Eibel H, Philippsen P
Mol Gen Genet. 1983;191(1):66-73. doi: 10.1007/BF00330891.
We isolated the LYS2 gene of S. cerevisiae on an autonomously replicating plasmid in a four-step procedure. First, we identified a recombinant plasmid which expressed a lys2 complementing activity upon yeast transformation of a lys2 mutant. Second, we determined the boundaries of the corresponding transcribed sequence in this plasmid by S1 nuclease mapping of the mRNA. Third, we inactivated the functional chromosomal copy coding for the lys2 complementing activity by directed integration of a plasmid that carried an internal fragment of the transcribed sequence. Fourth, we showed by a complementation test with an authentic lys2 mutant that the integration had inactivated the chromosomal LYS2 gene. This proved that the plasmid isolated in the first step indeed carried the LYS2 gene and not a suppressor of the lys2 mutation. The gene is unusually long (4.0 kb). It was used as hybridization probe in comparing LYS2 regions in various yeast strains and in a first construction of LYS2 based yeast vectors. Such vectors could be very useful because of the easy selection of lys2 mutants from any S. cerevisiae strain.
我们通过一个四步程序，在一个自主复制质粒上分离出了酿酒酵母的LYS2基因。首先，我们鉴定出一个重组质粒，该质粒在对lys2突变体进行酵母转化时表现出lys2互补活性。其次，我们通过对mRNA进行S1核酸酶作图，确定了该质粒中相应转录序列的边界。第三，我们通过定向整合一个携带转录序列内部片段的质粒，使编码lys2互补活性的功能性染色体拷贝失活。第四，我们通过与一个真正的lys2突变体进行互补试验表明，整合已使染色体LYS2基因失活。这证明第一步中分离出的质粒确实携带LYS2基因，而不是lys2突变的抑制子。该基因异常长（4.0 kb）。它被用作杂交探针，用于比较各种酵母菌株中的LYS2区域，并用于首次构建基于LYS2的酵母载体。由于可以很容易地从任何酿酒酵母菌株中筛选出lys2突变体，这样的载体可能非常有用。
