Introduction of foreign DNA into Chlorella saccharophila by electroporation

Summary Plasmid, pBI221, was introduced into protoplasts of Chlorella saccharophila c-211-1a prepared from the cells in the stationary phase by electroporation. Transient expression of the introduced plasmid was observed under a field strength of between 600 and 900 V/cm, and a pulse duration of around 400 ms, where high membrane permeability to 70-kDa FITC-dextran was ascertained.     

Photoreduction of sulfur dioxide by spinach leaves and isolated spinach chloroplasts

Labeled sulfur dioxide was found to be extensively absorbed by spinach (Spinacea oleracea L.) leaves. Labeled sulfides detected in leaf blades following fumigations with sulfur dioxide in light indicated that photoreduction of sulfur dioxide had occurred. Measurable proportions of this labeled sulfur was localized within the chloroplast fraction. Suspensions of isolated chloroplasts supplied with labeled sulfur dioxide contained labeled sulfides following a 30-minute illumination period in water-cooled reaction vessels. With reference to recent studies of the chloroplast sulfur reduction pathway, probable points of entry for sulfur dioxide and the subsequent release of hydrogen sulfide are discussed.     

Introduction and expression of DNA molecules in eukaryotic cells by electroporation

Exposing eukaryotic cells to brief, high voltage electrical fields can induce the uptake of exogenous materials, presumably through the transient formation of micropores in the cell membrane. This phenomenon has been exploited for the introduction of cloned DNA molecules into cells for permanent transformation or for transient expression and analysis of gene products. The magnitude and characteristics of the generated electrical field are critical for successful electroporation and simple, transient expression assays using indicator genes allow the calibration and optimization of electroporation conditions for a wide variety of eukaryotic cell types. These techniques may allow the genetic modification of a variety of host cells which cannot be easily transformed by other methods.     

Starch degradation in isolated spinach chloroplasts

A method for loading isolated intact spinach (Spinacia oleracea L.) chloroplasts with ¹⁴C-starch is described. These intact chloroplasts were incubated aerobically in the dark for 30 minutes. Radioactivity in starch declined and glyceric acid 3-phosphate and maltose were the major radioactive products. It is proposed that starch is degraded within the chloroplast to glyceric acid 3-phosphate and to maltose.     

Inhibition of photosynthesis by oxygen in isolated spinach chloroplasts

The inhibition of photosynthetic CO₂ fixation by O₂, commonly referred to as the Warburg effect, was examined in isolated intact spinach (Spinacia oleracea) chloroplasts. The major characteristics of this effect in isolated chloroplasts are rapid reversibility when O₂ is replaced by N₂, an increased inhibition by O₂ at low concentrations of CO₂ and a decreased effect of O₂ with increased concentrations of CO₂.     

Reversible abolition of enhancement in isolated spinach chloroplasts

The photosynthetic evolution of oxygen by isolated chloroplasts of Spinacia oleracea L. was studied using a modulated oxygen electrode. The enhancement effect, measured as the increase in the relative quantum yield of 650-nanometer light due to the presence of 710-nanometer light, was profoundly influenced by the concentration of inorganic cations in the bathing medium. Chloroplast fragments immersed in a solution containing a very low concentration of MgCl₂ or KCl, did not display enhancement but could be made to do so by raising the concentration of MgCl₂ to 3 mm, or that of KCl to 35 mm. This change in the enhancement properties was completely reversible. The maximum value of enhancement in a MgCl₂ solution appeared to occur at a concentration between 15 and 30 mm, while in KCl, the enhancement effect increased almost linearly up to a concentration of 100 mm.     

Reversible swelling and contraction of isolated spinach chloroplasts

By use of a micro technique for producing extracts of spinach mesophyll cells, chloroplasts were isolated in a state wherein they displayed microscopically visible, reversible osmotic properties. Swollen spherical chloroplasts treated with hypertonic sucrose or mannitol media, but not NaCl, could be shrunken to a state resembling their disk appearance in living cells. Reversible osmotic behavior was more easily demonstrated when the chloroplasts were initially isolated from cells in a relatively low osmolar concentration in contrast to using 0.25 m sucrose or more concentrated media. Individual chloroplasts could be swollen and contracted repeatedly through as many as 4 cycles. The relationship between the capacity for osmotic behavior and chloroplast appearance in cell extracts is discussed.     

Level of photosynthetic intermediates in isolated spinach chloroplasts

The level of intermediates of the photosynthetic carbon cycle was measured in intact spinach chloroplasts in an attempt to determine the cause of the induction lag in CO₂ assimilation. In addition, transient changes in the level of the intermediates were determined as affected by a light-dark period and by the addition of an excess amount of bicarbonate during a period of steady photosynthesis. Assayed enzymically were: ribulose 1,5-diphosphate, pentose monophosphates (mixture of ribose 5-phosphate, ribulose 5-phosphate and xylulose 5-phosphate, hexose monophosphates (mixture of glucose 6-phosphate, glucose 1-phosphate, and fructose 6-phosphate), glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, glycerate acid 3-phosphate, a mixture of fructose 1,6-diphosphate and sedoheptulose 1,7-diphosphate, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP).     

Synthesis of mono- and digalactosyldiacylglycerol in isolated spinach chloroplasts

Purified, intact chloroplasts of Spinacia oleracea L. synthesize galactose-labeled mono- and digalactosyldiacylglycerol (MGDG and DGDG) from UDP-[U-¹⁴C]galactose. In the presence of high concentrations of unchelated divalent cations they also synthesize tri- and tetra-galactosyldiacylglycerol. The acyl chains of galactose-labeled MGDG are strongly desaturated and such MGDG is a good precursor for DGDG and higher oligogalactolipids. The synthesis of MGDG is catalyzed by UDP-Gal:sn-1,2-diacylglycerol galactosyltransferase, and synthesis of DGDG and the oligogalactolipids is exclusively catalyzed by galactolipid:galactolipid galactosyltransferase. The content of diacylglycerol in chloroplasts remains low during UDP-Gal incorporation. This indicates that formation of diacylglycerol by galactolipid:galactolipid galactosyltransferase is balanced with diacylglycerol consumption by UDP-Gal:diacylglycerol galactosyltransferase for MGDG synthesis. Incubation of intact spinach chloroplasts with [2-¹⁴C]acetate or sn-[U-¹⁴C]glycerol-3-P in the presence of Mg²⁺ and unlabeled UDP-Gal resulted in high ¹⁴C incorporation into MGDG, while DGDG labeling was low. This de novo made MGDG is mainly oligoene. Its conversion into DGDG is also catalyzed, at least in part, by galactolipid:galactolipid galactosyltransferase.     

Characteristics of light-dependent inorganic carbon uptake by isolated spinach chloroplasts

The light-dependent accumulation of radioactively labeled inorganic carbon in isolated spinach (Spinacia oleracea L.) chloroplasts was determined by silicone oil filtering centrifugation. Intact chloroplasts, dark-incubated 60 seconds at pH 7.6 and 23°C with 0.5 millimolar sodium bicarbonate, contained 0.5 to 1.0 millimolar internal inorganic carbon. The stromal pool of inorganic carbon increased 5- to 7-fold after 2 to 3 minutes of light. The saturated internal bicarbonate concentration of illuminated spinach chloroplasts was 10- to 20-fold greater than that of the external medium. This ratio decreased at lower temperatures and with increasing external bicarbonate. Over one-half the inorganic carbon found in intact spinach chloroplasts after 2 minutes of light was retained during a subsequent 3-minute dark incubation at 5°C. Calculations of light-induced stromal alkalization based on the uptake of radioactively labeled bicarbonate were 0.4 to 0.5 pH units less than measurements performed with [¹⁴C]dimethyloxazolidine-dione. About one-third of the binding sites on the enzyme ribulose 1,5-bisphosphate carboxylase were radiolabeled when the enzyme was activated in situ and ¹⁴CO₂ bound to the activator site was trapped in the presence of carboxypentitol bisphosphates. Deleting orthophosphate from the incubation medium eliminated inorganic carbon accumulation in the stroma. Thus, bicarbonate ion distribution across the chloroplast envelope was not strictly pH dependent as predicted by the Henderson-Hasselbach formula. This finding is potentially explained by the presence of bound CO₂ in the chloroplast.     

Heterogeneous photosystem 2 activity in isolated spinach chloroplasts

A study was made of the fluorescence induction curves from gently-broken spinach chloroplasts inhibited with DCMU. It was found that there were four kinetically different phases associated with such curves of which only the fastest did not appear to follow exponential kinetics. A comparison of the effects of various concentrations of DCMU on the rate of oxygen evolution and on the fluorescence induction curve did not support the hypothesis that any of the kinetic phases was simply an artefact caused by incomplete inhibition of electron transport. It was also found that 5 min of dark incubation did not maximally oxidize the electron acceptors to photosystem 2 since some acceptors were only oxidized following far-red illumination, suggesting a heterogeneity among these acceptors with respect to their re-oxidation properties. Investigation of the effect of the Q₄₀₀ oxidation state on the fluorescence induction curve revealed that it only influenced the slowest kinetic phase and that Q₄₀₀ did not seem to be associated with the other phases.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1 - 1 dimethylurea - PS 1 photosystem 1 - PS2 photosystem 2 - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid - EDTA ethylene-diaminetetraacetic acid - F_max maximum yield of fluorescence emission - F₀ initial yield of fluorescence emission - F_v variable yield of fluorescence emission - N.E. non-exponential kinetics