Du Wenjin, Coaker Melisa, Sobel Jack D, Akins Robert A
Department of Biochemistry and Molecular biology, Wayne State University School of Medicine, Detroit, MI 48201, USA.
Curr Genet. 2004 Jun;45(6):390-8. doi: 10.1007/s00294-004-0499-3. Epub 2004 Mar 18.
Plasmids containing the inosine monophosphate dehydrogenase gene CaIMH3 from Candida albicans strain ATCC 32354 transform their host to resistance against mycophenolic acid (MPA). The transformants maintain the plasmids at a high copy number (20-40 per cell) and express the CaIMH3 gene at very high levels relative to untransformed controls. The plasmid copy number can be controlled by the concentration of MPA in the media. The transformation procedure is reproducible and the efficiency of transformation is high, up to 15,000 per microgram. Unrearranged plasmids are readily recovered by transforming total DNA from transformants back into Escherichia coli. C. albicans genes cloned into the plasmid are expressed at elevated levels relative to untransformed controls. A derivative vector containing the CaMAL2 promoter and termination sequences expresses the CaERG11 ORF at high levels and confers moderate resistance to fluconazole. These shuttle vectors should facilitate global genomics approaches in C. albicans that have been hampered by its diploid genome.
含有来自白色念珠菌菌株ATCC 32354的肌苷单磷酸脱氢酶基因CaIMH3的质粒可使宿主对霉酚酸（MPA）产生抗性。转化子以高拷贝数（每个细胞20 - 40个）维持质粒，并相对于未转化的对照以非常高的水平表达CaIMH3基因。质粒拷贝数可通过培养基中MPA的浓度来控制。转化过程具有可重复性，转化效率很高，可达每微克15000个。通过将转化子的总DNA再转化回大肠杆菌，可以很容易地回收未重排的质粒。克隆到质粒中的白色念珠菌基因相对于未转化的对照以更高的水平表达。一种含有CaMAL2启动子和终止序列的衍生载体可高水平表达CaERG11开放阅读框，并赋予对氟康唑的中度抗性。这些穿梭载体应有助于克服因白色念珠菌二倍体基因组而受阻的全基因组学研究方法。
