Preparation of Chloroplasts from Euglena Highly Active in Protein Synthesis

Abstract

Chloroplasts can be obtained by gentle lysis or mild shear of spheroplasts of vitamin B12-deficient Euglena gracilis and then purified by isopycnic sedimentation on gradients of Ludox AM or Percoll. The chloroplasts appear compact and highly refractile by phase contrast or Hoffmann contrast microscopy. Upon incubation with 13HIleucine or 135Slmethionine, the chloroplasts incorporate the amino acids into protein at rates that are 100-fold faster than we had previously observed with Euglena and up to 8-fold faster than with chloroplasts of spinach. Eugkna chloroplasts prepared by the current procedure are thus qualitatively superior to those previously available from Eugkna and at least as active in protein synthesis as chloroplasts from higher plants.Although Euglena gracilis offers many advantages in the study of chloroplast development, a serious limiting factor in pursuing the molecular biology of this process has been the difficulty in isolating pure, intact, and functional plastids from Euglena. The pioneer studies of Eisenstadt and Brawerman (5) were based on crude fractions obtained by differential centrifugation and differential flotation in sucrose. Although the plastids obtained were extensively contaminated with other subcellular particles and the integrity of the organelles was never assessed, their isolation procedures were nonetheless followed by a number of other laboratories. We now realize that the high osmotic pressures required for flotation in sucrose gradients are incompatible with the integrity of chloroplasts.Several investigators have used spheroplasts in an effort to isolate plastids under relatively mild conditions (3,8,11,13), but the chloroplasts obtained lacked stroma and the envelopes were broken or lost.We have described (18) a separation procedure in which the chloroplasts were separated by rate zonal centrifugation in isosmotic gradients of Ficoll. This method yielded chloroplasts that appeared intact by electron microscopy but were not functional. We then found (10) that pure, intact, and functional chloroplasts

Nucleotide sequence of aEuglena gracilischloroplast genome region coding for the elongation factor Tu; evidence for a spliced mRNA

“…Total RNA was extracted from purified chloroplasts (8,9) according to the procedure of Sagher et al (10).…”

Protein synthesis by developing plastids isolated from Euglena gracilis

“…Euglena gracilis (Klebs) strain z was grown in the dark at 22°C on a glucose-containing medium that was limiting in cyanocobalamin [8] and transferred at different times to light of 2 x 1015 quanta . s-t .…”

“…Mature chloroplasts were isolated from spheroplasts of Euglena by mild shear in a Waring Blendor followed by sedimentation into density gradients of silica sol, as in [8,9]. For cells exposed to <6 h light, centrifugation in the density gradient step was increased to 10 000 rev./min for 40 min.…”

Analysis of Euglena gracilis chloroplast DNA

“…Chloroplasts were isolated following the basic procedure in [14] and the modifications outlined in [9].…”