Real Sebastián M, Marzese Diego M, Gomez Laura C, Mayorga Luis S, Roqué María
Cellular and Molecular Laboratory -IHEM-, Faculty of Medical Sciences, National University of Cuyo, Mendoza, Argentina.
BMC Biotechnol. 2006 Sep 2;6:38. doi: 10.1186/1472-6750-6-38.
The detection of Premature Stop Codons (PSCs) in human genes is very useful for the genetic diagnosis of different hereditary cancers, e.g. Familial Breast Cancer and Hereditary Non-Polyposis Colorectal Cancer (HNPCC). The products of these PSCs are truncated proteins, detectable in vitro by the Protein Truncation Test and in vivo by using the living translation machinery of yeast or bacteria. These living strategies are based on the construction of recombinant plasmids where the human sequence of interest is inserted upstream of a reporter gene. Although simple, these assays have their limitations. The yeast system requires extensive work to enhance its specificity, and the bacterial systems yield many false results due to translation re-initiation events occurring post PSCs. Our aim was to design a recombinant plasmid useful for detecting PSCs in human genes and resistant to bacterial translation re-initiation interferences.
A functional recombinant plasmid (pREAL) was designed based on a bacterial two-hybrid system. In our design, the in vivo translation of fused fragments of the Bordetella pertussis adenylate cyclase triggers the production of cAMP giving rise to a selectable bacterial phenotype. When a gene of interest is inserted between the two fragments, any PSC inhibits the enzymatic activity of the product, and translation re-initiation events post-PSC yield separated inactive fragments. We demonstrated that the system can accurately detect PSCs in human genes by inserting mutated fragments of the brca1 and msh2 gene. Western Blot assays revealed translation re-initiation events in all the tested colonies, implying that a simpler plasmid would not be resistant to this source of false negative results. The application of the system to a HNPCC family with a nonsense mutation in the msh2 gene correctly diagnosed wild type homozygous and heterozygous patients.
The developed pREAL is applicable to the detection of PSCs in human genes related to different diseases and is resistant to translation re-initiation events. The diagnosis steps are easy, have a low cost, detect only pathologic mutations, and allow the analysis of separated alleles.
检测人类基因中的提前终止密码子（PSC）对于不同遗传性癌症的基因诊断非常有用，例如家族性乳腺癌和遗传性非息肉病性结直肠癌（HNPCC）。这些PSC的产物是截短的蛋白质，可通过蛋白质截短试验在体外检测到，并可通过利用酵母或细菌的活体翻译机制在体内检测到。这些活体策略基于构建重组质粒，其中将感兴趣的人类序列插入报告基因的上游。尽管这些检测方法很简单，但也有其局限性。酵母系统需要大量工作来提高其特异性，而细菌系统由于在PSC后发生翻译重新起始事件而产生许多假阳性结果。我们的目标是设计一种重组质粒，可用于检测人类基因中的PSC，并能抵抗细菌翻译重新起始干扰。
基于细菌双杂交系统设计了一种功能性重组质粒（pREAL）。在我们的设计中，百日咳博德特氏菌腺苷酸环化酶融合片段的体内翻译触发了cAMP的产生，从而产生了一种可选择的细菌表型。当将感兴趣的基因插入两个片段之间时，任何PSC都会抑制产物的酶活性，并且PSC后的翻译重新起始事件会产生分离的无活性片段。我们通过插入brca1和msh2基因的突变片段证明该系统可以准确检测人类基因中的PSC。蛋白质印迹分析揭示了所有测试菌落中的翻译重新起始事件，这意味着更简单的质粒无法抵抗这种假阴性结果来源。该系统应用于一个msh2基因存在无义突变的HNPCC家族，正确诊断出野生型纯合子和杂合子患者。
开发的pREAL适用于检测与不同疾病相关的人类基因中的PSC，并且能抵抗翻译重新起始事件。诊断步骤简单、成本低、仅检测病理性突变，并允许对分离的等位基因进行分析。
