Analysis of nucleoid proteins in tobacco chloroplasts

叶绿体是植物细胞特有的细胞器，是质体的分化形式之一。虽然叶绿体基因组结构、多肽和转录调控已被深入研究，但叶绿体分化的调控机制仍不清楚。本文以烟草（Nicotiana tabacum cv. Samsun NN）细胞的叶绿体分化的调控机制。通过连续密度梯度离心使用metrizamid从纯化的叶绿体纯化类核。电镜观察纯化的类核组分和蛋白酶或2 M NaCl处理表明，类核由DNA和蛋白质组成。与~（32）P标记的叶绿体DNA的Southwestern杂交表明，类核组分中的某些多肽能与DNA结合。从培养的烟草细胞中分离到一种具有DNA结合亲和力的多肽，其变性形式的分子量约为41 kDa。与鲑鱼DNA的西南杂交竞争实验表明，DNA结合是相当特异的叶绿体DNA。对41 kD DNA结合多肽N端氨基酸序列的测定表明，该多肽不是叶绿体基因组编码的。

Chloroplasts are cellular organelles, specific to plant cells and one of the differentiated form of plastids. While the genome structure, polypeptides and transcription regulation in chloroplasts have been investigated intensively, the controlling mechanism of chloroplast differentiation is still not clear. Here, we characterized the DNA binding proteins in chloroplast nucleoid(DNA-protein complex)of cultured tobacco(Nicotiana tabacum cv. Samsun NN)cells to get some information about the controlling mechanism of chloroplast differentiation. Nucleoids were purified by sequential density gradient centrifugation using metrizamid from purified chloroplasts. Electron microscopic observation of the purified nucleoid fraction and the treatment with proteinase or 2M NaCl indicated that nucleoids consist of DNAs and proteins. Southwestern hybridization with 32P-labeled chloroplast DNA indicated that some polypeptides in nucleoid fraction could bind DNA. The molecular weight of a polypeptide with DNA binding affinity isolated from cultured tobacco cells was about 4lkDa in denatured form. Competition experiment in southwestern hybridization with salmon DNA suggested that DNA binding was rather specific to cholroplast DNA. Determination on N-terminal amino acid sequence of 41kD DNA-binding polypeptide indicated that this polypeptide was not coded by choloroplast geneme.