Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10065, USA.
Genetics. 2011 Oct;189(2):423-39. doi: 10.1534/genetics.111.131326. Epub 2011 Aug 11.
The budding yeast Saccharomyces cerevisiae has proven to be a rich source of information about the mechanisms and regulation of homologous recombination during meiosis. A common technique for studying this process involves microdissecting the four products (ascospores) of a single meiosis and analyzing the configuration of genetic markers in the spores that are viable. Although this type of analysis is powerful, it can be laborious and time-consuming to characterize the large numbers of meioses needed to generate statistically robust data sets. Moreover, the reliance on viable (euploid) spores has the potential to introduce selection bias, especially when analyzing mutants with elevated frequencies of meiotic chromosome missegregation. To overcome these limitations, we developed a versatile, portable set of reporter constructs that drive fluorescent protein expression specifically in only those spores that inherit the reporter. These spore-autonomous fluorescence constructs allow direct visualization of inheritance patterns in intact tetrads, eliminating the need for microdissection and permitting meiotic segregation patterns to be ascertained even in aneuploid spores. As proof of principle, we demonstrate how different arrangements of reporters can be used to quantify crossover frequency, crossover interference, gene conversion, crossover/noncrossover ratios, and chromosome missegregation.
budding yeast Saccharomyces cerevisiae 已被证明是同源重组在减数分裂过程中的机制和调控的丰富信息来源。研究这个过程的一种常见技术涉及从单个减数分裂的四个产物（子囊孢子）中进行显微解剖，并分析可育孢子中遗传标记的构型。尽管这种类型的分析很强大，但要生成具有统计学稳健性数据集所需的大量减数分裂，其特征描述既费力又耗时。此外，依赖于可育（整倍体）孢子有可能引入选择偏差，尤其是在分析具有较高减数分裂染色体错误分离频率的突变体时。为了克服这些限制，我们开发了一套通用的、便携式报告基因构建体，这些构建体专门在继承报告基因的那些孢子中驱动荧光蛋白表达。这些孢子自主荧光构建体允许直接观察完整四分体中的遗传模式，无需进行显微解剖，并允许即使在非整倍体孢子中也能确定减数分裂分离模式。作为原理验证，我们展示了如何使用不同的报告基因排列来定量交叉频率、交叉干扰、基因转换、交叉/非交叉比以及染色体错误分离。
