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

细胞在非分裂条件下经历诱变事件，从而为进化增加了不依赖生长的过程。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。有益突变体可以随机方式积累的一种方式是通过转录相关诱变。在这个项目中，研究人员试图测试转录去抑制和转录诱导的DNA二级结构的形成有助于适应性突变的产生的假设。 该项目的目标1是确定转录水平的降低是否与选择中基因突变频率的降低相关。实验系统是表达leuC 427突变等位基因的亮氨酸营养缺陷型枯草芽孢杆菌菌株。该突变基因由效应子（S-腺苷甲硫氨酸，SAM）响应性核糖开关控制机制控制，使得SAM水平的增加导致leuC 417基因的逐渐更大的抑制。预期是转录的降低将成比例地降低突变体积累并逃避亮氨酸饥饿。 目的2试图证实相关的假设，即转录诱导的二级DNA茎环结构特别容易发生突变，从而提高选择下的突变体积累。 为了验证这一假设，研究人员将创建诱导型thiF和argF等位基因，其差异仅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。预期与具有稳定二级结构（高突变性）的菌株相比，携带具有不稳定茎环结构（低突变性）的等位基因的菌株中Thi+和Arg+回复突变体的数量显著减少。总之，这些具体的目标将提供潜在的新见解的分子机制的压力诱导的适应性诱变，从而提供了一个更好的理解的分子过程，一直在进化的核心。更广泛的影响这项工作将促进和教育公众在进化的过程中。此外，这项研究将推进并为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不断增长。这些目标将通过实施向当地高中提供的讲座计划来实现。这项工作将与当地高中学区一起实施和协调。最后，这项研究计划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.