UPSTREAM ACTIVATION OF BACILLUS PROMOTERS

芽孢杆菌启动子的上游激活

基本信息

批准号：
3296438
负责人：
JAMES Alfred HOCH
金额：
$ 15.26万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-02-01 至 1991-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3296438
关键词：
Bacillus subtilis DNA binding protein bacterial genetics enzyme biosynthesis gene expression gene mutation genetic manipulation genetic mapping genetic promoter element genetic transcription messenger RNA molecular cloning nucleic acid probes nucleic acid sequence peptidases regulatory gene site directed mutagenesis transposon /insertion element

项目摘要

A number of genes including those for the major degradative enzymes of Bacillus subtilis appear to be controlled by several regulatory genes which may comprise a global regulatory system. The regulatory genes, sacU, sacQ, prtR, and hpr pleiotropically affect the synthesis of several enzymes including levansucrase, sacB, and alkaline protease, aprE. The target of these regulatory genes on either sacB or aprE is at least 100 basepairs upstream of the promoter for the genes and they stimulate transcription from the normal start site. Studies are proposed to isolate the only remaining uncloned regulatory gene, sacU and characterize mutations in the gene. The molecular basis for the phenotypes of mutations in each gene will be probed. Other genes in which mutation leads to an alteration of this regulatory network will be isolated and characterized. The site of action of the regulatory genes will be determined for the aprE (subtilisin) promoter. Extensive deletion analyses from both directions will be used to define the target sites. Site-directed and cassette mutagenesis experiments will further implicate important bases. Messenger RNA will be isolated from mutant strains and transcription start sites found by primer extension to determine if all the regulatory mutations stimulate transcription from the normal start site. Northern analysis will ascertain if messenger RNA levels in all mutants are elevated. Large quantities of purified proteins will be produced in expression vectors. These proteins will be used in binding studies and footprints analyses to determine which proteins bind to the targets and where. Studies are proposed using lac fusions to each regulatory gene to uncover the regulatory interactions among them. The relationship between these genes and catabolite repression will be probed.

许多基因，包括主要降解的基因枯草芽孢杆菌的酶似乎由几个可能构成全球监管系统的监管基因。调节基因，SACU，SACQ，PRTR和HPR PLEIOTIOTIOTIC 影响几种酶的合成，包括左千非酶， SACB和碱性蛋白酶，Apre。这些监管的目标 SACB或APRE上的基因至少是100底游基因的启动子，它们刺激了转录正常的开始站点。提出了研究以隔离唯一的剩余的未粘结的调节基因，SACU和特征基因中的突变。表型的分子基础每个基因中的突变将被探测。其他基因突变会导致该监管网络的改变孤立和特征。监管的作用部位将确定针对APRE（枯草菌素）启动子的基因。来自两个方向的大量删除分析将用于定义目标位点。定向和盒式诱变实验将进一步暗示重要的基础。信使 RNA将从突变菌株中分离出来，转录开始引物扩展发现的位点以确定是否所有调节突变刺激正常起始位点的转录。北方分析将确定Messenger RNA是否水平突变体升高。大量纯化蛋白将在表达载体中产生。这些蛋白质将用于结合研究和足迹分析，以确定哪个蛋白质结合到靶标和靶标。提出了研究与每个调节基因的lac融合以发现调节他们之间的互动。这些基因之间的关系将探测分解代谢物的抑制作用。