Summary
A method has been developed for the isolation of chloroplasts from Euglena gracilis grown under mixotrophic conditions. This method utilizes sucrose density-gradient centrifugation in the AXII zonal rotor and allows the rapid preparation of large amounts of chloroplasts free from contaminating whole cells and other cytoplasmic materials. The majority of the isolated chloroplasts appear intact in phase contrast and electron micrographs. The purified chloroplast fraction contains DNA, the major species of which has a density of 1.682 g/cm3. The species of DNA having a density of 1.707 g/cm3 seemed to result from the presence of contaminating nuclear fragments which could be removed by isopycnic flotation.
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Anderson, N. G.: Studies on isolated cell components. Exp. Cell Res. 9, 446–459 (1955).
Brawerman, G.: The isolation of a specific species of ribosomes associated with chloroplast development in Euglena gracilis. Biochim. biophys. Acta (Amst.) 72, 317–331 (1963).
Brawerman, G., Eisenstadt, J. M.: Template and ribosomal ribonucleic acids associated with the chloroplasts and the cytoplasm of Euglena gracilis. J. molec. Biol. 10, 403–411 (1964).
Brawerman, G., Eisenstadt, J. M.: Deoxyribonucleic acid from the chooroplasts of Euglena gracilis. Biochim. biophys. Acta (Amst.) 91, 477–485 (1964).
Brown, R. D., Haselkorn, R.: The isolation of Euglena gracilis chloroplasts un contaminated by nuclear DNA. BIochim. biophys. Acta (Amst.) 259, 1–4 (1972)
Carell, E. F., Kahn, J. S.: Synthesis of prophyrins by isolated chloroplasts of Euglena. Arch. Biochem. 108, 1–6 (1964).
Crampton, C. F., Lipshitz, R., Chargaff, E.: Studies on nucleoproteins. I. Dissociation and reassociation of the deoxyribonucleohistone of calf thymus. J. biol. Chem. 206, 499–510 (1954).
Edelman, M., Schiff, J. A., Epstein, H. T.: Studies of chloroplast development in Euglena. XII. Two types of satellite DNA. J. molec. Biol. 11, 769–774 (1965).
Eisenstadt, J. M., Brawerman, G.: Isolation of chloroplasts from Euglena gracilis. In: Methods in enzymology, vol. XII, pt. A, p. 476–478, Colowick, S. P., Kaplan, N. O., Grossman, L., Moldave, K., eds. New York-London: Academic Press 1968.
Greenblatt, C. L., Schiff, J. A.: A pheophytin-like pigment in dark-adapted Euglena gracilis. J. Protozool. 6, 23–28 (1963).
Harris, E. H., Preston, J. F., Selsky, M. I., Eisenstadt, J. M.: RNA and protein synthesis by isolated chloroplasts of Euglena gracilis. Bact. Proc. 150 (1970)
Jacobson, A. B.: A procedure for isolation of proplastids from etiolated maize leaves. J. Cell Biol. 38, 238–244 (1968).
Kirk, J. T. O.: Studies on the dependence of chlorophyll synthesis of protein synthesis in Euglena gracilis, together with a nomogram for determination of chlorophyll concentration. Planta (Berl.) 78, 200–207 (1968).
Knight, G. J., Price, C. A.: Measurements of s- coordinates during the development of Euglena chloroplasts. Biochim. biophys. Acta (Amst.) 158, 283–285 (1968).
Krawiec, S., Eisenstadt, J. M.: Ribonucleic acids from the mitochondria of bleached Euglena gracilis z. I. Isolation of mitochondria and extraction of nucleic acids. Biochim. biophys. Acta (Amst.) 217, 120–131 (1970).
Mandel, M., Shildkraut, C. L., Marmur, J.: Use of CsCl density gradient analysis for determining the guanine plus cytosine content of DNA. In: Methods in enzymology, vol. XII, pt. B, p. 184–195, Colowick, S. P., Kaplan, N. O., Grossman, L., Moldave, K., eds. New York-London: Acad. Press. 1968.
Mollenhauer, H. H.: Plastic embedding materials for use in electron microseopy. Stain Technol. 39, 111–114 (1964).
Preston, J. F., Harris, E. H., Eisenstadt, J. M.: Ribosomes and RNA of the chloroplasts of Euglena gracilis. Bact. Proc. 48 (1970).
Ray, D. S., Hanawalt, P. C.: Properties of the satellite DNA associated with the chloroplasts of Euglena gracilis. J. molec. Biol. 9, 812–824 (1964).
Reynolds, E. S.: The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol. 17, 208–212 (1963).
Shildkraut, C. L., Marmur, J., Doty, P.: Determination of the base composition of deoxyribonucleic acid from its buoyant density in CsCl. J. molec. Biol. 4, 430–443 (1962).
Still, C. C., Price, C. A.: Bulk separation of chloroplasts with intact membranes in the zonal centrifuge. Biochim. biophys. Acta (Amst.) 141, 176–178 (1967).
Stutz, E., Vandrey, J. P.: Ribosomal DNA satellite of Euglena gracilis chloroplast DNA. FEBS Lett. 17, 277–280 (1971).
Vasconcelos, A., Pollack, M., Mendiola, L. R., Hoffmann, H. P., Brown, D. H. Price, C. A.: Isolation of intact chloroplasts from Euglena gracilis by zonal centrifugation. Plant Physiol. 47, 217–221 (1971).
Wolken, J. J., Palade, G. E.: An electron microscope study of two flagellates. Chloroplast structure and variation. Ann. N. Y. Acad. Sci. 56, 873–881 (1953).
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This research was supported in part by a grant from USPHS No. AM-07189 (to J. M. E.), American Cancer Society Fellowship No. PF-443 (to J.F.P.) and grant No. 2972E06 from the Office of Sponsored Research, University of Florida (to J.F.P.). Univ. of Fla. Agr. Exp. Stat. No. 4538.
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Preston, J.F., Parenti, F. & Eisenstadt, J.M. Studies on the isolation and purification of chloroplasts from Euglena gracilis . Planta 107, 351–367 (1972). https://doi.org/10.1007/BF00386396
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DOI: https://doi.org/10.1007/BF00386396