REGULATION OF SPORULATION-ASSOCIATED GENE TRANSCRIPTION

环境胁迫诱导基因转录 信号是遗传学的一个核心但仍然知之甚少的方面， 调控 芽孢杆菌枯草芽孢杆菌 激活多种孢子形成相关基因， 营养不足，并提供了一个良好的系统， 研究转录调控机制， 对营养环境的反应。 我目前 研究三个孢子形成相关基因的转录; 早期诱导孢子形成基因。 spoOH和spoVG以及一个基因 参与合成肽类抗生素tycA。 均出现 处于一种常见的消极控制之下， 一个叫做abrB的基因位点 细胞内GTP水平也 似乎在转录调控中发挥作用， 产孢基因 本提案中概述的主要目标 是： 1)定义顺式作用转录调控位点， spoVG和spoOH基因启动子的突变分析。 我们 我们也将研究其他基因的表达， 期望受到abrB的影响（即，编码基因 中性蛋白酶NPR）。 2)确定与GTP-和abrB-相关的调节因子， 依赖性阴性对照，最终目标是建立 连接促进孢子形成的代谢事件的途径 基因转录的激活。 我们将开始 分离引起spoVG，tycA， 和spoOH是组成性的和/或独立于负的 控制 3)利用转座子Tn分离abrB基因， 在abrB基因座中发现了917插入。 克隆DNA 转座子的侧翼将用于进行二倍体分析， 为了确定abrB基因座是否由一个以上的 基因 我们还将使用该DNA获得完整的abrB基因 然后将其排序，以确定主要的 产品结构（S）。 克隆的DNA将被用来制造 abrB缺失突变体。 我们还将使用abrB DNA 监测abrB的表达，并确定基因 表达的要求。 abrB产品将 在B subtilis细胞中过量产生，以检查高浓度的 AbrB水平对spoVG spoOH和tycA转录的影响 对孢子形成的影响。 abrB DNA将被诱变 并重新引入B。枯草，希望获得和 表征干扰孢子形成的abrB突变 入会仪式

The induction of gene transcription in response to environmental signals is a central but still poorly understand aspect of genetic regulation. The spore-forming bacterium Bacillus subtilis will activate a diverse collection of sporulation-associated genes when nutritionally deprived and provides an excellent system with which to study transcriptional regulatory mechanisms which are responsive to the nutritional environment. I am currently studying the transcription of three sporulation-associated genes; the early-induced sporulation genes. spoOH and spoVG and a gene involved in the synthesis of a peptide antibiotic, tycA. All appear to be under a common form of negative control that is dependent on a genetic locus known as abrB. Intracellular levels of GTP also appear to play a role in the transcriptional regulation of sporulation genes. The main objectives outlined in this proposal are to: 1) define cis-acting transcriptional regulatory sites through mutational analysis of the spoVG and spoOH gene promoters. We will also examine the expression of other genes which we would expect to be influenced by abrB (i.e., the gene encoding the neutral protease npr). 2) identify regulators that are associated with GTP- and abrB- dependent negative control with the ultimate goal of establishing a pathway linking the metabolic events which promote sporulation with the activation of gene transcription. We will begin by isolating mutations which cause the expression of spoVG, tycA, and spoOH to be constitutive and/or independent of negative control. 3) isolate the abrB gene(s) by taking advantage of a transposon Tn 917 insertion we have discovered in the abrB locus. Cloned DNA flanking the transposon will be used to perform diploid analysis in order to determine if the abrB locus is composed of more than one gene. We will also use this DNA to obtain the intact abrB gene(s) which will then be sequenced in order to determine the primary structure of its product(s). The cloned DNA will be used to create a abrB deletion mutant. We will also use the abrB DNA to monitor the expression of abrB and to determine the genetic requirements for its expression. The abrB product(s) will be overproduced in B subtilis cells to examine the effect of high AbrB levels on the transcription of spoVG spoOH and tycA as well as the effect on sporulation. The abrB DNA will be mutagenized and reintroduced into B. subtilis in hopes of obtaining and characterizing abrB mutations which interfere with sporulation initiation.