Sidhu R S, Bollon A P
Department of Molecular Genetics, Wadley Institutes of Molecular Medicine, Dallas, TX 75235.
Gene. 1987;54(2-3):175-84. doi: 10.1016/0378-1119(87)90485-9.
In yeast, Saccharomyces cerevisiae, the PHO5 gene encodes the repressible acid phosphatase (APase) whose activity can be easily monitored by either the staining of colonies or by colorimetric assay. Therefore, gene fusions to PHO5 provide a convenient system for structural and functional analysis of yeast genes. We have constructed fusions of the PHO5 gene with a MF alpha 1 gene of yeast to delineate the secretion signal(s) in the alpha-factor leader peptide. Gene fusion between MF alpha 1 and PHO5 codes for a hybrid protein in which the alpha-factor leader peptide of 89 amino acids (aa) directed the export of APase, a periplasmic protein, into the medium. Since the hybrid gene is transcribed from the alpha-factor promoter, expression of the APase activity from these hybrid genes showed cell type-specific regulation. Further analyses of another MF alpha 1-PHO5 fusion showed that only the first 22 aa of the 89-aa alpha-factor leader peptide contained sufficient information for the secretion of APase into the medium. This shows that, in addition to the analysis of gene regulation, PHO5 fusions can be used to study signals involved in the proper localization of proteins.
在酵母酿酒酵母中，PHO5基因编码可阻遏酸性磷酸酶（APase），其活性可通过菌落染色或比色测定法轻松监测。因此，与PHO5的基因融合为酵母基因的结构和功能分析提供了一个便捷的系统。我们构建了PHO5基因与酵母MFα1基因的融合体，以描绘α因子前导肽中的分泌信号。MFα1和PHO5之间的基因融合编码一种杂合蛋白，其中89个氨基酸（aa）的α因子前导肽将周质蛋白APase引导至培养基中。由于杂合基因是从α因子启动子转录而来的，这些杂合基因的APase活性表达显示出细胞类型特异性调控。对另一个MFα1-PHO5融合体的进一步分析表明，89个氨基酸的α因子前导肽中只有前22个氨基酸包含将APase分泌到培养基中的足够信息。这表明，除了基因调控分析外，PHO5融合体还可用于研究参与蛋白质正确定位的信号。
