Brunelli J P, Pall M L
Department of Genetics and Cell Biology, Washington State University, Pullman 99164-4234.
Yeast. 1993 Dec;9(12):1299-308. doi: 10.1002/yea.320091203.
Expression/shuttle vectors for the yeast Saccharomyces cerevisiae have usually been large plasmids with only one or a small number of sites that are suitable for cloning and expression. We report here the construction and properties of a series of 12 expression vectors with multiple (four to eight) unique sites in their polylinkers which allow directional cloning and expression of DNA sequences under four different promoters. Eleven of these plasmids replicate at high copy number in Escherichia coli, and all have the yeast TRP1 gene, and the 2 microns origin including REP3 sequence, allowing selection and high copy number replication in yeast. Six of the plasmids are designed for the construction and selection and high copy number replication in yeast. Six of the plasmids are designed for the construction and selection of cDNA libraries from various eukaryotic organisms, allowing directional cloning and expression of cDNAs. All of these six have similar polylinkers containing a unique promoter proximal EcoRI site and a unique promoter distal XhoI site, allowing for directional cloning and expression of 'ZAP'-type cDNAs. cDNAs that complement a wide variety of yeast mutants can be selected from libraries constructed in this way. The four alternative promoters, ADH2, PGK, GAL10 and SV40 were compared for their relative activity, both in E. coli and in yeast. All yeast promoters showed substantial activity in E. coli with ADH2 showing the highest activity. ADH2 also was well-regulated in yeast, showing very high relative activity under derepressing conditions. cDNAs selected by genetic complementation from libraries constructed in these vectors should be easily subclonable into other vectors, allowing expression in different eukaryotic organisms, DNA sequencing or site-directed mutagenesis.
用于酿酒酵母的表达/穿梭载体通常是大型质粒，只有一个或少数几个适合克隆和表达的位点。我们在此报告了一系列12种表达载体的构建及其特性，这些载体的多克隆位点中有多个（四到八个）独特位点，可实现DNA序列在四种不同启动子下的定向克隆和表达。其中11种质粒在大肠杆菌中以高拷贝数复制，并且都含有酵母TRP1基因以及包括REP3序列的2μm 原点，从而允许在酵母中进行选择和高拷贝数复制。其中六种质粒设计用于在酵母中进行构建、选择和高拷贝数复制。另外六种质粒设计用于构建和选择来自各种真核生物的cDNA文库，可实现cDNA的定向克隆和表达。所有这六种质粒都具有相似的多克隆位点，包含一个独特的靠近启动子的EcoRI位点和一个独特的远离启动子的XhoI位点，可实现“ZAP”型cDNA的定向克隆和表达。可以从以这种方式构建的文库中选择能够互补多种酵母突变体的cDNA。比较了四种替代启动子ADH2、PGK、GAL10和SV40在大肠杆菌和酵母中的相对活性。所有酵母启动子在大肠杆菌中均表现出显著活性，其中ADH2活性最高。ADH2在酵母中也受到良好调控，在去阻遏条件下显示出非常高的相对活性。通过遗传互补从这些载体构建的文库中选择的cDNA应该很容易亚克隆到其他载体中，从而允许在不同的真核生物中表达、进行DNA测序或定点诱变。
