Connolly B, White C I, Haber J E
Department of Biology, Brandeis University, Waltham, Massachusetts 02254.
Mol Cell Biol. 1988 Jun;8(6):2342-9. doi: 10.1128/mcb.8.6.2342-2349.1988.
The kinetics of mating type switching in Saccharomyces cerevisiae can be followed at the DNA level by using a galactose-inducible HO (GAL-HO) gene to initiate the event in synchronously growing cells. From the time that HO endonuclease cleaves MAT a until the detection of MAT alpha DNA took 60 min. When unbudded G1-phase cells were induced, switched to the opposite mating type in "pairs." In the presence of the DNA synthesis inhibitor hydroxyurea, HO-induced cleavage occurred but cells failed to complete switching. In these blocked cells, the HO-cut ends of MATa remained stable for at least 3 h. Upon removal of hydroxyurea, the cells completed the switch in approximately 1 h. The same kinetics of MAT switching were also seen in asynchronous cultures and when synchronously growing cells were induced at different times of the cell cycle. Thus, the only restriction that confined normal homothallic switching to the G1 phase of the cell cycle was the expression of HO endonuclease. Further evidence that galactose-induced cells can switch in the G2 phase of the cell cycle was the observation that these cells did not always switch in pairs. This suggests that two chromatids, both cleaved with HO endonuclease, can interact independently with the donors HML alpha and HMRa.
通过使用半乳糖诱导型HO（GAL-HO）基因在同步生长的细胞中引发该事件，酿酒酵母中交配型转换的动力学可以在DNA水平上进行追踪。从HO内切酶切割MAT a到检测到MATα DNA需要60分钟。当未出芽的G1期细胞被诱导时，会“成对”转换为相反的交配型。在DNA合成抑制剂羟基脲存在的情况下，HO诱导的切割发生，但细胞未能完成转换。在这些受阻的细胞中，MATa的HO切割末端至少保持稳定3小时。去除羟基脲后，细胞在大约1小时内完成转换。在异步培养物中以及在细胞周期的不同时间诱导同步生长的细胞时，也观察到了相同的MAT转换动力学。因此，将正常同宗配合转换限制在细胞周期G1期的唯一限制因素是HO内切酶的表达。半乳糖诱导的细胞可以在细胞周期的G2期进行转换的进一步证据是观察到这些细胞并不总是成对转换。这表明两条都被HO内切酶切割的染色单体可以独立地与供体HMLα和HMRa相互作用。
