Sasnauskas K V, Gedminene G K, Markiavichius A I, Naktinis V I, Ianulaĭtis A A
Genetika. 1986 Apr;22(4):549-56.
ADE1 gene of Saccharomyces cerevisiae codes for the primary structure of SAICAR-synthetase. Mutational changes of ADE1 gene result in the accumulation of red pigment in cells. Colour differences, thus, serve as a basis for the selection of mutants or transformants. ADE1 gene was cloned as a 4.0 kb HindIII fragment of yeast DNA in a shuttle vector by complementing the ade1 mutation in yeast. The study of ADE1 gene expression in Escherichia coli showed that the 4.0 kb fragment containing the ADE1 gene does not complement purC mutations in E. coli. However, prototrophic colonies appeared at a frequency of 10(-7)-10(-8) after incubating clones bearing the recombinant plasmid with ADE1 gene on selective media. The plasmid DNA isolated from such clones complements the purC mutation in E. coli and the ade1 mutation in S. cerevisiae. Structural analysis of the plasmid demonstrated that the cloned DNA fragment contained an additional insertion of the bacterial origin. Further restriction enzyme analysis proved the insertion to be the bacterial element IS1. Expression of the cloned ADE1 gene in S. cerevisiae is controlled by its own promoter, whereas in E. coli it is controlled by the IS1 bacterial element.
酿酒酵母的ADE1基因编码SAICAR合成酶的一级结构。ADE1基因的突变会导致细胞中红色色素的积累。因此，颜色差异可作为筛选突变体或转化体的基础。通过互补酵母中的ade1突变，将ADE1基因作为酵母DNA的4.0 kb HindIII片段克隆到穿梭载体中。对ADE1基因在大肠杆菌中的表达研究表明，含有ADE1基因的4.0 kb片段不能互补大肠杆菌中的purC突变。然而，将携带含ADE1基因重组质粒的克隆在选择性培养基上培养后，原养型菌落出现的频率为10^(-7)-10^(-8)。从这些克隆中分离出的质粒DNA可互补大肠杆菌中的purC突变和酿酒酵母中的ade1突变。对该质粒的结构分析表明，克隆的DNA片段包含一个额外的细菌来源插入片段。进一步的限制性内切酶分析证明该插入片段是细菌元件IS1。克隆的ADE1基因在酿酒酵母中的表达受其自身启动子控制，而在大肠杆菌中则受IS1细菌元件控制。
