TRANSCRIPTION ASSOCIATED MUTAGENESIS IN CELLS UNDER CONDITIONS OF ARRESTED GROWT

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 该提案研究了在生长停滞条件下细胞中发生突变的新机制。这些机制与细胞生长和分化以及人类健康相关，因为它们可能产生绕过生长细胞周期检查点的突变，这导致基因组不稳定，以及肿瘤或细胞死亡的形成，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，细胞中选择的基因的转录（基因产物的产生）增加导致促进从生长停滞中逃逸的突变的积累（转录相关诱变）。为了检验这一假设，我们使用了一个细菌模型的细胞生长和分化，其中枯草芽孢杆菌细胞进行氨基酸饥饿。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限制性氨基酸合成的基因（在选择压力下的基因）中的功能突变的获得来逃避生长停滞。我们提出这样一个问题：改变基因的转录水平是否会导致突变的积累发生变化，从而促进从生长停滞中逃脱？为了回答这个问题，我们将使用两种实验方法来测定在选择压力下调节基因转录的效果：1）我们将遗传地检测介导转录的因子，并确定它们的失活是否导致靶基因中突变的表达和积累的减少。2)我们将构建基因构建体，其中靶基因的表达可以被诱导或抑制，并分析突变的积累是否与转录水平相关。最后，这里提出的实验提供了一个更好的理解突变的机制，影响所有的生物体，是特别重要的细胞生长和分化的调节和人类健康。

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.