Meijer A H, Ouwerkerk P B, Hoge J H
Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory, The Netherlands.
Yeast. 1998 Nov;14(15):1407-15. doi: 10.1002/(SICI)1097-0061(199811)14:15<1407::AID-YEA325>3.0.CO;2-M.
We describe the construction of a number of vectors that can be used in yeast genetic selection systems for cloning of transcription factors or other DNA-binding proteins and for identification of the target sites recognized by transcription factors. For transcription factor cloning we have designed an integration vector with two HIS3 reporter gene cassettes to stably integrate reporter gene constructs at the non-essential yeast PDC6 locus. This set of plasmids was tested in a one-hybrid assay with the rice transcription factor Oshox1, a member of the class of homeodomain leucine zipper proteins. A hybrid protein of Oshox1 and the Gal4 transcriptional activation domain was shown to specifically activate a reporter gene construct with upstream Oshox1 binding sites, which had been integrated at the PDC6 locus using the described vector system. Target site identification by genetic selection in yeast employs a transcriptional activator construct and a library of genomic or random DNA fragments upstream of a reporter gene. We have constructed two variants of a bacteriophage lambda vector which facilitates the construction of the required reporter gene library because of high cloning efficiency and easy conversion into a yeast/Escherichia coli shuttle vector library by Cre-loxP-mediated automatic subcloning. Tests with Oxhox1 as transcriptional activator demonstrated the usefulness of the deprived reporter gene vector.
我们描述了多种载体的构建方法，这些载体可用于酵母遗传选择系统，用于克隆转录因子或其他DNA结合蛋白，以及鉴定转录因子识别的靶位点。为了克隆转录因子，我们设计了一种带有两个HIS3报告基因盒的整合载体，以便将报告基因构建体稳定整合到酵母非必需的PDC6位点。这套质粒在与水稻转录因子Oshox1（一种同源域亮氨酸拉链蛋白家族成员）的单杂交试验中进行了测试。结果表明，Oshox1与Gal4转录激活域的融合蛋白能够特异性激活一个带有上游Oshox1结合位点的报告基因构建体，该构建体已使用所述载体系统整合到PDC6位点。通过酵母中的遗传选择来鉴定靶位点，采用一个转录激活构建体和一个位于报告基因上游的基因组或随机DNA片段文库。我们构建了两种噬菌体λ载体变体，由于其克隆效率高且易于通过Cre-loxP介导的自动亚克隆转化为酵母/大肠杆菌穿梭载体文库，因此便于构建所需的报告基因文库。以Oxhox1作为转录激活剂进行的测试证明了这种缺失报告基因载体的实用性。
