NEW HETEROLOGOUS MODULES FOR CLASSICAL OR PCR-BASED GENE DISRUPTIONS IN SACCHAROMYCES-CEREVISIAE
NEW HETEROLOGOUS MODULES FOR CLASSICAL OR PCR-BASED GENE DISRUPTIONS IN SACCHAROMYCES-CEREVISIAE
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DOI：
10.1002/yea.320101310
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发表时间：
1994-12-01
期刊：
影响因子：
2.6
通讯作者：
PHILIPPSEN, P
中科院分区：
文献类型：
--
作者：
WACH, A;BRACHAT, A;PHILIPPSEN, P
我们构建并测试了一个优势抗性模块，用于酿酒葡萄球菌转化体的选择，该转化体完全由异源DNA组成。该kanMX模块包含已知的大肠杆菌转座子Tn903的kan(r)开放阅读框，该框架融合了丝状真菌Ashbya gossypii的TEF基因的转录和翻译控制序列。该杂交模块允许有效选择抗遗传素（G418）的转化子。我们还构建了一个lacZMT报告模块，其中大肠杆菌lacZ基因的开放阅读框（缺少前9个密码子）在其3'端与S， cerevisiae ADH1终止子融合。KanMX和lacZMT模块，或两个模块一起，被克隆在一个新的多重克隆序列的中心，该序列由18个独特的限制性内切位点组成，两侧是Not r位点。使用双模块构建框架内基因替换，只需要一个转化实验来测试启动子的活性，并寻找由于该基因失活而导致的表型。为了允许重复使用G418选择，一些kanMX模块的两侧有470 bp的直接重复序列，以10(-3)-10（-4）的频率促进体内切除。1.4 kb的kanMX模块也被证明对基于PCR的基因破坏非常有用。在一项实验中，用携带pcr添加的末端序列的DNA分子进行基因破坏，这些DNA分子与每个目标位点只有35个碱基同源，所有12个被测试的抗基因菌落都携带了正确整合的kanMX模块。
We have constructed and tested a dominant resistance module, for selection of S. cerevisiae transformants, which entirely consists of heterologous DNA. This kanMX module contains the known kan(r) open reading-frame of the E. coli transposon Tn903 fused to transcriptional and translational control sequences of the TEF gene of the filamentous fungus Ashbya gossypii. This hybrid module permits efficient selection of transformants resistant against geneticin (G418). We also constructed a lacZMT reporter module in which the open reading-frame of the E. coli lacZ gene (lacking the first 9 codons) is fused at its 3' end to the S, cerevisiae ADH1 terminator. KanMX and the lacZMT module, or both modules together, were cloned in the center of a new multiple cloning sequence comprising 18 unique restriction sites flanked by Not r sites. Using the double module for constructions of in-frame substitutions of genes, only one transformation experiment is necessary to test the activity of the promotor and to search for phenotypes due to inactivation of this gene. To allow for repeated use of the G418 selection some kanMX modules are flanked by 470 bp direct repeats, promoting in vivo excision with frequencies of 10(-3)-10(-4). The 1.4 kb kanMX module was also shown to be very useful for PCR based gene disruptions. In an experiment in which a gene disruption was done with DNA molecules carrying PCR-added terminal sequences of only 35 bases homology to each target site, all twelve tested geneticin-resistant colonies carried the correctly integrated kanMX module.