REGULATION OF A COMPETENCE GENE LOCUS IN H INFLUENZAE

H 型流感病毒活性基因位点的调控

• 批准号: 2185744
• 负责人: HAMILTON O SMITH
• 金额: $ 14.06万
• 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/2185744
• 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 分子和生化的流感h。流感菌的遗传转化 等级。 特别是，我们想了解供体DNA的机械 通过细胞膜和复杂的调节途径的运输 控制能力诱导。 正是针对后一个目标 该赠款提案是指导的。 在隔离许多转换突变体的过程中 迷你TN10KAN转座子诱变方法，我们发现了一群 跨染色体的4-kb区域的突变。 我们最近测序了 该区域和侧翼序列，发现了六个串联的基因 在能力发展过程中是协调诱导的。 我们的研究 首先将重点放在确定这些基因是否被共转印为 操纵子，或单独转录。 此外，我们将确定 并研究了与 基因座的转录调节。 我们的具体目的和方法可以总结如下：（i）最高 确定转录方式的优先级 使用质粒融合和mRNA映射的表达。 5'和3' 成绩单的末端将使用S1-核酸酶保护精确地位于 测定和底漆扩展映射。 （ii）接下来我们将确定哪个 六个基因是转化并检查其他基因的基因 测序的区域可能使用插入或位点 - 可能参与 定向诱变。 （iii）定义了转录模式 表达，我们将寻求在面前确定调节性DNA序列 使用报告基因测定的基因。 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 乙酰基转移酶（CAT）基因和测定其活性响应于 能力诱导。 包含监管地点的5'区域将是 通过缺失分析和定向诱变进一步分析 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。 （v） 我们将调查昏迷的产物是第一个基因 基因座，调节其自身的表达以及其他基因的表达 含有昏迷操纵子或蛋白质融合的菌株与 完整的昏迷基因。 （vi）我们将搜索其他影响的调节基因 基因座的表达。 （vii）最后，我们将搜索 全球可能涉及的特定能力特定的Sigma因素 从本构基因转换为能力基因表达 能力诱导。 为此，我们将检查蛋白质提取物 来自使用抗sigma抗体的能力和非竞争细胞 蛋白质印迹。 另一种方法将使用PCR扩大使用 高度保守区域的Sigma因子特异性退化引物。