Kumar Anuj
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109-1048.
Cold Spring Harb Protoc. 2016 Jun 1;2016(6):2016/6/pdb.top080259. doi: 10.1101/pdb.top080259.
Libraries of transposon-insertion alleles constitute powerful and versatile tools for large-scale analysis of yeast gene function. Transposon-insertion libraries are constructed most simply through mutagenesis of a plasmid-based genomic DNA library; modification of the mutagenizing transposon by incorporation of yeast selectable markers, recombination sites, and an epitope tag enables the application of insertion alleles for phenotypic screening and protein localization. In particular, yeast genomic DNA libraries have been mutagenized with modified bacterial transposons carrying the URA3 marker, lox recombination sites, and sequence encoding multiple copies of the hemagglutinin (HA) epitope. Mutagenesis with these transposons has yielded a large resource of insertion alleles affecting nearly 4000 yeast genes in total. Through well-established protocols, these insertion libraries can be introduced into the desired strain backgrounds and the resulting insertional mutants can be screened or systematically analyzed. Relative to alternative methods of UV irradiation or chemical mutagenesis, transposon-insertion alleles can be easily identified by PCR-based approaches or high-throughput sequencing. Transposon-insertion libraries also provide a cost-effective alternative to targeted deletion approaches, although, in contrast to start-codon to stop-codon deletions, insertion alleles might not represent true null-mutants. For protein-localization studies, transposon-insertion alleles can provide encoded epitope tags in-frame with internal codons; in many cases, these transposon-encoded epitope tags can provide a more accurate localization for proteins in which terminal sequences are crucial for intracellular targeting. Thus, overall, transposon-insertion libraries can be used quickly and economically and have a particular utility in screening for desired phenotypes and localization patterns in nonstandard genetic backgrounds.
转座子插入等位基因文库是用于大规模分析酵母基因功能的强大且通用的工具。构建转座子插入文库最简单的方法是对基于质粒的基因组DNA文库进行诱变；通过掺入酵母选择标记、重组位点和表位标签来修饰诱变转座子，可将插入等位基因应用于表型筛选和蛋白质定位。特别是，酵母基因组DNA文库已用携带URA3标记、lox重组位点和编码多个血凝素（HA）表位拷贝的序列的修饰细菌转座子进行了诱变。用这些转座子进行诱变已产生了大量的插入等位基因资源，总共影响了近4000个酵母基因。通过成熟的方案，这些插入文库可被导入所需的菌株背景中，并且可以对产生的插入突变体进行筛选或系统分析。相对于紫外线照射或化学诱变的替代方法，转座子插入等位基因可以通过基于PCR的方法或高通量测序轻松鉴定。转座子插入文库也为靶向缺失方法提供了一种经济高效的替代方案，尽管与从起始密码子到终止密码子的缺失相比，插入等位基因可能不代表真正的无效突变体。对于蛋白质定位研究，转座子插入等位基因可以提供与内部密码子读框内的编码表位标签；在许多情况下，这些转座子编码的表位标签可以为那些末端序列对细胞内靶向至关重要的蛋白质提供更准确的定位。因此，总体而言，转座子插入文库可以快速且经济地使用，并且在筛选非标准遗传背景中的所需表型和定位模式方面具有特殊用途。
