Phelps D E, Dressler G R
NICMD, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 1996 Apr 5;271(14):7978-85. doi: 10.1074/jbc.271.14.7978.
The Pax genes encode a family of developmental transcription factors that bind to specific DNA sequences via the paired domain and are necessary for the morphogenesis of a variety of tissues. The murine Pax-2 gene, through alternative splicing, encodes two nuclear proteins, Pax-2A and Pax-2B, which are transiently expressed during the differentiation of specific neural cell types and early kidney formation. In order to identify potential in vivo Pax-2 target sequences, chromatin from embryonic neural tube was immunoprecipitated with Pax-2 specific antibodies and cloned. Two unique immunoprecipitated clones containing three specific Pax-2 binding sites were identified by functional binding assays using Pax-2 proteins produced in both Escherichia coli and eukaryotic cells. In vitro DNA binding assays, using Pax-5 and Pax-8 DNA recognition sequences as well as the three immunopurified Pax-2 binding sites, demonstrated that both forms of the Pax-2 protein bind DNA with a similar specificity and that this binding is mediated by the paired domain. The binding sites identified in this report share significant homology among themselves and with previously defined consensus sequences for Pax-5 and Pax-2. The genomic clones can now be used as sequence tags to identify potential target loci.
Pax基因编码一类发育转录因子，它们通过配对结构域与特定DNA序列结合，是多种组织形态发生所必需的。小鼠Pax-2基因通过可变剪接编码两种核蛋白，即Pax-2A和Pax-2B，它们在特定神经细胞类型分化和早期肾脏形成过程中短暂表达。为了鉴定潜在的体内Pax-2靶序列，用Pax-2特异性抗体对胚胎神经管的染色质进行免疫沉淀并克隆。通过使用在大肠杆菌和真核细胞中产生的Pax-2蛋白进行功能结合测定，鉴定出两个含有三个特定Pax-2结合位点的独特免疫沉淀克隆。使用Pax-5和Pax-8 DNA识别序列以及三个免疫纯化的Pax-2结合位点进行的体外DNA结合测定表明，两种形式的Pax-2蛋白以相似的特异性结合DNA，并且这种结合由配对结构域介导。本报告中鉴定的结合位点彼此之间以及与先前定义的Pax-5和Pax-2共有序列具有显著同源性。现在可以将基因组克隆用作序列标签来鉴定潜在的靶基因座。
