TRANSCRIPTION ASSOCIATED MUTAGENESIS IN CELLS UNDER CONDITIONS OF ARRESTED GROWT

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 这项提议研究了细胞在生长停滞条件下发生的新的突变机制。这些机制与细胞生长和分化以及人类健康相关，因为它们可能产生突变，绕过生长细胞周期检查点，导致基因组不稳定，形成肿瘤或细胞死亡，所有这些过程都与癌症和其他退行性疾病有关。这里测试的总体假设是，在生长停滞条件下，细胞中被选择的基因的转录(基因产物的产生)增加会导致促进逃避生长停滞的突变的积累(转录相关突变)。为了验证这一假设，我们使用了一个细菌细胞生长和分化模型，在该模型中，枯草芽孢杆菌细胞受到氨基酸饥饿。在这些条件下，携带氨基酸合成遗传缺陷的细胞通过获得编码合成限制性氨基酸的基因(处于选择压力下的基因)的功能突变来逃脱生长受阻。我们提出的问题是：改变被选择基因的转录水平是否会导致促进逃脱生长停滞的突变积累的变化？为了回答这个问题，我们将使用两种实验方法来测试在选择压力下调节基因转录的效果：1)我们将从基因上灭活介导转录的因子，并确定它们的失活是否会导致目标基因表达的减少和突变的积累。2)我们将构建可以诱导或抑制目标基因表达的遗传结构，并分析突变的积累是否与转录水平相关。最终，这里提出的实验提供了对影响所有生物体的突变机制的更好的理解，并且对调节细胞生长和分化以及对人类健康特别重要。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This proposal studies novel mechanisms of mutation that take place in cells under conditions of growth arrest. These mechanisms are of relevance to cell growth and differentiation and human health as they may generate mutations that circumvent growth cell cycle check points, which lead to genome instability, and the formation of tumors or cell death, all processes associated with cancer and other degenerative diseases. The overall hypothesis tested here is that increases in transcription (the making of gene products) of genes under selection in cells in conditions of growth arrest results in accumulation of mutations that promote escape from arrested growth (transcription associated mutagenesis). To examine this hypothesis, we use a bacterial model for cell growth and differentiation in which Bacillus subtilis cells are subjected to amino acid starvation. Under these conditions, cells carrying genetic deficiencies in amino acid synthesis escape arrested growth by acquiring gain of function mutations in the genes coding for synthesis of the limiting amino acid (genes under selective pressure). We ask the question: does altering transcription levels of genes under selection lead to changes in the accumulation of mutations that promote escape from growth arrest? To answer this question, we will use two experimental approaches to assay the effects of modulating transcription of genes under selective pressure: 1) We will genetically inactivate factors mediating transcription and determine whether their inactivation leads to a decrease in expression and accumulation of mutations in a target gene. 2) We will build genetic constructs where expression of target gene can be induced or repressed and assay whether the accumulation of mutations correlates with levels of transcription. Ultimately, the experiments proposed here provide a better understanding of mechanisms of mutation that affect all organisms and are of particular importance to regulation of cell growth and differentiation and to human health.