Structure and function of DNA-binding proteins that are present in plastid envelope.

1.cpDNA的动态：荧光显微镜观察不同发育阶段叶绿体和叶绿体cpDNA与叶绿体被膜的结合情况表明，在分裂活跃的幼体中，cpDNA与叶绿体被膜结合，这种结合不受光和类囊体膜的影响。2.叶绿体被膜中cpDNA特异的DNA结合蛋白的鉴定。利用西南向杂交技术在叶绿体被膜中检测到一个130 kDa的DNA结合蛋白。PEND蛋白定位于内被膜，能与cpDNA的几个(至少四个)不同区域结合，幼体被膜中有PEND蛋白表达，成熟叶绿体被膜中未检测到PEND蛋白。用生物素标记的DNA探针和包被链霉亲和素的磁性颗粒对pend蛋白进行部分亲和纯化。在标准的SDS-PAGE中检测到该蛋白为115 kDa的多肽。4.质粒DNA结合蛋白的克隆。经一系列西南筛选得到3个克隆，其中一种蛋白(PD1)的抗体检测到了50 kDa和87 kDa的多肽。这些多肽的积累被细胞分裂素显著减少。

1.Dynamics of cpDNA.Observation by fluorescence microscopy of the binding of cpDNA with plastid envelope at various stages of development of etioplast and chloroplast indicated that cpDNA binds to plastid envelope in young plastids active in division and that this binding was not affected by light or the presence of thylakoid membrane.2.Identification of a cpDNA-specific DNA-binding protein in plastid envelope. A 130 kDa DNA-binding protein was detected in plastid envelop by South-western technique. The protein named PEND protein was localized in the inner envelope, and was able to bind to several (at least four) distinct regions of cpDNA.PEND protein was detected in the envelope of young plastid but not in the envelop of mature chloroplast.3.Effort to purify PEND protein. PEND protein was partially affinty-purified using a biotin-labelled DNA probe and streptavidin-coated magnetic particles. The protein was detected as a 115kDa polypeptide in the standard SDS-PAGE.4.cDNA cloning of plastid DNA-binding proteins. A series of South-western screening yielded 3 clones of cDNA.The antibody against one of these proteins (PD1) detected 50kDa and 87kDa polypeptides in the plastid. The accumulation of these polypeptides was markedly reduced by a cytokinin.