Ross-Macdonald P, Sheehan A, Roeder G S, Snyder M
Department of Biology, Yale University, New Haven, CT 06520-8103, USA.
Proc Natl Acad Sci U S A. 1997 Jan 7;94(1):190-5. doi: 10.1073/pnas.94.1.190.
Analysis of the function of a particular gene product typically involves determining the expression profile of the gene, the subcellular location of the protein, and the phenotype of a null strain lacking the protein. Conditional alleles of the gene are often created as an additional tool. We have developed a multifunctional, transposon-based system that simultaneously generates constructs for all the above analyses and is suitable for mutagenesis of any given Saccharomyces cerevisiae gene. Depending on the transposon used, the yeast gene is fused to a coding region for beta-galactosidase or green fluorescent protein. Gene expression can therefore be monitored by chemical or fluorescence assays. The transposons create insertion mutations in the target gene, allowing phenotypic analysis. The transposon can be reduced by cre-lox site-specific recombination to a smaller element that leaves an epitope tag inserted in the encoded protein. In addition to its utility for a variety of immunodetection purposes, the epitope tag element also has the potential to create conditional alleles of the target gene. We demonstrate these features of the transposons by mutagenesis of the SPA2, ARP100, SER1, and BDF1 genes.
对特定基因产物功能的分析通常包括确定该基因的表达谱、蛋白质的亚细胞定位以及缺乏该蛋白质的缺失菌株的表型。该基因的条件等位基因通常作为一种额外的工具来创建。我们开发了一种基于转座子的多功能系统，该系统能同时生成用于上述所有分析的构建体，并且适用于对任何给定的酿酒酵母基因进行诱变。根据所使用的转座子，酵母基因会与β-半乳糖苷酶或绿色荧光蛋白的编码区域融合。因此，可以通过化学或荧光测定法监测基因表达。转座子会在靶基因中产生插入突变，从而实现表型分析。转座子可以通过cre-lox位点特异性重组减少为一个较小的元件，该元件会在编码的蛋白质中留下一个表位标签。除了可用于多种免疫检测目的外，表位标签元件还有可能创建靶基因的条件等位基因。我们通过对SPA2、ARP100、SER1和BDF1基因进行诱变来证明这些转座子的特性。
