Photocontrol of organelle and cell type specific changes in the polypeptide composition of Euglena and sorghum.

Two-dimensional (2-D) gel electrophoresis has been used to follow changes in cell type specific and organelle localized polypeptides upon exposure of etiolated sorghum shoots and dark-grown resting Euglena to light. Total protein extracted from isolated bundle sheath strands and mesophyll protoplasts was resolved by 2-D gel electrophoresis. The cell type specific polypeptides were localized on the whole shoot 2-D gel map in order to determine changes in the levels of these polypeptides upon light exposure. An image analyzer was used to analyze fluorographs of 2-D gels of total Euglena protein pulse-labeled with [35S]sulfate in the dark, immediately upon light exposure and 1, 4, 14, 24, 48 and 72 h after light exposure. The subset of polypeptides whose relative rate of synthesis changes more than threefold immediately upon light exposure was identified. The different patterns of changes in the rate of synthesis of this subset of polypeptides were followed.

A flow cytometric study of DNA staining in situ in exponentially growing and stationary Euglena gracilis.

DNA stainability by different fluorochromes has been compared in exponentially dividing and stationary Euglena cells. With the intercalating fluorochromes, ethidium bromide, acridine orange and DAPI, a decrease of fluorescence intensity of the G1 cells is observed when cells enter stationary stage. However this decrease of fluorescence is not obtained with the nonintercalating fluorochrome Hoechst 33258. If nuclear basic proteins are extracted, however, the intensity of staining by either Hoechst 33258 or ethidium-bromide is comparable in stationary and dividing cells. Therefore, the decrease of fluorescence intensity of the G1 cells observed during the transition from exponential to stationary phase is not due to a loss of DNA but is related to the exposure of chromatin binding sites for ethidium bromide. In Euglena cells, DNA accessibility for intercalating fluorochromes depends upon chromatin structure and consequently upon cell age.

DNA flow cytometry of control Euglena and cell cycle blockade of vitamin B12-starved cells.

Vitamin B12 starvation in Euglena induces a cell cycle arrest that leads to unbalanced growth. Microfluorometry and flow cytometry analyses of cellular DNA fluorescence after Hoechst 33258 staining were performed on control and vitamin B12-deficient cells. Convergent results are obtained with both methods. Histograms that represent arrested cells are unimodal, with a mode channel value nearly twice that of the G1 control cell peak. Dispersion of fluorescence values is great, and values from 2C and over 4C are observed and discussed. It appears that vitamin B12 starvation in Euglena leads to defective DNA synthesis. Blocked cells have different DNA content, corresponding to blockade of DNA replication during the S phase. A second block prevents the onset of mitosis even for 4C cells.

Occurrence of phosphatidylsulfocholine, the sulfonium analog of phosphatidylcholine in some diatoms and algae.

A survey of seven species of diatoms, one Euglena sp. and one dinoflagellate sp. for the presence of phosphatidylsulfocholine (PSC), the sulfonium analog of phosphatidylcholine (PC), was carried out using 1H-NMR spectroscopy and ammonia desorption chemical ionization mass spectrometry. PSC alone was found only in a non-photosynthetic diatom, Nitzschia alba. PSC, together with PC, was found in four of the diatoms (Nitzschia angularis, Cylindrotheca fusiformis, Phaeodactylum tricornutum and Navicula pelliculosa) in proportions of 6-24% of the total PC + PSC fraction, but little or no PSC (less than 2%) was detected in the remaining two (Cyclotella nana and Navicula incerta). Little or no PSC (less than 2%) was detected in a Euglena sp. by 1H-NMR but its presence was confirmed by 35S-labeling. The amount of PSC, if any, in the dinoflagellate (Amphidinium carterae) was below the level of detection by 1H-NMR.

Sterols of the cultured euglenid Eutreptia viridis: a novel delta 23-unsaturated sterol.

The sterol mixture isolated from the marine alga Eutreptia viridis consists mainly of delta 5,7-dienes which account for 80% of the free sterols. Eighteen different sterols were detected, including a novel sterol with the rare delta 23-unsaturation, viz. 24-ethylcholesta-5,7,23Z-trien-3 beta-o1.

The proteins of the cytoplasmic ribosomes of Euglena gracilis.

Two types of cytoplasmic ribosomes of Euglena gracilis have been found when the stability in vitro of the large subunit at 25 degrees C for 30 min has been considered. Sucrose density gradient analysis have revealed that the large subunit of ribosomes obtained from cells harvested in the stationary phase of growth was degraded when heated at 25 degrees C. The large subunit of ribosomes obtained from exponentially growing cells was stable in the same experimental conditions. Studies of the ribosomal RNAs have not explained this differential stability observed in vitro. In fact, after SDS-extraction, the largest RNA species always appears degraded in both types of ribosomes. Therefore, an explanation to this phenomenon have been searched for through the analysis of the ribosomal proteins. One and two dimensional gel electrophoresis techniques have been employed. At least one difference between the two ribosomal protein groups has been observed and this could be associated to the differential stability of the ribosomes. Approximately 72 proteins molecules have been found in Euglena's cytoplasmic ribosome. About 42 proteins belong to the large subunit and 30 to the small one.

Immunological quantitation of chloroplast ferredoxin.

An immunosassy for the quantitative determinations of ferredoxins in cell free extracts from plant tissues is described. The method is accurate for the assay of 0.3-1.5 nmol ferredoxin directly from the extracts. The following average values (nmol ferredoxin/mg extractable protein) were obtained: 3.9, 1.8. 5.90, 14.8 and 10.9 for Euglena gracilis, spinach, parsley, lettuce, and broccoli, respectively. Specific factors affecting the method are discussed in detail.

Growth in volume of Euglena gracilis during the division cycle.

The distribution of volumes of Euglena gracilis cells was measured conductimetrically. The volume spectrum of cultures in balanced growth was analyzed by the method of Collins and Richmond. The kinetics of volume increase of Euglena is neither linear nor exponential; the growth rate of small and large cells is low, but intermediate size cells show the largest growth rate.