Frameshift Mutations in Yeast

9405953 Jinks-Robertson突变通常可分为自发突变或诱变剂诱导突变。 虽然自发突变被认为是以随机方式发生的，而不考虑功能，但最近的工作表明，适应性的，选择性有利的突变也可能发生。 该项目的重点是一种特殊类型的自发突变的起源和性质：移码突变。 酵母酿酒酵母将被用作研究真核生物中移码突变产生的模型系统。 移码突变将通过选择确定的移码等位基因（lys 2deltaBgl等位基因）的回复来鉴定。 初步分析表明，95%的回复事件对应于基因内的第二位点补偿突变，其恢复基因的正确阅读框。 lys 2deltaBgl等位基因将用于三组相互关联的实验中。 首先，将通过DNA序列分析确定随机与适应性基因内移码回复事件的谱。 从这种分析中获得的分子数据可能会产生有关酵母适应性突变机制的信息。 第二，酵母错配修复系统在复制错误去除中的作用将通过比较野生型与错配修复缺陷型酵母菌株中移码事件的频率和谱来检查。 第三，DNA聚合酶在产生移码突变中的作用将使用聚合酶受损的酵母突变体进行评估。 对于第一组实验，将使用先前适应性突变研究中使用的lys 2deltaBgl菌株。 对于后两组实验，将lys 2deltaBgl等位基因的一部分置于其中去除对编码基因产物的功能性限制的背景中。 唯一的限制是发生补偿性移码突变，允许翻译通过整个lys 2区域，导致产生可测定的完整基因产物。 这种方法应该允许仅具有移码突变的DNA的限定区域饱和，从而模拟如果移码特异性正向突变系统可用时将看到的突变谱。 基因突变的过程几乎影响着生物科学的每一个领域。 突变是进化力量作用的原材料，突变变体的存在使遗传学作为一门学科的发展成为可能。 突变分析是现代生物学家的一个重要工具，因为它提供了一种分析复杂生物过程的方法。 在本项目中，PI将继续研究突变形成过程。 ***

9405953 Jinks-Robertson Mutations can be generally classified as either spontaneous or mutagen-induced. While spontaneous mutations are considered to occur in a random manner without regard to function, recent work has indicated that adaptive, selectively advantageous mutations can occur as well. The focus of this project is on the origin and nature of a particular type of spontaneous mutation: the frameshift mutation. The yeast Saccharomyces cerevisiae will be used as a model system to study the generation of frameshift mutations in eukaryotes. Frameshift mutations will be identified by selecting for the reversion of a defined frameshift allele, the lys2deltaBgl allele. Preliminary analyses have demonstrated that 95% of reversion events correspond to intragenic, second site compensatory mutations that restore the correct reading frame of the gene. The lys2deltaBgl allele will be used in three interrelated sets of experiments. First, the spectrum of random versus adaptive intragenic frameshift reversion events will be determined by DNA sequence analysis. Molecular data derived from this analysis may yield information concerning the mechanism of adaptive mutation in yeast. Second, the role of the yeast mismatch repair system(s) in the removal of replication errors will be examined by comparing the frequency and spectrum of frameshift events in wild type versus mismatch repair defective yeast strains. Third, the role of DNA polymerase in generating frameshift mutations will be assessed using polymerase-impaired yeast mutants. For the first set of experiments the lys2deltaBgl strain used in previous adaptive mutation studies will be used. For the latter two sets of experiments, a portion of the lys2deltaBgl allele will be placed in a context where functionality constraints on the encoded gene product are removed. The only constraint is that a compensatory frameshift mutation occur that allows translation through the entire lys2 region, resulting in the production of a full-lengt h, assayable gene product. This approach should allow saturation of a defined region of DNA with frameshift mutations only, thus mimicking the spectrum of mutations that would be seen if a frameshift-specific forward mutation system were available. %%% The process of gene mutation influences virtually every area of biological science. Mutation is the raw material upon which evolutionary forces act and the existence of mutant variants made possible the development of genetics as a discipline. Mutational analysis is an essential tool in the repertoire of the modern biologist since it affords a way to dissect complex biological processes. In this project the PI will continue her investigation into the processes involved in the formation of mutations. ***