Van Houten J V, Newlon C S
Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark 07103.
Mol Cell Biol. 1990 Aug;10(8):3917-25. doi: 10.1128/mcb.10.8.3917-3925.1990.
Yeast autonomously replicating sequence (ARS) elements contain an 11-base-pair core consensus sequence (5'-[A/T]TTTAT[A/G]TTT[A/T]-3') that is required for function. The contribution of each position within this sequence to ARS activity was tested by creating all possible single-base mutations within the core consensus sequence of ARS307 (formerly called the C2G1 ARS) and testing their effects on high-frequency transformation and on plasmid stability. Of the 33 mutations, 22 abolished ARS function as measured by high-frequency transformation, 7 caused more than twofold reductions in plasmid stability, and 4 had no effect on plasmid stability. Mutations that reduced or abolished ARS activity occurred at each position in the consensus sequence, demonstrating that each position of this sequence contributes to ARS function. Of the four mutations that had no effect on ARS activity, three created alternative perfect matches to the core consensus sequence, demonstrating that the alternate bases allowed by the consensus sequence are, indeed, interchangeable. In addition, a change from T to C at position 6 did not perturb wild-type efficiency. To test whether the essential region extends beyond the 11-base-pair consensus sequence, the effects on plasmid stability of point mutations one base 3' to the T-rich strand of the core consensus sequence (position 12) and deletion mutations that altered bases 5' to the T-rich strand of the core consensus sequence were examined. An A at position 12 or the removal of three T residues 5' to the core consensus sequence severely diminished ARS efficiency, showing that the region required for full ARS efficiency extends beyond the core consensus sequence in both directions.
酵母自主复制序列（ARS）元件包含一个11个碱基对的核心共有序列（5'-[A/T]TTTAT[A/G]TTT[A/T]-3'），该序列是功能所必需的。通过在ARS307（以前称为C2G1 ARS）的核心共有序列内创建所有可能的单碱基突变，并测试它们对高频转化和质粒稳定性的影响，来检验该序列内每个位置对ARS活性的贡献。在33个突变中，22个通过高频转化检测发现消除了ARS功能，7个导致质粒稳定性降低两倍以上，4个对质粒稳定性没有影响。降低或消除ARS活性的突变发生在共有序列的每个位置，表明该序列的每个位置都对ARS功能有贡献。在对ARS活性没有影响的四个突变中，有三个与核心共有序列形成了替代的完美匹配，表明共有序列允许的替代碱基确实是可互换的。此外，第6位从T到C的变化并没有干扰野生型效率。为了测试必需区域是否延伸到11个碱基对的共有序列之外，研究了核心共有序列富含T链3'端一个碱基的点突变（第12位）以及改变核心共有序列富含T链5'端碱基的缺失突变对质粒稳定性的影响。第12位的A或核心共有序列5'端三个T残基的缺失严重降低了ARS效率，表明完整ARS效率所需的区域在两个方向上都延伸到核心共有序列之外。
