Transcription-Associated Mutagenesis in Bacillus Subtilis Cells in Conditions of Stress

应激条件下枯草芽孢杆菌细胞中转录相关的突变

• 批准号: 0843606
• 负责人: Eduardo Robleto
• 金额: $ 37.5万
• 依托单位: University of Nevada Las Vegas
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-15 至 2013-02-28
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0843606&HistoricalAwards=false
• 关键词: Transcription; Associated; Mutagenesis; Bacillus; Subtilis

Cells undergo mutagenic events under non-dividing conditions, thereby adding growth-independent processes to evolution. This prediction is highly debated because of the implication that the organism directs the accumulation of beneficial mutations. One way beneficial mutants may accumulate in a stochastic manner is by transcription-associated mutagenesis. In this project, the investigators seek to test the hypothesis that transcription de-repression and formation of transcription-induced DNA secondary structures contribute to the generation of adaptive mutations. Aim 1 of the project is to establish whether a decrease in the level of transcription correlates with a reduction in mutation frequency in a gene under selection. The experimental system is a leucine auxotrophic Bacillus subtilis strain that expresses a leuC427 mutant allele. This mutant gene is governed by an effector (S-adenosyl-methionine, SAM)-responsive riboswitch control mechanism such that increasing levels of SAM lead to incrementally greater repression of the leuC417 gene. The expectation is that a decrease in transcription will proportionally lower mutant accumulation and escape from leucine starvation. Aim 2 seeks to confirm the related hypothesis that transcription-induced secondary DNA stem-loop structures are particularly prone to mutagenesis, thereby enhancing the mutant accumulation under selection. To test this hypothesis, the investigators will create inducible thiF and argF alleles that differ only in the potential to form stable stem-loop structures to measure their relative accumulation of mutants under inducing and non-inducing conditions. The expectation is that the number of Thi+ and Arg+ revertants decreases significantly in the strains carrying the alleles with destabilized stem-loop structures (low mutability) compared to those with stable secondary structures (high mutability). Together, these specific aims will provide potential novel insights into the molecular mechanism of stress-induced adaptive mutagenesis, and consequently provide a better understanding of the molecular processes that have been at the core of evolution.Broader ImpactsThis work will promote and educate the public in the process of evolution. In addition,this study will advance and provide opportunities in science for students in a state that historically has been under-funded for science initiatives and education. The sponsoring state has a growing population of minorities. These objectives will be accomplished by implementing lecture programs offered to local high schools. This effort will be implemented and coordinated with the local high school district. Lastly, this research program will also provide much needed science hands-on experience to college students.

细胞在非分裂条件下发生诱变事件，从而为进化增加了生长独立的过程。这一预测备受争议，因为它暗示生物体指导有益突变的积累。有益突变体以随机方式积累的一种方式是通过转录相关的诱变。在这个项目中，研究人员试图验证转录去抑制和转录诱导的DNA二级结构的形成有助于产生适应性突变的假设。该项目的目的1是确定转录水平的降低是否与选择下基因突变频率的降低相关。实验系统是一种表达leuC427突变等位基因的亮氨酸营养不良枯草芽孢杆菌菌株。该突变基因受一种效应物（s -腺苷-蛋氨酸，SAM）反应性核开关控制机制控制，SAM水平的增加导致leuC417基因的逐渐抑制。预期转录的减少将成比例地降低突变体的积累和逃避亮氨酸饥饿。Aim 2试图证实相关假设，即转录诱导的次级DNA茎环结构特别容易发生突变，从而增强了选择下的突变积累。为了验证这一假设，研究人员将创建可诱导的thiF和argF等位基因，它们的不同之处在于形成稳定茎环结构的潜力，以测量它们在诱导和非诱导条件下突变体的相对积累。结果表明，与具有稳定二级结构（高易变性）的菌株相比，携带具有不稳定茎环结构（低易变性）的等位基因的菌株中Thi+和Arg+回复体的数量显著减少。总之，这些特定的目标将为应激诱导适应性诱变的分子机制提供潜在的新见解，从而更好地理解进化核心的分子过程。更广泛的影响这项工作将在进化的过程中促进和教育公众。此外，这项研究将推动并为该州的学生提供科学机会，该州历史上一直缺乏科学计划和教育的资金。赞助国的少数民族人口不断增长。这些目标将通过在当地高中实施讲座项目来实现。这项工作将与当地高中学区一起实施和协调。最后，这个研究项目也将为大学生提供急需的科学实践经验。