白色念珠菌抗藥性基因MDR1調控序列之分析研究

Abstract

先前實驗室(Lo, 2002)在麵包酵母菌系統下利用刪減序列分析以及定點突變方式指出對白色念珠菌MDR1 promoter 的M12 (-736 ~-734)位置突變後可使活性明顯下降。為了找出其他在MDR1 promoter 的cis-element序列，我將不同臨床菌株帶有不同序列的MDR1 promoter與Lac Z的open reading frame建構成重組基因。藉由測量b-galactosidase活性，找到了-640、-617以及-259的序列可能會調節MDR1 promoter的活性。為了確認這些點以及M12對MDR1 promoter活性的重要性，我將含有這些突變點的MDR1 promoter接上水母冷光酵素基因並送回白色念珠菌的基因體內，測量冷光活性。正如預期的，MDR1 promoter在4-NQO藥物的誘導下，活性增加約二十倍。平均看來這些含有突變的MDR1 promoters比野生株的活性高，但這個結果並不明顯。出乎意料的，在cph1/cph1 homozygous 菌株內，MDR1 promoter 的活性增加將近十倍，此資料顯示Cph1 蛋白質在白色念珠菌內可能扮演負向調控因子角色。

Previously research in the laboratory (Lo, 2002) has used deletion analysis and mutagenesis method to demonstrate that the activity of the promoter of Candida albicans MDR1 gene was greatly decreased when it contains a mutation at the sequence named “M12”(-734 to -736；A of translation initiation site ATG as +1) in S. cerevisiae. To identify other potential cis-acting regulatory elements on the MDR1 promoter, I have constructed MDR1 promoter-lacZ (MDR1p-lac Z) fusion plasmids of which MDR1 promoters were from different clinical C. albicans strains. The –640, -617, and –259 bp were identified as potential sites for regulating the activity of the MDR1 promoter by comparing the activity of b-galactosidase of different MDR1p-lac Z fusions. The importance of these sites and the M12 site for the activity of the MDR1 promoter in C. albicans were further investigated by integrating MDR1 promoter-Renilla luciferase gene (MDR1p-RLUC) fusion of which the MDR1 promoters contained different mutations into C. albicans. As expected, the activity of the MDR1 promoter has increased approximately 20-fold under the 4-nitroquinoline oxide (4-NQO) induction. By average, the MDR1 promoters with mutations have higher activities than the wild type MDR1 promoter under 4-NQO induction. Nevertheless, the increase is not dramatically. Surprisingly, the activity of MDR1p-RLUC was increased approximately 10-fold in the cph1/cph1 homozygous mutant strain. This data suggests that the Cph1 acts as a negative regulator of the MDR1 promoter in C. albicans.