Raveh D, Hughes S H, Shafer B K, Strathern J N
Biology Department, Ben Gurion University, Beersheba, Israel.
Mol Gen Genet. 1989 Dec;220(1):33-42.
A galactose-inducible HO gene was used to induce mating type switching in heterothallic Saccharomyces cerevisiae cells arrested in G1, in rad52 mutants defective in DNA damage repair, and in cells lacking the donor cassettes. The HO-cleaved MAT intermediate is stable over significant lengths of time, i.e. HO cleavage is not coupled to the subsequent gene conversion event. The in vivo cleavage site was mapped to single base resolution by primer extension experiments on total genomic DNA. Cells arrested in G1 with alpha-factor switched mating type thus demonstrating that switches can occur in the absence of replication of the genome. rad52 mutants did not produce MAT DNA of the opposite mating type indicating that the block is prior to the gene duplication stage of the switch. In strains in which the HM donor cassettes are deleted the cut MAT DNA was degraded after induction of the HO gene.
利用半乳糖诱导型HO基因，在停滞于G1期的异宗配合酿酒酵母细胞、DNA损伤修复缺陷的rad52突变体以及缺乏供体盒的细胞中诱导交配型转换。HO切割产生的MAT中间体在相当长的时间内是稳定的，即HO切割与随后的基因转换事件不相关联。通过对总基因组DNA进行引物延伸实验，将体内切割位点定位到单碱基分辨率。用α因子使停滞于G1期的细胞转换交配型，从而证明在基因组不复制的情况下也能发生转换。rad52突变体未产生相反交配型的MAT DNA，这表明阻断发生在转换的基因复制阶段之前。在缺失HM供体盒的菌株中，诱导HO基因后，切割后的MAT DNA会被降解。
