REGULATION OF A COMPETENCE GENE LOCUS IN H. INFLUENZAE

流感嗜血杆菌中活性基因座的调控

• 批准号: 3307738
• 负责人: HAMILTON O SMITH
• 金额: $ 14.39万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3307738
• 关键词: Haemophilus influenzae; RNase protection assay; bacterial genetics; fusion gene; gene complementation; gene expression; genetic mapping; genetic regulation; genetic regulatory element; genetic transcription; lac operon; molecular cloning; reporter genes; site directed mutagenesis; western blottings

The long term objective of our laboratory is to understand the mechanism of genetic transformation in H. influenzae at a molecular and biochemical level. In particular, we want to understand the machinery of donor DNA transport through the cell membranes and the complex regulatory pathways that control competence induction. It is toward the latter objective that this grant proposal is directed. In the course of isolating a number of transformation mutants using the mini-Tn10kan transposon mutagenesis approach, we discovered a cluster of mutations spanning a 4-kb region of the chromosome. We recently sequenced this region and flanking sequences and found six tandemly arranged genes which are coordinately induced during competence development. Our studies will focus first on determining whether these genes are cotranscribed as in an operon, or are individually transcribed. In addition, we will identify and study the regulatory genes and cis DNA sequences involved in the transcriptional regulation of the locus. Our specific aims and methods can be summarized as follows: (i) Highest priority will be given to determining the mode of transcriptional expression using plasmid-complementation and mRNA mapping. The 5' and 3' ends of transcripts will be precisely located using S1-nuclease protection assays and primer extension mapping. (ii) Next we will determine which of the six genes are required for transformation and check other genes in the sequenced region for their possible involvement using insertional or site- directed mutagenesis. (iii) Having defined the mode of transcriptional expression, we will seek to identify regulatory DNA sequences in front of the genes using a reporter gene assay. Specifically, we will clone the 5' regions preceding each gene upstream of a promoterless chloramphenicol acetyltransferase (CAT) gene and assay for its activity in response to competence induction. The 5' regions containing regulatory sites will be further analyzed by deletion analysis and site-directed mutagenesis to locate and identify the sites. (iv) CAT activity will also be determined in strains mutant for the known competence regulatory genes crp and sxy1. (v) We will investigate whether the product of comA, the first gene in the locus, regulates its own expression, and that of the other genes, in strains containing a comA-lacZ operon or protein fusion in tandem with an intact comA gene. (vi) We will search for other regulatory genes affecting the expression of the locus. (vii) Finally, we will search for competence-specific sigma factors that might be involved in global switching from constitutive-gene to competence gene expression during competence induction. For this purpose, we will examine protein extracts from competent and non-competent cells using anti-sigma antibodies in Western blots. An alternate approach will employ PCR-amplification using sigma factor-specific degenerate primers for highly conserved regions.

我们实验室的长期目标是了解其机制 流感嗜血杆菌的分子和生化遗传转化 等级。 特别是，我们想了解供体 DNA 的机制 通过细胞膜和复杂的调节途径运输 控制能力诱导。 正是为了实现后一个目标 该赠款提案是有针对性的。 在使用分离许多转化突变体的过程中 mini-Tn10kan转座子诱变方法，我们发现了一组 跨越染色体 4 kb 区域的突变。 我们最近测序了 该区域和侧翼序列发现了六个串联排列的基因 这些是在能力发展过程中协调诱导的。 我们的研究 将首先关注确定这些基因是否是共转录的，如 操纵子，或单独转录。 此外，我们将确定 并研究参与该过程的调控基因和顺式DNA序列 位点的转录调控。 我们的具体目标和方法可概括如下： (i) 最高 将优先考虑确定转录模式 使用质粒互补和 mRNA 作图进行表达。 5' 和 3' 使用 S1 核酸酶保护精确定位转录本末端 测定和引物延伸图谱。 (ii) 接下来我们将确定哪一个 转化需要六个基因，并检查其他基因 使用插入或位点测序可能参与的区域 定向诱变。 (iii) 定义了转录模式 表达，我们将寻求识别前面的调控DNA序列 使用报告基因测定法检测基因。 具体来说，我们将克隆 5' 无启动子氯霉素上游每个基因之前的区域 乙酰转移酶（CAT）基因及其响应活性的测定 能力诱导。 包含调控位点的 5' 区域将是 通过缺失分析和定点突变进一步分析 找到并识别站点。 (iv) CAT 活动也将在 已知能力调节基因 crp 和 sxy1 的菌株突变体。 （五） 我们将研究 comA（第一个基因）的产物是否 位点，调节其自身的表达以及其他基因的表达， 含有 comA-lacZ 操纵子或蛋白融合蛋白的菌株 完整的 comA 基因。 (vi) 我们将寻找其他影响的调控基因 轨迹的表达。 (vii) 最后，我们将寻找 可能涉及全球能力的特定能力西格玛因素 期间从组成型基因表达转变为能力基因表达 能力诱导。 为此，我们将检查蛋白质提取物 使用抗 Sigma 抗体从感受态和非感受态细胞中分离 蛋白质印迹。 另一种方法是采用 PCR 扩增 用于高度保守区域的 sigma 因子特异性简并引物。