Cid V J, Alvarez A M, Santos A I, Nombela C, Sanchez M
Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Spain.
Yeast. 1994 Jun;10(6):747-56. doi: 10.1002/yea.320100606.
Yeast exo-1,3-beta-glucanases are secretable proteins whose function is basically trophic and may also be involved in cell wall glucan hydrolytic processes. Since fluorescein di(beta-D-glucopyranoside) is a fluorogenic substrate detectable and quantifiable by flow cytometry, it was used for testing the ability of the EXG1 gene product of Saccharomyces cerevisiae and its homologous gene in Candida albicans to function as reporter genes. These open reading frames were coupled to different promoters in multicopy plasmids, and exoglucanase activity quantified at flow cytometry. Exoglucanases were found to be useful tools for the study of promoter regions in S. cerevisiae. This technique has the advantage over other reporter gene systems--such as beta-galactosidase fusions--that it does not require permeabilization of yeast cells and therefore it allows the recovery of viable cells--by sorting--after flow cytometry analysis.
酵母外切 -1,3-β-葡聚糖酶是可分泌蛋白，其功能主要是营养性的，也可能参与细胞壁葡聚糖的水解过程。由于荧光素二（β-D-吡喃葡萄糖苷）是一种可通过流式细胞术检测和定量的荧光底物，因此它被用于测试酿酒酵母EXG1基因产物及其白色念珠菌同源基因作为报告基因的功能。这些开放阅读框与多拷贝质粒中的不同启动子相连，并通过流式细胞术对外切葡聚糖酶活性进行定量。结果发现外切葡聚糖酶是研究酿酒酵母启动子区域的有用工具。该技术相对于其他报告基因系统（如β-半乳糖苷酶融合系统）具有优势，即它不需要对酵母细胞进行通透处理，因此在流式细胞术分析后可通过分选回收活细胞。
