Role of transcription in adaptive mutagenesis

描述（由申请人提供）：由突变蛋白引起的细胞功能改变可能增强病原体克服宿主反应和抗生素治疗的潜力。本提案的主要目标是在分子水平上表征产生突变蛋白的新细胞机制，这些突变蛋白是应激诱导的遗传多样性增加的一部分。这些机制有可能影响各种过程，如肿瘤的演变和发展。枯草芽孢杆菌为研究突变体转录本的形成、细胞分化的控制和胁迫诱导的突变提供了一个容易获得的范例。本研究的具体假设是枯草芽孢杆菌中的mfd基因有助于在非生长细胞中产生突变的转录本。据推测，Mfd，一种参与转录DNA修复的因子，通过增加转录并允许在DNA损伤存在下形成突变的转录物来介导突变的产生。具体目的是研究mfd对枯草芽孢杆菌转录的影响，包括两类实验：1）我们将研究转录对稳定期诱变的影响速率：我们将通过诱导系统在饥饿/胁迫条件下操纵转录水平，并测量选择下基因中突变的转录和积累。预期高水平的转录与突变频率的增加相关。2)我们将研究稳定期的转录旁路：我们将使用最近开发的萤火虫荧光素酶（luc）-报告系统，研究在饥饿/应激条件下突变转录本的产生。在该系统中，非突变的信息不产生荧光素酶活性，而RNAP的旁路或误读产生了可以在不存在生长的情况下检测到的功能性信息。此外，这些实验将确定该基因对这些过程的影响。这项新的研究将有助于我们理解压力下细胞的遗传程序。这项工作的主要目标是在分子水平上了解当细胞处于应激条件下时产生突变的新过程。此类机制有可能影响进化、癌症发展以及病原体如何克服宿主反应和抗生素治疗等多种过程。

DESCRIPTION (provided by applicant): Altered cellular functions, originated by mutant proteins, may enhance the potential for pathogens to overcome host responses and antibiotic therapy. The main goal in this proposal is to characterize, at the molecular level, novel cellular mechanisms that generate mutant proteins as part of the stress-induced increase in genetic diversity. Such mechanisms have the potential for influencing processes as diverse as evolution and the development of neoplasms. Bacillus subitlis provides a readily accessible paradigm for the study of formation of mutant transcripts, the control of cell differentiation and stress induced mutagenesis. The specific hypothesis in this research is that the mfd gene in Bacillus subtilis contributes to the generation of mutated transcripts in non-growing cells. It is speculated that Mfd, a factor involved in repair of transcribed DNA, mediates the generation of mutations by increasing transcription and allowing the formation of mutated transcripts in the presence of DNA lesions. The specific aims are to investigate the effects of mfd on transcription in Bacillus subtilis, and involve two kinds of experiments: 1) We will investigate rate of transcription effects on stationary phase mutagenesis: We will manipulate levels of transcription, by inducible systems, in conditions of starvation/stress and measure transcription and accumulation of mutations in genes under selection. It is expected that high levels of transcription correlate with increases in frequency of mutation. 2) We will investigate transcriptional bypass in stationary phase: We will examine the generation of mutated transcripts in conditions of starvation/stress by using a firefly luciferase (luc)-reporter system recently developed. In this system, non-mutated messages generate no luciferase activity, whereas bypass or misreading by RNAP generates a functional message that could be detected in the absence of growth. Also, these experiments will determine the effect of this gene on these processes. This novel research will contribute to our understanding of genetic programs of cells under stress. The main goal of this work is to understand, at the molecular level, new processes that generate mutations when cells are in stress conditions. Such mechanisms have the potential to influence processes as diverse as evolution, the development of cancer and how pathogens overcome host responses and antibiotic therapy.