Kitamoto T, Ikeda K, Salvaterra P M
Division of Neurosciences, Beckman Research Institute of the City of Hope, Duarte, California 91010.
J Neurosci. 1992 May;12(5):1628-39. doi: 10.1523/JNEUROSCI.12-05-01628.1992.
We have analyzed the cis-regulatory elements in the 5' flanking region of the Drosophila choline acetyltransferase gene (ChAT, E.C.2.3.1.6). DNA fragments were fused to the Escherichia coli lacZ reporter gene and introduced into the Drosophila germ line by P-element-mediated transformation. A 7.4 kb 5' flanking sequence directed beta-galactosidase expression in the adult optic lobes and other well-defined CNS structures with a pattern very similar to the distribution of endogenous ChAT protein. In contrast, the proximal 3.3 kb and 1.2 kb of 5' flanking DNA directed lacZ expression in only selected subsets of the structures seen with the 7.4 kb lacZ construct. Our results indicate that both qualitative and quantitative regulatory elements are present in the 5' flanking DNA and that these elements distinguish various subsets of cholinergic neurons. We have also fused the same 5' flanking DNA sequences to wild-type ChAT cDNA and used these constructs to transform Chatsl mutant flies. Not only the 7.4 kb cDNA construct, but also the 3.3 and 1.2 kb constructs, rescued Chatsl from temperature-dependent paralysis and adult lethality, indicating that the regulatory information in any of these genomic fragments can drive sufficient wild-type ChAT expression to overcome these mutant phenotypes.
我们分析了果蝇胆碱乙酰转移酶基因（ChAT，E.C.2.3.1.6）5'侧翼区域的顺式调控元件。将DNA片段与大肠杆菌lacZ报告基因融合，并通过P元件介导的转化引入果蝇种系。一段7.4 kb的5'侧翼序列在成年视叶和其他明确的中枢神经系统结构中指导β-半乳糖苷酶表达，其模式与内源性ChAT蛋白的分布非常相似。相比之下，5'侧翼DNA近端的3.3 kb和1.2 kb仅在7.4 kb lacZ构建体所见结构的选定子集中指导lacZ表达。我们的结果表明，5'侧翼DNA中存在定性和定量调控元件，这些元件区分胆碱能神经元的不同子集。我们还将相同的5'侧翼DNA序列与野生型ChAT cDNA融合，并使用这些构建体转化Chatsl突变果蝇。不仅7.4 kb的cDNA构建体，而且3.3 kb和1.2 kb的构建体都使Chatsl从温度依赖性麻痹和成年致死性中恢复过来，表明这些基因组片段中的任何一个的调控信息都可以驱动足够的野生型ChAT表达以克服这些突变表型。
