Koob M, Szybalski W
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Science. 1990 Oct 12;250(4978):271-3. doi: 10.1126/science.2218529.
The 15-megabase pair Saccharomyces cerevisiae and the 4.7-megabase pair Escherichia coli genomes were completely cleaved at a single predetermined site by means of the Achilles' heel cleavage (AC) procedure. The symmetric lac operator (lacOs) was introduced into the circular Escherichia coli genome and into one of the 16 yeast chromosomes. Intact chromosomes from the resulting strains were prepared in agarose microbeads and methylated with Hha I (5'-GCGC) methyltransferase (M.Hha I) in the presence of lac repressor (LacI). All Hae II sites (5'-[sequence: see text]) with the exception of the one in lacOs, which was protected by LacI, were modified and thus no longer recognized by Hae II. After inactivation of M.Hha I and LacI, Hae II was used to completely cleave the chromosomes specifically at the inserted lacOs. These experiments demonstrate the feasibility of using the AC approach to efficiently extend the specificity of naturally occurring restriction enzymes and create new tools for the mapping and precise molecular dissection of multimegabase genomes.
通过“阿喀琉斯之踵切割”（AC）程序，15兆碱基对的酿酒酵母基因组和4.7兆碱基对的大肠杆菌基因组在单个预定位点被完全切割。将对称的乳糖操纵子（lacOs）导入环状大肠杆菌基因组以及16条酵母染色体之一中。从所得菌株中制备完整的染色体，并在琼脂糖微珠中用Hha I（5'-GCGC）甲基转移酶（M.Hha I）在乳糖阻遏物（LacI）存在的情况下进行甲基化。除了被LacI保护的lacOs中的那个位点外，所有Hae II位点（5'-[序列：见正文]）都被修饰，因此不再被Hae II识别。在M.Hha I和LacI失活后，使用Hae II特异性地在插入的lacOs处完全切割染色体。这些实验证明了使用AC方法有效扩展天然存在的限制酶的特异性并为多兆碱基基因组的图谱绘制和精确分子剖析创建新工具的可行性。
