Cloning and sequence analysis of the promoter region of the MRP gene of HL60 cells isolated for resistance to adriamycin.
Cloning and sequence analysis of the promoter region of the MRP gene of HL60 cells isolated for resistance to adriamycin.
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DOI：
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发表时间：
1994-08
期刊：
影响因子：
11.2
通讯作者：
Q. Zhu;M. S. Center
中科院分区：
文献类型：
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作者：
Q. Zhu;M. S. Center
HL60/ADR细胞的非P-糖蛋白多药耐药性似乎与MRP基因的过度表达有关。最近的研究表明，该基因可能在细胞对某些化疗药物的新形式耐药性中发挥重要作用。为了检查调节该基因转录活性的机制，从 HL60/ADR 细胞制备的基因组文库中分离出 MRP 的 2.2 KB 5' 侧翼序列。在瞬时转染实验中，发现与报告质粒中的氯霉素乙酰转移酶 (CAT) 基因连接的 2.2 KB DNA 片段能够驱动该基因的表达。这种含有启动子活性的 DNA 已被完整测序，并发现含有多个假定的调控位点。使用一系列与 CAT 报告基因连接的缺失突变体来检查 2.2 KB 序列的功能域。结果表明，启动子活性包含在 MRP 基因组富含 GC 区域的核苷酸 -91 至 +103 中。然而，该序列中包含的启动子活性受到正向和负向调节元件的调节。某些调控位点包含在其他基因的启动子区域中发现的正调控元件和负调控元件的共有序列。引物延伸分析表明存在来自 MRP 启动子的多个主要转录起始位点。 MRP 基因组和互补 DNA 的序列分析定义了外显子/内含子边界以及 MRP 基因组 5' 端区域的一部分的组织。因此，这些研究的结果为可能在 MRP 基因表达调节中发挥作用的位点特异性结构域提供了新的见解。
Non-P-glycoprotein multidrug resistance of HL60/ADR cells appears to be related to overexpression of the MRP gene. Recent studies suggest that this gene may play an important role in a new form of cell resistance to certain chemotherapeutic agents. To examine mechanisms regulating transcriptional activity of this gene, a 2.2-kilobase 5'-flanking sequence of MRP has been isolated from a genomic library prepared from HL60/ADR cells. The 2.2-kilobase DNA fragment linked to the chloramphenicol acetyltransferase (CAT) gene in a reporter plasmid was found to be capable of driving expression of this gene in transient transfection experiments. This DNA containing promoter activity has been sequenced in its entirety and found to contain multiple putative regulatory sites. A series of deletion mutants linked to the CAT reporter gene was used to examine functional domains of the 2.2-kilobase sequence. The results suggest that promoter activity is contained in nucleotides -91 to +103 in a GC-rich region of the MRP genome. Promoter activity contained within this sequence, however, is modulated by both positive and negative regulatory elements. Certain of the regulatory sites contain consensus sequences for positive and negative regulatory elements which have been found in the promoter regions of other genes. Primer extension analysis indicates the presence of multiple major transcriptional start sites from the MRP promoter. Sequence analysis of MRP genomic and complementary DNAs has defined the exon/intron boundaries and the organization of a portion of the 5'-end region of the MRP genome. The results of these studies thus provide new insight in site-specific domains which may function in the regulation of MRP gene expression.