Zvonok N M, Horváth E, Bajszár G
Laboratory of Molecular Biology, VEPEX-BIOTECHNIKA Ltd., Szeged, Hungary.
Gene. 1988 Jun 30;66(2):313-8. doi: 10.1016/0378-1119(88)90368-x.
The promoterless PHO5 gene of the yeast Saccharomyces cerevisiae, encoding the repressible acid phosphatase (AP) was utilized as a reporter gene for the construction of a novel vector system for selection and functional analysis of yeast promoters. The Escherichia coli-yeast shuttle plasmids, pZHB81 and pZHB82, contain different arrays of unique restriction sites located upstream of the PHO5 coding region. Yeast promoters could be screened from random DNA fragments (cloned in the upstream sites) for their ability to direct the expression of the PHO5 gene in transformed (AP-deficient) yeast host cells. AP-expressing transformants were selected directly on agar plates by using a routine colony staining method. Relative promoter strength was assessed by direct assay for AP activity in cell lysates.
酿酒酵母的无启动子PHO5基因编码可阻遏酸性磷酸酶（AP），该基因被用作报告基因，用于构建一种新型载体系统，以筛选酵母启动子并对其进行功能分析。大肠杆菌-酵母穿梭质粒pZHB81和pZHB82在PHO5编码区上游含有不同排列的独特限制酶切位点。可以从随机DNA片段（克隆在上游位点）中筛选酵母启动子，看其指导PHO5基因在转化的（缺乏AP）酵母宿主细胞中表达的能力。通过常规菌落染色法在琼脂平板上直接筛选表达AP的转化体。通过直接检测细胞裂解物中的AP活性来评估相对启动子强度。
