Smith D J, Proudfoot A E, Detiani M, Wells T N, Payton M A
Glaxo Institute for Molecular Biology, Geneva, Switzerland.
Yeast. 1995 Apr 15;11(4):301-10. doi: 10.1002/yea.320110402.
Using a DNA fragment derived from the Saccharomyces cerevisiae phosphomannose isomerase (PMI) structural gene as a probe against a random ordered array library of genomic DNA from the pathogenic fungus Candida albicans, we have cloned the C. albicans PMI 1 gene. This gene, which is unique in the C. albicans genome, can functionally complement PMI-deficient mutants of both S. cerevisiae and Escherichia coli. The DNA sequence of the PMI 1 gene predicts a protein with 64.1% identity to PMI from S. cerevisiae. Sequential gene disruption of PMI 1 produces a strain with an auxotrophic requirement for D-mannose. The heterologous expression of the PMI 1 gene at levels up to 45% of total cell protein in E. coli leads to partitioning of the enzyme between the soluble and particulate fractions. The protein produced in the soluble fraction is indistinguishable in kinetic properties from the material isolated from C. albicans cells.
我们使用来自酿酒酵母磷酸甘露糖异构酶（PMI）结构基因的DNA片段作为探针，与致病性真菌白色念珠菌基因组DNA的随机有序阵列文库进行杂交，从而克隆出了白色念珠菌PMI 1基因。该基因在白色念珠菌基因组中是独一无二的，它能够在功能上互补酿酒酵母和大肠杆菌的PMI缺陷型突变体。PMI 1基因的DNA序列预测其编码的蛋白质与酿酒酵母的PMI有64.1%的同一性。对PMI 1进行连续基因破坏会产生一株对D-甘露糖有营养缺陷需求的菌株。PMI 1基因在大肠杆菌中以高达总细胞蛋白45%的水平进行异源表达时，该酶会在可溶部分和颗粒部分之间进行分配。在可溶部分产生的蛋白质在动力学性质上与从白色念珠菌细胞中分离出的物质没有区别。
