Stearns T, Ma H, Botstein D
Methods Enzymol. 1990;185:280-97. doi: 10.1016/0076-6879(90)85025-j.
The vectors and techniques described here enable one to manipulate the yeast genome to meet specific needs. Genes can be cloned, and the clone used to delete the wild-type gene from the chromosome, or replace it with mutant versions. Mutants derived by classical methods, such as mutagenesis of whole cells, or by reversion of a phenotype, can be cloned and analyzed in vitro. Yeast genes and foreign genes can either be inserted into autonomously replicating plasmid vectors that are reasonably stable or integrated into a yeast chromosome where they are maintained at one copy per genome. The combination of these techniques with the characterized promoter systems available in yeast make it possible to express almost any gene in yeast. Once this is achieved, the entire repertoire of yeast genetics is available to probe the function of the gene, or to engineer the expression in useful ways.
本文所述的载体和技术使人们能够操纵酵母基因组以满足特定需求。基因可以被克隆，克隆的基因可用于从染色体上删除野生型基因，或用突变体版本取代它。通过经典方法获得的突变体，如全细胞诱变或表型回复突变产生的突变体，可以在体外进行克隆和分析。酵母基因和外源基因既可以插入相当稳定的自主复制质粒载体中，也可以整合到酵母染色体中，在每个基因组中以单拷贝形式维持。这些技术与酵母中可用的特征化启动子系统相结合，使得在酵母中表达几乎任何基因成为可能。一旦实现这一点，就可以利用酵母遗传学的全部方法来探究该基因的功能，或以有用的方式设计其表达。
