Mallonee D H, Hylemon P B
Department of Microbiology and Immunology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0678, USA.
Mol Biotechnol. 1999 Feb;11(1):27-35. doi: 10.1007/BF02789174.
A short (43-bp) A/T-rich stretch of DNA located in the intergenic region between the baiA2 and baiF genes from Eubacterium sp. strain VPI 12708 was amplified by polymerase chain reaction (PCR) and inserted in front of the Shine-Dalgarno (SD) sequences of three inefficiently-expressed Eubacterium sp. strain VPI 12708 genes cloned in Escherichia coli plasmids. Insertion of this A/T-rich cassette increased gene expression in all cases tested. Deletion of part of the A/T-rich region from a baiF clone in pUC19 resulted in decreased gene expression. Synthesis of specific mRNA was increased with addition of the A/T-rich cassette to constructs containing the baiC gene from Eubacterium sp. strain VPI 12708, but mRNA synthesis was not significantly changed in cells containing plasmid constructs with the baiF and baiG genes. Enhanced translation resulting from a decrease in mRNA secondary structure in the ribosome binding site region is discussed as a possible reason for increased gene expression with the A/T-rich cassette.
通过聚合酶链反应（PCR）扩增了位于真杆菌属菌株VPI 12708的baiA2和baiF基因之间基因间隔区的一段短的（43个碱基对）富含A/T的DNA片段，并将其插入克隆于大肠杆菌质粒中的三个表达效率低下的真杆菌属菌株VPI 12708基因的Shine-Dalgarno（SD）序列之前。在所有测试案例中，插入这个富含A/T的盒式结构均增加了基因表达。从pUC19中的一个baiF克隆中缺失部分富含A/T的区域导致基因表达下降。向含有真杆菌属菌株VPI 12708的baiC基因的构建体中添加富含A/T的盒式结构可增加特异性mRNA的合成，但在含有带有baiF和baiG基因的质粒构建体的细胞中，mRNA合成没有显著变化。核糖体结合位点区域mRNA二级结构的减少导致翻译增强，这被认为是富含A/T的盒式结构使基因表达增加的一个可能原因。
