Schründer J, Meinhardt F
Institut für Mikrobiologie, Westfälische Wilhelms-Universität Münster, Federal Republic of Germany.
Plasmid. 1995 Mar;33(2):139-51. doi: 10.1006/plas.1995.1015.
Based on the cytoplasmically localized killer plasmids pGKL1 and pGKL2 of Kluyveromyces lactis two new linear hybrid plasmids were constructed which consist of pGKL1, into which in addition to the previously developed cytoplasmically expressible LEU2* selectable marker a glucose dehydrogenase-encoding bacterial gene (gdh A) has been integrated. One of the hybrid plasmids carries the bacterial gene preceded by an arbitrarily placed cytoplasmic promoter (upstream conserved sequence) in front of the coding region (pRKL121). The other plasmid was constructed in such a way that the ATG start codon of the gdh A gene was fused in frame to the ATG start codon of the killer plasmid's open reading frame 5 (pRKL122). The structures of both linear hybrid plasmids were confirmed by restriction analysis, Southern hybridization, and sequencing of the junction sites. Yeast strains carrying either of the plasmids expressed the glucose dehydrogenase gene; however, expression of the in phase fused gene was 40-fold higher compared to the arbitrarily placed cytoplasmic promoter. In general, an in phase fusion was not required for expression, but efficiency is dramatically enhanced when the 5' noncoding sequences in front of the heterologous genes are the same as those found on the native killer plasmids. The developed system can serve as a reporter for determining the efficiency of the different cytoplasmic promoters present on both linear plasmids. Hybrid plasmids were stably maintained without selective pressure in K. lactis and they were transferred and expressed also in Saccharomyces cerevisiae.
基于乳酸克鲁维酵母的细胞质定位杀伤质粒pGKL1和pGKL2，构建了两个新的线性杂交质粒，它们由pGKL1组成，除了先前开发的可在细胞质中表达的LEU2*选择标记外，还整合了一个编码葡萄糖脱氢酶的细菌基因（gdhA）。其中一个杂交质粒携带的细菌基因前面有一个位于编码区之前的任意位置的细胞质启动子（上游保守序列）（pRKL121）。另一个质粒的构建方式是，gdhA基因的ATG起始密码子与杀伤质粒开放阅读框5的ATG起始密码子读框融合（pRKL122）。通过限制性分析、Southern杂交和连接位点测序证实了两个线性杂交质粒的结构。携带任一质粒的酵母菌株都表达了葡萄糖脱氢酶基因；然而，与任意放置的细胞质启动子相比，读框融合基因的表达高出40倍。一般来说，表达不需要读框融合，但当异源基因前面的5'非编码序列与天然杀伤质粒上的序列相同时，表达效率会显著提高。所开发的系统可作为一种报告系统，用于确定两种线性质粒上不同细胞质启动子的效率。杂交质粒在乳酸克鲁维酵母中无需选择压力即可稳定维持，并且它们也能在酿酒酵母中转移和表达。
