped and so produced normal dhfr mRNA. In contrast to the parental cell line, the splicing mutants display a DHFR-positive growth phenotype. Splicing mutants were isolated from this cell line after treatment with four different mutagens (racemic benzo[c]phenanthrene diol epoxide, ethyl methanesulfonate, ethyl nitrosourea, and UV irradiation). By polymerase chain reaction amplification and direct DNA sequencing, we determined the base changes in 66 mutants. Each of the mutagens generated highly specific base changes. All mutations were single-base substitutions and comprised 24 different changes distributed over 16 positions. Most of the mutations were within the consensus sequences at the exon 2 splice donor, acceptor, and branch sites. The RNA splicing patterns in the mutants were analyzed by quantitative reverse transcription-polymerase chain reaction. The recruitment of cryptic sites was rarely seen; simple exon skipping was the predominant mutant phenotype. The wide variety of mutations that produced exon skipping suggests that this phenotype is the typical consequence of splice site damage and supports the exon definition model of splice site selection. A few mutations were located outside the consensus sequences, in the exon or between the branch point and the polypyrimidine tract, identifying additional positions that play a role in splice site definition. That most of these 66 mutations fell within consensus sequences in this near-saturation mutagenesis suggests that splicing signals beyond the consensus may consist of robust RNA structures.
机译：构建了一个中国仓鼠细胞系，该细胞系含有一个插入到二氢叶酸还原酶（dhfr）小基因的单个内含子中的外显子2（50 bp）。额外的外显子2被有效地剪接到RNA中，导致无法编码DHFR酶的mRNA。减少此额外外显子2剪接的突变导致其被跳过，因此产生正常的dhfr mRNA。与亲本细胞系相反，剪接突变体显示DHFR阳性生长表型。在用四种不同的诱变剂（外消旋苯并[c]菲二醇环氧化物，甲磺酸乙酯，亚硝基乙基脲和紫外线照射）处理后，从该细胞系中分离出剪接突变体。通过聚合酶链反应扩增和直接DNA测序，我们确定了66个突变体的碱基变化。每个诱变剂均产生高度特异性的碱基变化。所有突变均为单碱基取代，包括分布在16个位置的24个不同变化。大多数突变都在外显子2剪接供体，受体和分支位点的共有序列内。通过定量逆转录-聚合酶链反应分析突变体中的RNA剪接模式。很少看到秘密地点的招募;简单的外显子跳跃是主要的突变表型。产生外显子跳跃的突变种类繁多，表明该表型是剪接位点损伤的典型结果，并支持剪接位点选择的外显子定义模型。一些突变位于共有序列之外，外显子中或在分支点与聚嘧啶束之间，从而鉴定了在剪接位点定义中起作用的其他位置。在这种接近饱和的诱变中，这66个突变中的大多数都位于共有序列内，这表明超出共有序列的剪接信号可能由坚固的RNA结构组成。

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