DEGRADATION REACTION IN SPORE GERMINATION

孢子萌发中的降解反应

• 批准号: 3269748
• 负责人: Peter Setlow
• 金额: $ 31.83万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-09-01 至 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3269748
• 关键词: Bacillus subtilis; Clostridium; DNA directed RNA polymerase; Escherichia coli; bacterial genetics; bacterial proteins; fusion gene; gene expression; genetic promoter element; genetic transcription; laboratory mouse; messenger RNA; microorganism metabolism; molecular cloning; mutant; nucleic acid sequence; operon; peptidases; protein biosynthesis; protein sequence; protein structure function; radiotracer; spores; sporogenesis

The long term goals of this project are to achieve detailed understanding of the in vivo function of small, acid-soluble spore proteins (SASP) of Bacillus subtilis, and the regulation of the genes (ssp genes) which code for SASP as well as other co-regulated genes. Specific aims are 1) with genes coding for major SASP determine the regions required for the initiation of their transcription in sporulation, by constructing upstream deletion mutations and analyzing their transcription in vivo and in vitro with oG RNA polymerase; 2) Test the specificity of oG RNA polymerase by constructing mutations in the -10 and -35 regions of ssp genes, and analyzing the transcription of these mutant genes in vivo and in vitro; 3) Determine those parts of major B subtilis SASP, ssp gene mRNAs or ssp genes which are involved in the feedback regulation of SASP synthesis, by analyzing the feedback regulation of ssp genes with various mutations, as well as different ssp-lacZ fusions. 4) Investigate the mechanism whereby oG synthesis is regulated by using an anti oG antibody to monitor oG synthesis during sporulation and by measuring the oG mRNA level and transcription start site(s) during sporulation of various B.subtilis strains; 5) Determine if the spore protease gene is part of an operon, and study the regulation of this gene (or operon) by measuring its in vivo transcription start site and expression in various asporogenous mutants, and its transcription in vitro with oG RNA polymerase; 6) Determine the supercoiling of various plasmids in cells and spores of various B.subtilis strains as well as the kinetics of change in these values during germination and sporulation. 7) In E.coli measure the supercoiling of plasmids carrying spp genes which can be expressed from an inducible promoter; 8) Determine the effect(s) of purified major and minor SASP on the supercoiling of DNA in vitro; 9) Determine the effects of various degrees of plasmid supercoiling on the expression of various forespore specific genes including ssp genes; 10) Continue studies on the killing of E.coli by induction of synthesis of high levels of oB type SASP; 11) Purify and characterize the minor SASP from B.subtilis spores, in particular those which are not antigenically related to major SASP, and attempt to correlate these proteins with genes and functions; 12) Test the possibility that the extreme conservation of the amino acid sequences of a/b type SASP is due to the interference of even a minor mutant a/b type SASP with the changes in DNA structure needed to give spore UV resistance; 13) Clone and sequence several a/b type ssp genes from Clostridial species.

该项目的长期目标是实现详细的理解 小酸性孢子蛋白（SASP）的体内功能 枯草芽孢杆菌和基因（SSP基因）的调节 用于SASP以及其他共同调节的基因。 具体目的是1） 编码主要SASP的基因确定 通过构建启动其在孢子中的转录 上游删除突变并在体内分析其转录 体外与OG RNA聚合酶； 2）测试OG RNA的特异性 通过在SSP的-10和-35区域构建突变来聚合酶 基因，并在体内分析这些突变基因的转录 体外; 3）确定主要B枯草SASP，SSP基因的那些部分 参与SASP反馈调节的mRNA或SSP基因 合成，通过分析具有不同的SSP基因的反馈调节 突变以及不同的SSP-LACZ融合。 4）调查 通过使用抗OG抗体调节OG合成的机制 监测孢子形成过程中的OG合成和测量OG mRNA 各种孢子形成期间的水平和转录起始位点 B.subtilis菌株； 5）确定孢子蛋白酶基因是否是 操纵子，并通过测量该基因（或操纵子）研究该基因的调节 其体内转录启动位点和各种表达 亚波源突变体及其在体外用OG RNA转录 聚合酶； 6）确定细胞中各种质粒的超串联 以及各种b.subtilis菌株的孢子以及动力学 在发芽和孢子形成过程中这些值的变化。 7）在大肠杆菌中 测量携带SPP基因的质粒的超串联 由诱导启动子表示； 8）确定效果 在体外DNA的超涂层上纯化的主要和次要SASP； 9） 确定各种程度的质粒超螺旋对 包括SSP基因在内的各种特定基因的表达； 10） 继续研究通过诱导的合成来杀死大肠杆菌 高水平的OB类型SASP； 11）纯化和表征次要的SASP 来自B.subtilis孢子，尤其是那些不是抗原的孢子 与主要SASP相关，并试图将这些蛋白质与 基因和功能； 12）测试极端的可能性 A/B类型SASP的氨基酸序列的保护是由于 即使是次要突变体A/B类型SASP的干扰DNA的变化 赋予孢子紫外线抗性所需的结构； 13）克隆和序列 来自梭菌种类的几个A/B型SSP基因。