Frameshift Mutations in Yeast

[405953]金克斯-罗伯逊突变一般可分为自发突变和诱变剂诱导突变。虽然自发突变被认为是随机发生的，而不考虑功能，但最近的研究表明，适应性的、选择性的有利突变也可以发生。这个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。酵母将被用作研究真核生物移码突变产生的模型系统。移码突变将通过选择一个已定义的移码等位基因lys2deltaBgl等位基因的反转来识别。初步分析表明，95%的逆转事件对应于基因内的第二位点补偿性突变，这些突变恢复了基因的正确阅读框。lys2deltaBgl等位基因将用于三组相互关联的实验。首先，随机和自适应基因内移码反转事件的频谱将通过DNA序列分析确定。从这一分析中得到的分子数据可能提供有关酵母适应性突变机制的信息。其次，酵母错配修复系统在消除复制错误中的作用将通过比较野生型与错配修复缺陷酵母菌株移码事件的频率和频谱来检验。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。在第一组实验中，将使用先前适应性突变研究中使用的lys2deltaBgl菌株。对于后两组实验，将lys2deltaBgl等位基因的一部分置于去除对编码基因产物的功能限制的环境中。唯一的限制是发生了一个补偿性移码突变，允许翻译通过整个lys2区域，导致产生全长h，可测定的基因产物。这种方法应该允许只有移码突变的特定DNA区域饱和，从而模拟如果有特定移码的正向突变系统，将会看到的突变谱。基因突变的过程几乎影响着生物科学的每一个领域。突变是进化力量发挥作用的原料，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重要工具，因为它提供了一种剖析复杂生物过程的方法。在这个项目中，PI将继续她对突变形成过程的研究。＊＊＊

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. ***