Carú M, Cifuentes V, Pincheira G, Jiménez A
Departmento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Santiago.
J Appl Bacteriol. 1989 Oct;67(4):401-10. doi: 10.1111/j.1365-2672.1989.tb02510.x.
A plasmid (named pCN2) carrying a 7.6 kb BamHI DNA insert was isolated from a Neurospora crassa genomic library raised in the yeast vector YRp7. Saccharomyces cerevisiae suco and N. crassa inv strains transformed with pNC2 were able to grow on sucrose-based media and expressed invertase activity. Saccharomyces cerevisiae suco (pNC2) expressed a product which immunoreacted with antibody raised against purified invertase from wild type N. crassa, although S. cerevisiae suc+ did not. The cloned DNA hybridized with a 7.6 kb DNA fragment from BamHI-restricted wild type N. crassa DNA. Plasmid pNC2 transformed N. crassa Inv- to Inv+ by integration either near to the endogenous inv locus (40% events) or at other genomic sites (60% events). It appears therefore that the cloned DNA piece encodes the N. crassa invertase enzyme. A 3.8 kb XhoI DNA fragment, derived from pNC2, inserted in YRp7, in both orientation, was able to express invertase activity in yeast, suggesting that it contains an intact invertase gene which is not expressed from a vector promoter.
从以酵母载体YRp7构建的粗糙脉孢菌基因组文库中分离出一个携带7.6 kb BamHI DNA插入片段的质粒（命名为pCN2）。用pNC2转化的酿酒酵母suco和粗糙脉孢菌inv菌株能够在以蔗糖为基础的培养基上生长并表达转化酶活性。酿酒酵母suco（pNC2）表达了一种与针对野生型粗糙脉孢菌纯化转化酶产生的抗体发生免疫反应的产物，而酿酒酵母suc+则没有。克隆的DNA与来自BamHI酶切的野生型粗糙脉孢菌DNA的7.6 kb DNA片段杂交。质粒pNC2通过整合在内源inv基因座附近（40%的事件）或其他基因组位点（60%的事件）将粗糙脉孢菌Inv-转化为Inv+。因此，似乎克隆的DNA片段编码粗糙脉孢菌转化酶。源自pNC2的一个3.8 kb XhoI DNA片段以两种方向插入YRp7，能够在酵母中表达转化酶活性，这表明它包含一个完整的转化酶基因，该基因不是从载体启动子表达的。
