Isolation of chloroplasts and chloroplast-nuclei(nucleoids)from Chlamydomonas reinhardtii

Abstract

Summary: We developed a method for isolating chloroplast-nuclei(nucleoids)from a unicellular green alga, Chlamydomonas reinhardtii, using cell-wall-deficient mutant(cw15)cells. Because the cells drastically change the structure of their chloroplast-nuclei during the cell division cycle, we synchronized the cell division under a 12: 12-h light: dark regimen, and collected cells in the 8th h during the dark period for use as starting material, when chloroplast-nuclei were granular in shape and scattered randomly within individual chloroplasts. The cells were converted to protoplasts and disrupted by repeated passages through a narrow-bore needle, and the intact chloroplasts were purified by Percoll density-gradient centrifugation. The chloroplast-nuclei were isolated from the purified chloroplasts following lysis with Nonidet P-40. The results of Southern and northern hybridization analyses suggested that not only structure, but also DNA synthesis and transcriptional activities of the chloroplast-nuclei might fluctuate during the cell division cycle of C. reinhardtii. Thus, the chloroplast-nuclei of C. reinhardtii may provide an ideal system for analyzing the structure-function relationships of DNA-protein complexes.