Scorpione R C, De Camargo S S, Schenberg A C, Astolfi-Filho S
Departamento de Biologia Celular, Universidade de Brasília, DF.
Yeast. 1993 Jun;9(6):599-605. doi: 10.1002/yea.320090606.
A system is described for the selection of DNA sequences showing promoter activity in the yeast Saccharomyces cerevisiae using a heterologous reporter enzyme which is efficiently secreted by the yeast host. A multicopy shuttle plasmid of the YEp-type was constructed so as to carry multiple unique cloning sites at the 5' end of the Aspergillus awamori glucoamylase cDNA. Glucoamylase can only be expressed upon insertion at one of these unique cloning sites of a DNA fragment from any source, provided it is endowed with promoter function in S. cerevisiae. As the glucoamylase signal-peptide is functional in S. cerevisiae, the enzyme is efficiently secreted by the yeast transformants. This phenotype can be very easily detected on plate assays and accurately quantified by spectrophotometric analysis of the culture supernatant. Since S. cerevisiae naturally lacks amylolytic activity, any wild-type strain can be used as a host in this system. To evaluate the system, a DNA pool of random fusions was created by ligating sau 3A digested S. cerevisiae genomic DNA to the BglII-linearized vector. The resulting hybrid plasmids were transformed into S. cerevisiae and several transformants secreting glucoamylase to varying degrees were obtained.
描述了一种利用酵母宿主有效分泌的异源报告酶来选择在酿酒酵母中显示启动子活性的DNA序列的系统。构建了YEp型多拷贝穿梭质粒，以便在泡盛曲霉葡糖淀粉酶cDNA的5′端携带多个独特的克隆位点。只有当来自任何来源的DNA片段插入这些独特克隆位点之一时，葡糖淀粉酶才能表达，前提是该片段在酿酒酵母中具有启动子功能。由于葡糖淀粉酶信号肽在酿酒酵母中具有功能，该酶可被酵母转化体有效分泌。这种表型在平板分析中很容易检测到，并通过对培养上清液的分光光度分析进行准确量化。由于酿酒酵母天然缺乏淀粉分解活性，任何野生型菌株都可以用作该系统的宿主。为了评估该系统，通过将经Sau 3A消化的酿酒酵母基因组DNA连接到经BglII线性化的载体上，创建了一个随机融合的DNA文库。将所得的杂交质粒转化到酿酒酵母中，获得了几个不同程度分泌葡糖淀粉酶的转化体。
