Hsiao C L, Carbon J
Proc Natl Acad Sci U S A. 1979 Aug;76(8):3829-33. doi: 10.1073/pnas.76.8.3829.
Hybrid ColE1 plasmids, containing cloned DNA from the yeast ARG4 region [e.g., pYe(arg4)1], transform yeast arg4 mutants to ARG4(+) with a frequency of 10(-4) (about 10(3) transformants per mug of plasmid DNA) and can replicate autonomously without integrating into the yeast genome. The yeast transformants are genetically unstable when grown on nonselective medium, but can be readily grown and maintained on minimal medium lacking arginine. The existence of unintegrated replicating plasmid DNA in the yeast transformants was demonstrated by Southern gel hybridization and by transformation of Escherichia coli argH mutants with DNA preparations from yeast transformants and subsequent recovery of intact plasmid DNA from the bacterial transformants. Plasmid DNAs recovered from the E. coli-yeast-E. coli "shuttle" remain essentially unchanged, as judged by DNA restriction fragment patterns. Some plasmid mutations leading to increased efficiency of expression of the ARG4 gene in E. coli do not appear to affect expression of the cloned ARG4 gene in yeast. Appropriate derivatives of these ARG4 plasmids are of potential usefulness as vectors for cloning genes in yeast and for studying the mechanism of yeast DNA replication.
含有来自酵母ARG4区域克隆DNA的杂种ColE1质粒（例如pYe(arg4)1），能以10^(-4)的频率将酵母arg4突变体转化为ARG4(+)（每微克质粒DNA约有10^3个转化体），并且能自主复制而不整合到酵母基因组中。酵母转化体在非选择性培养基上生长时遗传不稳定，但能在缺乏精氨酸的基本培养基上容易地生长和维持。通过Southern凝胶杂交以及用酵母转化体的DNA制剂转化大肠杆菌argH突变体并随后从细菌转化体中回收完整质粒DNA，证明了酵母转化体中存在未整合的复制性质粒DNA。从大肠杆菌-酵母-大肠杆菌“穿梭”中回收的质粒DNA，根据DNA限制性片段图谱判断，基本上保持不变。一些导致ARG4基因在大肠杆菌中表达效率提高的质粒突变，似乎不影响酵母中克隆的ARG4基因的表达。这些ARG4质粒的合适衍生物作为在酵母中克隆基因和研究酵母DNA复制机制的载体具有潜在用途。
