Bonner J J
Department of Biology, Indiana University, Bloomington 47405.
Gene. 1991 Jul 31;104(1):113-8. doi: 10.1016/0378-1119(91)90475-q.
A series of 13 vectors is described. All are yeast centromere plasmids with the LEU2 gene for selection in yeast, and pUC19 sequences for growth in Escherichia coli. All contain the GAL1 promoter directing transcription into a multiple cloning site (MCS). For twelve of the plasmids, synthetic oligodeoxyribonucleotides create an ATG start codon, in a productive context for yeast, prior to the MCS. Spacing between the ATG and the MCS is variable, to facilitate the cloning of gene fragments in the appropriate reading frame. Nine of the plasmids also contain the strong transcriptional activator from the herpes simplex virus VP16 gene, joined downstream from the MCS. In these nine vectors, all possible combinations of reading frames are available. The suitability of these plasmids for the expression and analysis of DNA-binding domains is tested by cloning into them fragments of the yeast HSF1 gene, encoding the heat shock transcription factor (HSF). The regulation of reporter gene expression by the chimeric HSF-VP16 fusions is described, as is the utility of these vectors for other applications.
本文描述了一系列13种载体。所有载体均为酵母着丝粒质粒，带有用于在酵母中筛选的LEU2基因以及用于在大肠杆菌中生长的pUC19序列。所有载体都含有GAL1启动子，该启动子可将转录导向多克隆位点（MCS）。对于其中12种质粒，合成的寡脱氧核糖核苷酸在MCS之前的酵母有效环境中产生一个ATG起始密码子。ATG与MCS之间的间隔是可变的，以便于以合适的阅读框克隆基因片段。其中9种质粒还含有来自单纯疱疹病毒VP16基因的强转录激活因子，连接在MCS的下游。在这9种载体中，可以使用所有可能的阅读框组合。通过将编码热休克转录因子（HSF）的酵母HSF1基因片段克隆到这些质粒中，测试了它们用于DNA结合结构域表达和分析的适用性。本文描述了嵌合HSF-VP16融合蛋白对报告基因表达的调控，以及这些载体在其他应用中的效用。
