Use of a halobacterial bgaH reporter gene to analyse the regulation of gene expression in halophilic archaea
Use of a halobacterial bgaH reporter gene to analyse the regulation of gene expression in halophilic archaea
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DOI：
10.1099/00221287-147-7-1745
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发表时间：
2001-07-01
期刊：
影响因子：
2.8
通讯作者：
Pfeifer, F
中科院分区：
文献类型：
--
作者：
Gregor, D;Pfeifer, F
将编码“Haloferax palantei”的β-半乳糖苷酶的bgaH阅读框用作报告基因，以研究来自Haloferax volcanii转化体中的地中海盐生盐菌（Mc-gvpA）和盐生盐杆菌（C-gvpA和P-gvpA）的gvpA基因的三个不同启动子区。bgaH在每个gvpA阅读框的起始密码子处的融合（A1-bgaH融合基因）在某些情况下引起翻译问题。含有在gvpA阅读框（A-bgaH）的更下游具有融合的构建体的转化体产生β-半乳糖苷酶，并且当用X-Gal喷雾时，琼脂平板上的菌落变成蓝色。通过标准ONPG测定法定量的β-半乳糖苷酶活性与用含有相应gvpA基因的转化体测定的mRNA数据良好相关：cA-bgaH融合基因完全失活，mcA-bgaH转化体显示少量产物，而pA-bgaH融合基因在相应转化体中组成型表达。每个A-bgaH基因的转录被同源转录激活蛋白GvpE激活。cGvpE、pGvpE和mcGvpE蛋白能够激活pA-kgaH和mcA-bgaH的启动子，而cA-bgaH的启动子仅被cGvpE激活。在所测试的三种GvpE蛋白中，cGvpE似乎是最强的转录激活因子。
The bgaH reading frame encoding a beta -galactosidase of 'Haloferax alicantei' was used as a reporter gene to investigate three different promoter regions derived from gvpA genes of Haloferax mediterranei (mc-gvpA) and Halobacterium salinarum (c-gvpA and p-gvpA) in Haloferax volcanii INTRODUCTION transformants. The fusion of bgaH at the start codon of each gvpA reading frame (A1-bgaH fusion genes) caused translational problems in some cases. Transformants containing constructs with fusions further downstream in the gvpA reading frame (A-bgaH) produced beta -galactosidase, and colonies on agar plates turned blue when sprayed with X-Gal. The beta -galactosidase activities quantified by standard ONPG assays correlated well with the mRNA data determined with transformants containing the respective gvpA genes: the cA-bgaH fusion gene was completely inactive, the mcA-bgaH transformants showed low amounts of products whereas the pA-bgaH fusion gene was constitutively expressed in the respective transformants. The transcription of each A-bgaH gene was activated by the homologous transcriptional activator protein GvpE. The cGvpE, pGvpE and mcGvpE proteins were able to activate the promoter of pA-kgaH and mcA-bgaH, whereas the promoter of cA-bgaH was only activated by cGvpE. Among the three GvpE proteins tested, cGvpE appeared to be the strongest transcriptional activator.