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

细胞在不分裂的条件下经历突变事件，从而增加了进化中不依赖生长的过程。这一预测引起了激烈的争论，因为这意味着有机体指导着有益突变的积累。有益的突变体可能以随机方式积累的一种方式是通过转录相关突变。在这个项目中，研究人员试图验证这样一个假设，即转录抑制和转录诱导的DNA二级结构的形成有助于产生适应性突变。该项目的目标1是确定转录水平的降低是否与正在选择的基因中突变频率的降低相关。实验系统是一株表达LeuC427突变等位基因的亮氨酸营养缺陷型枯草芽孢杆菌。该突变基因受S-腺苷-蛋氨酸(SAM)反应的核糖开关调控机制的控制，使SAM水平的增加导致LeuC417基因逐渐受到更大的抑制。人们的预期是，转录水平的下降将按比例减少突变的积累，并逃脱亮氨酸饥饿。目的2旨在证实转录诱导的二级DNA茎环结构特别容易发生突变的相关假设，从而增强选择下的突变积累。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，这些等位基因只有在形成稳定的茎环结构的可能性方面不同，以测量它们在诱导和非诱导条件下突变的相对积累。预期携带具有不稳定茎环结构的等位基因(低突变性)的菌株的Thi+和Arg+逆转株的数量比那些具有稳定的二级结构(高突变性)的菌株显著减少。总之，这些特定的目标将为应激诱导适应性突变的分子机制提供潜在的新见解，从而更好地理解处于进化核心的分子过程。此外，这项研究将推进并为处于历史上科学倡议和教育资金不足的州的学生提供科学机会。赞助国的少数民族人口在不断增加。这些目标将通过实施向当地高中提供的讲座计划来实现。这一努力将得到实施，并与当地高中学区进行协调。最后，这个研究项目还将为大学生提供急需的科学实践经验。

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.