筛选抑制酿酒酵母生长的人类基因

【目的】基于人类基因文库,构建一个筛选抑制酿酒酵母生长的人类基因的方法,并运用此方法筛选含有生长抑制性人源蛋白质的酿酒酵母,用于分析人类基因的生理功能及其抑制剂的寻找。【方法】利用Gateway~(TM)重组技术将人类蛋白质编码基因构建到酿酒酵母表达质粒中。得到的质粒分别转化酿酒酵母细胞中,分析哪些基因的表达会抑制酿酒酵母的生长,并用绿色荧光蛋白标签对典型候选基因在酿酒酵母中的定位进行观察。【结果与结论】本研究建立了抑制酿酒酵母生长的人类基因的筛选方法,并运用此方法成功地从2991个人类蛋白质编码基因中筛选到29个显著抑制酿酒酵母生长的基因。其中一些是引起人类疾病的致病基因。例如,PDLIM4参与到骨质疏松症和前列腺癌的形成和发展,但其生理功能尚不清楚。我们的研究可能为揭示这些候选基因的功能和调节机制提供新的途径。

Screening of human genes that inhibit vegetative growth of budding yeast

Objective] Heterologous expression of some human genes in yeast cells leads a severe growth defect. This phenomenon can occur when intrinsic yeast regulatory mechanisms are perturbed by the physiological function of the ectopically expressed human protein. Yeast cells are amenable to genetic studies and used as a platform for high-throughput screening. Thus, yeast cells harboring such growth inhibitory human proteins may be useful to analyze physiological function of the human protein and applied to find its inhibitor. In the present study, we constructed a collection of yeast expression plasmid harboring human protein-coding genes. Using the human gene library, we established a screening system to identify genes whose expression cause a growth defect in yeast cells. Methods] Using the Gateway recombination technology, human protein-cording genes were cloned into a yeast expression plasmid. The resulting plasmids were individually transformed into yeast cells and analyzed whether their expression effects on yeast growth. Furthermore, identified inhibitory genes were expressed as GFP fusion proteins and their localization in yeast cells were analyzed. Results and Conclusion] Among 2991 human protein-cording genes, we identified 29 genes whose expression caused a sever growth defect in yeast cells. Some of them are causative genes to develop human disorders. For example, PDLIM4 is involved in development of osteoporosis and prostate cancer. Physiological function of PDLIM4 has not been understand very well. Our study may provide another option to investigate human proteins.