Trapping developmental promoters in Dictyostelium.
Trapping developmental promoters in Dictyostelium.
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DOI：
10.1006/plas.1995.0003
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发表时间：
1995-11
期刊：
影响因子：
2.6
通讯作者：
W. Chang;J. Gross;P. Newell
中科院分区：
文献类型：
--
作者：
W. Chang;J. Gross;P. Newell
最近，一种插入诱变程序已经开发出来，允许克隆在盘状盘基骨菌发育突变体中受影响的基因（Kuspa和Loomis， Proc. Natl）。学会科学。中国科学院学报（自然科学版），1992)。在此过程中，携带URA （pyr5-6）基因的质粒与限制性内切酶线性化，并与相应的限制性内切酶一起电穿孔到URA-变形虫（尿嘧啶的营养不良）中。在没有尿嘧啶的情况下也能生长的转化子被筛选为由于将质粒插入对发育有重要意义的基因而导致的发育缺陷。我们修改了这一程序，以允许表征启动子和基因的结构序列，这些基因的启动子和结构序列将被标准程序遗漏，因为它们的破坏不会产生明显的表型。设计了携带无启动子的大肠杆菌lacZ基因的构建体，使lacZ的表达需要插入到活性宿主转录单元中。通过筛选限制性内切酶生成的转化子，我们分离出了几种lacZ受发育激活启动子控制的菌株，并克隆了插入位点附近的5'侧链DNA。质粒与宿主基因组连接的测序证实了与lacZ基因的框架内融合，将克隆的质粒重新引入亲本细胞表明克隆的序列确实含有相关的启动子。这一程序应能使我们随时获得广泛的发育启动子，而无需事先鉴定所涉及的发育基因。
Recently an insertional mutagenesis procedure has been developed to permit cloning of genes affected in developmental mutants of Dictyostelium discoideum (Kuspa and Loomis, Proc. Natl. Acad. Sci. USA 89, 8803-8807, 1992). In this procedure a plasmid bearing the URA (pyr5-6) gene is linearized with a restriction enzyme and electroporated into URA- amoebae (auxotrophic for uracil) together with the corresponding restriction enzyme. Transformants that can grow without uracil are screened for developmental defects resulting from insertion of the plasmid into a gene of developmental importance. We have modified this procedure to permit characterization of the promoters and structural sequences of genes that would be missed by the standard procedure because their disruption produces no obvious phenotype. Constructs carrying a promoter-less Escherichia coli lacZ gene were designed so that expression of lacZ requires insertion into an active host transcription unit. By screening restriction enzyme-generated transformants we have isolated several strains in which lacZ is under the control of a developmentally activated promoter and have cloned the 5' flanking DNA adjacent to the insertion site. Sequencing the junction between plasmid and host genome has confirmed in-frame fusion with the lacZ gene, and reintroduction of the cloned plasmids into parental cells has shown that the cloned sequences do actually contain the relevant promoters. This procedure should give ready access to a wide range of developmental promoters without the need for prior identification of the developmental genes involved.