Gaur Naseem Akhtar, Puri Neeti, Karnani Neerja, Mukhopadhyay Gauranga, Goswami Shyamal K, Prasad Rajendra
Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110-067, India.
FEMS Yeast Res. 2004 Jan;4(4-5):389-99. doi: 10.1016/S1567-1356(03)00204-6.
We have earlier shown that transcriptional activation of the Candida drug resistance gene, CDR1, is linked to various stresses wherein a proximal promoter (-345 bp from the transcription start point (TSP)) was found to be predominantly more responsive. In this study we have examined basal expression of the CDR1 proximal promoter by employing a Renilla luciferase reporter system. We observed that upon sequential deletion of the proximal promoter, there was modulation in basal reporter activity. The reporter activity was highest (2.3-fold) in NGY261 (-261 bp from TSP), and was reduced upon subsequent deletions. DNase I footprinting revealed four protected regions (W1, W2, W3 and W4) in the proximal promoter which could represent possible trans-acting factor binding sites and thus might be involved in CDR1 expression. Site-directed mutational analysis of three of these protected regions did not significantly affect the basal reporter activity, however, the mutation of W1 led to a considerable enhancement in reporter activity (approximately 4-fold) and was designated a negative regulatory element (NRE). Mutation as well as deletion of the W1 sequence in the native promoter (-1147 bp from TSP) and sequential deletion of the 5'-flanking region-harboring W1 (NRE) also resulted in enhanced promoter reporter activity. When the reporter activity of native (NPY1147) and NRE-mutated (NGYM1147) promoter integrants was monitored throughout the growth phase of Candida albicans, there was modulation in reporter activity in both integrants, but interestingly the level of basal reporter activity of the NRE-mutated promoter was always approximately 3-fold higher than that of the native promoter. UV cross-linking and affinity purification confirmed that a purified approximately 55-kDa nuclear protein specifically interacts with the NRE. Taken together, we have identified a NRE and purified its interactive protein, which may be involved in controlling basal expression of CDR1.
我们之前已经表明，念珠菌耐药基因CDR1的转录激活与多种应激相关，其中发现近端启动子（距转录起始点（TSP）-345 bp）对这些应激反应更为显著。在本研究中，我们采用海肾荧光素酶报告系统检测了CDR1近端启动子的基础表达。我们观察到，随着近端启动子的逐步缺失，报告基因的基础活性发生了变化。在NGY261（距TSP -261 bp）中报告基因活性最高（2.3倍），随后的缺失使其降低。DNase I足迹分析揭示了近端启动子中的四个保护区域（W1、W2、W3和W4），这些区域可能代表潜在的反式作用因子结合位点，因此可能参与CDR1的表达。对其中三个保护区域进行定点突变分析并未显著影响报告基因的基础活性，然而，W1的突变导致报告基因活性显著增强（约4倍），并被指定为负调控元件（NRE）。天然启动子（距TSP -1147 bp）中W1序列的突变和缺失，以及包含W1（NRE）的5'侧翼区域的逐步缺失，也导致启动子报告基因活性增强。当在白色念珠菌的整个生长阶段监测天然（NPY1147）和NRE突变（NGYM1147）启动子整合体的报告基因活性时，两个整合体的报告基因活性均有变化，但有趣的是，NRE突变启动子的基础报告基因活性水平始终比天然启动子高约3倍。紫外线交联和亲和纯化证实，一种纯化的约55 kDa核蛋白与NRE特异性相互作用。综上所述，我们鉴定出了一个NRE并纯化了其相互作用蛋白，它们可能参与控制CDR1的基础表达。
