Analysis of nucleoid proteins in tobacco chloroplasts

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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.