Zhao Ji-lin, Chen Yang-xi, Bao Lang, Wu Tuo-jiang, Zhou Li
Department of Orthodontics, West China College of Stomatology, Sichuan University, Chengdu, Sichuan, 610041 PR China.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2005 Aug;22(4):419-22.
To gain new insights into the molecular pathogenesis of the 109(InsG) and 139(C--> T) mutations and their roles in familial oligodontia.
The region of PAX9 paired domain (PAX9PD) was amplified and the expression plasmids were constructed in pGEXlambda -1T by PCR-based cloning. PAX9PD proteins were prepared on the basis of GST instruction. The binding of wild type and two novely mutant PAX9 paired domain to double-stranded DNA targets were analyzed by gel mobility shift assay.
Wild type PAX9PD protein bind to the high affinity paired domain recognition sequences, CD19-2(A-ins) and Pax6CON, the 109(InsG) and 139(C--> T) mutant PAX9PD protein were unable to bind to these cognate DNA-binding sites.
The functional defects in DNA binding of mutant 109(InsG) PAX9 and 139(C--> T) PAX9, as well as loss-of-function of PAX9 most likely result in its haploinsufficiency during the patterning of dentition and the subsequent loss of posterior teeth.
深入了解109（InsG）和139（C→T）突变的分子发病机制及其在家族性少牙症中的作用。
通过基于聚合酶链反应（PCR）的克隆技术扩增PAX9配对结构域（PAX9PD）区域，并构建于pGEXlambda -1T表达质粒中。根据谷胱甘肽S-转移酶（GST）说明书制备PAX9PD蛋白。采用凝胶迁移率变动分析（gel mobility shift assay）检测野生型和两种新型突变型PAX9配对结构域与双链DNA靶点的结合情况。
野生型PAX9PD蛋白可与高亲和力的配对结构域识别序列CD19-2（A-ins）和Pax6CON结合，而109（InsG）和139（C→T）突变型PAX9PD蛋白则无法与这些同源DNA结合位点结合。
109（InsG）PAX9和139（C→T）PAX9突变体在DNA结合方面的功能缺陷，以及PAX9的功能丧失，很可能导致其在牙列形成过程中出现单倍剂量不足，进而导致后牙缺失。
