Jain Vikas, Sujatha Subbanna, Ojha Anil Kumar, Chatterji Dipankar
Molecular Biophysics Unit, Indian Institute of Science, Bangalore.
Gene. 2005 May 23;351:149-57. doi: 10.1016/j.gene.2005.03.038.
The rel gene is responsible for the maintenance of the level of (p)ppGpp in bacteria under nutrient starvation. This phenomenon known as stringent response plays an important role during survival of the microorganisms in stationary phase. We have cloned 1.6 kb upstream sequence of rel gene of Mycobacterium tuberculosis in a shuttle vector pSD5B containing promoterless lacZ gene and promoter activity was observed in Mycobacterium smegmatis cells by blue/white selection and was measured by beta-galactosidase assay. In order to delineate the minimal promoter element of rel gene, a 200 bp fragment from this 1.6 kb upstream sequence was further cloned in promoterless lacZ shuttle vector pSD5B and promoter activity was observed in M. smegmatis cells in similar way. The 200 bp promoter fragment was found to be mycobacterium specific and did not respond when transformed in Escherichia coli. The +1 transcription start site was determined by primer extension method. The -10 promoter region was identified to be TATCCT. The three T bases when mutated, showed a remarkable decrease in the lacZ expression thus confirming the -10 region. The translation start site has also been identified by site directed frame shift mutagenesis. It appears that this rel promoter can be used for expression of proteins in mycobacteria.
rel基因负责在营养饥饿情况下维持细菌中（p）ppGpp的水平。这种被称为严谨反应的现象在微生物处于稳定期存活期间发挥重要作用。我们已将结核分枝杆菌rel基因的1.6 kb上游序列克隆到含有无启动子lacZ基因的穿梭载体pSD5B中，并通过蓝/白筛选在耻垢分枝杆菌细胞中观察到启动子活性，且通过β-半乳糖苷酶测定法进行了测量。为了确定rel基因的最小启动子元件，将来自该1.6 kb上游序列的一个200 bp片段进一步克隆到无启动子lacZ穿梭载体pSD5B中，并以类似方式在耻垢分枝杆菌细胞中观察到启动子活性。发现该200 bp启动子片段是分枝杆菌特异性的，在转化到大肠杆菌中时无反应。通过引物延伸法确定了+1转录起始位点。鉴定出-10启动子区域为TATCCT。这三个T碱基发生突变时，lacZ表达显著下降，从而证实了-10区域。还通过定点移码诱变确定了翻译起始位点。看来这个rel启动子可用于在分枝杆菌中表达蛋白质。
