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.
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