Jayaram M, Sumida S, Young L J
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, CA 92037.
Curr Genet. 1986;11(2):85-91. doi: 10.1007/BF00378198.
The yeast plasmid, 2 micron circle, encodes a stability system consisting of the plasmid replication origin, a cis-active locus designated REP3 and two trans-active functions--the products of the REP1 and REP2 genes. We have constructed 2 micron circle derivatives in which the expression of the REP1 gene is placed under the control of the yeast GAL10 promoter. We show that in such plasmids the stability-system is inducible, being turned off by glucose and turned on by galactose. Further, our results unequivocally demonstrate that, of the two potential in-frame ATG codons at which REP1 translation might initiate (as inferred from the 2 micron circle DNA sequence and from the cap site of the major REP1 transcript), the upstream ATG is dispensable without affecting REP1 function. We also illustrate here a simple and general method for constructing in vivo in yeast 2 micron circle analogs which contain desired alterations within specific regions of the 2 micron circle genome.
酵母质粒2μm环编码一个稳定系统，该系统由质粒复制起点、一个称为REP3的顺式作用位点和两种反式作用功能——REP1和REP2基因的产物组成。我们构建了2μm环衍生物，其中REP1基因的表达置于酵母GAL10启动子的控制之下。我们表明，在这类质粒中，稳定系统是可诱导的，在葡萄糖存在时关闭，在半乳糖存在时开启。此外，我们的结果明确证明，从2μm环DNA序列和主要REP1转录本的帽位点推断，REP1翻译可能起始的两个潜在读码框内ATG密码子中，上游ATG在不影响REP1功能的情况下是可有可无的。我们在此还阐述了一种简单通用的方法，用于在酵母体内构建2μm环类似物，这些类似物在2μm环基因组的特定区域内包含所需的改变。
