Functional promoter analysis using an approach based on an in vitro evolution strategy
Functional promoter analysis using an approach based on an in vitro evolution strategy
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DOI：
10.2144/05382st01
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发表时间：
2005-02-01
期刊：
影响因子：
2.7
通讯作者：
Schenk, PM
中科院分区：
文献类型：
--
作者：
Remans, T;Grof, CPL;Schenk, PM
体外进化模仿基因的自然进化，已非常成功地应用于编码序列的修饰，但尚未应用于启动子序列。我们提出了一种替代方法，通过应用在体外进化方案组成的轮易错PCR，然后通过DNA改组和选择的突变启动子活性的功能启动子分析。我们修改了香蕉线条病毒“金手指”分离物的启动子区域在胚性甘蔗细胞中的活性，并获得了突变的启动子序列，该序列在应用一轮易错PCR和DNA改组后显示出平均2.5%的突变率。选择和测序的启动子序列的活性降低或不变，使我们能够迅速映射的位置的一个顺式作用元件，影响启动子的活性，在胚性甘蔗细胞，并发现中性突变，不影响启动子连接。该启动子分析方法的“选择性鸟枪法“在启动子边界已经通过5'缺失分析限定之后立即进行，显著地减少了与传统的“接头扫描“缺失分析相关的劳动，以揭示功能性启动子结构域的位置。此外，该方法允许一次研究整个启动子，而不是选择的结构域或核苷酸，增加了鉴定相互作用的启动子区域的前景。
In vitro evolution imitates the natural evolution of genes and has been very successfully applied to the modification of coding sequences, but it has not yet been applied to promoter sequences. We propose an alternative method for functional promoter analysis by applying an in vitro evolution scheme consisting of rounds of error-prone PCR, followed by DNA shuffling and selection of mutant promoter activities. We modified the activity in embryogenic sugarcane cells of the promoter region of the "Goldfinger" isolate of banana streak virus and obtained mutant promoter sequences that showed an average mutation rate of 2.5% after applying one round of error-prone PCR and DNA shuffling. Selection and sequencing of promoter sequences with decreased or unaltered activity allowed us to rapidly map the position of one cis-acting element that influenced promoter activity in embryogenic sugarcane cells and to discover neutral mutations that did not affect promoter Junction. The "selective-shotgun " approach of this promoter analysis method immediately after the promoter boundaries have been defined by 5' deletion analysis dramatically reduces the labor associated with traditional "linker-scanning " deletion analysis to reveal the position of functional promoter domains. Furthermore, this method allows the entire promoter to be investigated at once, rather than selected domains or nucleotides, increasing the, prospect of identifying interacting promoter regions.