Cloning of Genes Transcribed by Alternate Sigma Factors

9316114 Jonas本项目将鉴定革兰氏阳性原核生物枯草芽孢杆菌中由备用sigma因子转录的基因。具有特定sigma因子识别的启动子的基因将使用含有报告基因（Tn917lac）的转座子进行鉴定，该转座子应在枯草芽孢杆菌的染色体中产生随机插入突变（和转录融合）。这些突变将在枯草芽孢杆菌菌株中筛选，这些菌株含有活性形式的b或e基因的结构基因的诱导拷贝（在空间启动子的控制下），这些基因由IPTG诱导插入，在这样的系统中将被定位、克隆和测序。这些基因的启动子区域将被分析，提供关于由备选sigma因子使用的调控序列的信息。这些基因的功能可以通过插入突变体的表型或与已知功能基因序列的同源性来揭示。我们将尝试通过识别调控基因的功能来确定备选sigma因子在体内发挥的作用。获得的有关基因调控的知识可能对发酵工业有用，这可能受益于在细菌中过量生产基因产物时使用备用sigma因子。＊＊＊

9316114 Jonas This project will result in the identification of genes transcribed by alternate sigma factors in the Gram positive prokaryote, Bacillus subtilis. Genes having promoters recognized by a particular sigma factor will be identified using a transposon engineered to contain a reporter gene (Tn917lac), which should generate random insertional mutations (and transcriptional fusions) in the chromosome of B. subtilis. These mutations will be screened in strains of B. subtilis containing inducible copies (under control of the spac promoter) of the structural genes for the active forms of B. or E. genes bearing insertion that are induced by IPTG in such a system will be mapped, cloned, and sequenced. The promoter regions of these genes will be analyzed, providing information concerning regulatory sequences used by alternative sigma factors. The functions of these genes may be revealed by the phenotype of the insertional mutants, or by homology with the sequences of genes with known functions. We will attempt to determine the role the alternative sigma factor plays in vivo by recognition of the function of the genes in the regulon. %%% The knowledge gained concerning genetic regulation may prove useful to the fermentation industry, which could benefit from using alternate sigma factors in over producing gene product in bacteria. ***