Cytoskeletal structures inEuglena gracilis after triton X-100 extraction and dry cleaving

Summary A three-dimensional network of structural filaments was visible with common electron microscopes in the cytoplasm ofEuglena gracilis green cells extracted with buffers containing the nonionic detergent Triton X-100. A similar filamentous web was detected at the periphery of critical point dried cells cleaved on grids by means of an adhesive tape. SDS-polyacrylamide gel electrophoresis of the detergent-resistent cytoskeleton showed that actin or actin-like proteins of molecular weight in the range of 43–45 K are not among the components having a structural role inEuglena. The significance of these findings was discussed in relation to the capability of the alga to change the cell shape.The study was supported by grants from Consiglio Nazionale delle Ricerche (CNR) and Ministero della Pubblica Istruzione of Italy.     

Alterations induced by dimethyl sulfoxide inEuglena gracilis with emphasis on the side effects

Summary In a previous study, we demonstrated that 5% dimethyl sulfoxide (DMSO) alters the contractile system responsible for cell motility (euglenoid movements) and cytokinesis inEuglena gracilis. However, the nucleus continued to divide and most cells were larger than normal and binucleated.The present study reveals that DMSO, besides altering the cell functions requiring microfilaments, also affects other cell parts. More precisely, the materials normally covering the plasma membrane detach from it; the nucleus shows an irregular outline and aberrations in the nucleolus and chromosomes; the chloroplasts decompose the internal structures and, in a number of cells, transform into proplastid-like organelles. Also, the development of the proplastid into chloroplast in etiolated algae exposed to the light in the presence of DMSO is highly disturbed.These results show that DMSO has remarkable side effects like all the cytoskeletal poisons experimented up to now. An interpretation of the nuclear and chloroplast alterations is advanced.The study was supported by grants from Consiglio Nazionale delle Ricerche (CNR) and Ministero della Pubblica Istruzione of Italy.     

Pigments associated with the flagellum ofEuglena gracilis

The pigments associated with the flagellum of the phytoflagellateEuglena gracllis were characterized by HPLC. The pigment pattern of the wild-type strain was compared with a set of white mutants which did not display phototaxis and photoaccumulation in response to blue light. Flagella of the wild type contained FMN and FAD. Two mutants which lacked the stigma but retained a small paraxonemal body (PAB) contained less flavins. The whiteEuglena mutant FB, which retained a residual stigma and also a PAB, and the white phytoflagellateAstasia longa, a close relative ofEuglena, had normal amounts of flagellar flavins. Cells and flagella ofEuglena wild type contained an unldentified pterin-like pigment, called Pt₁₆, which was substantially reduced inAstasia and theEuglena mutants. A third pigment, designated P₅₂₈ with major absorption at 528 nm and fluorescence emission at 550 nm was present mainly in flagella. The association of the three pigment types with flagella and their respective alterations in the white strains indicates their possible role in photoreception. Dedicated to Pill-Soon Song on the occasion of his 60th birthday.     

The mechanism of growth inhibition by threonine inEuglena gracilis: Regulation of isoleucine-valine biosynthesis by 2-oxobutyrate

InEuglena gracilis the growth inhibition by threonine was accompanied by a rapid accumulation of isoleucine in the cells. Among threonine-catabolizing enzymes only threonine dehydratase was detected in high activity inEuglena, and 2-oxobutyrate, the dehydratase products of threonine, also inhibited as did threonine. Threonine dehydratase was located in the cytosol, and its activity was not affected by isoleucine and related amino acids. 2-Oxobutyrate strongly inhibited the synthesis of valine from pyruvate while augmented the synthesis of isoleucine in mitochondria.     

Structural alterations inEuglena gracilis exposed to the herbicide isopropyl-N-phenylcarbamate (IPC)

Summary Ultrastructural abnormalities of various kinds and severity appeared inEuglena gracilis green cells after a 48-hour exposure to 4 × 10^–4M of isoprophyl-N-phenylcarbamate (IPC), a widely used herbicide thought to affect microtubules and/or microtubule organizing centers in susceptible plant and animal species.A high proportion of cells contained nuclei in the G 2 phase of the cycle; in a significant percentage of organisms, however, structural aberrations of the nucleolus and chromosomes were evident. The pellicle outline, chloroplasts, mitochondria, and dictyosomes were also affected. The cytoplasm was rich in dense bodies which sometimes showed granular, fibrillar or tubular materials. Furthermore IPC partially inhibited flagellum regeneration after mechanical amputation.The mechanism by which IPC causes these responses remains unclear. Nevertheless, some indications suggest that the herbicide acts primarily on microtubule organizing centers. However, mitochondria, chloroplasts and nuclear constituents appear as possible additional targets of the compound.The study was supported by a grant from Italian Research Council (CNR), contract No. 80.00420.04.     

Indications about the mechanism of action and toxicity of netilmicin on the basis of the antiplastidial activity inEuglena gracilis

Summary The effects of the new antibiotic netilmicin (NT) were studied onEuglena gracilis green cells. It was found that, in the presence of the drug, chlorophyll synthesis was strongly inhibited and plastid structure dramatically altered as revealed by fluorescence and electron microscopic observations. Importantly, NT at low concentrations (10–20 g/ml) and for short periods of time (36–72 hours) induced a marked bleaching effect that was permanent and accompanied by the persistence in the colorless cells of poorly differentiated plastids. Other cell components were not influenced as shown by their appearance and also by the kinetics of growth and the cell viability. On the basis of the results and of the literature on the bleaching agents it is suggested that NT is an untoxic antibiotic which specifically inhibits protein synthesis at prokaryotic level.This work was supported by a grant from Italian Research Council (CNR), Contract N. 82.02016.04.     

Uptake and incorporation of pyrimidines in Euglena gracilis

In photoorganotrophically grown cells of Euglena gracilis the uptake and incorporation degree of 12 different pyrimidines were tested. The rate of uptake of pyrimidines has distinct maxima in the late log phase and in the stationary phase of cell multiplication. The kinetics of uptake are linear in the first 2 h, do not show saturation at various concentrations and increase with the concentrations. No accumulation of the pyrimidines at various concentrations could be observed in the first 2 h of incubation. Membrane inhibitors as uranyl acetate inhibit the uptake of the reference substance -AIB, which is wellknown transported by an active transport mechanism, but have no effect on uptake rate of uracil and cytosine. It could not be observed an energy requirement tested in temperature dependence and with electron transport inhibitors. Uptake of uridine, uracil, barbituric acid and -AIB is inhibited by cycloheximide in a different manner after 5–10 min.Abbreviations Barb barbituric acid - 5-BrU 5-bromouracil - Cyd cytidine - Cyt cytosine - DHU dihydrouracil - dUrd deoxyuridine - dThd thymidine - 5-FU 5-fluorouracil - Ora orotic acid - Thy thymidine - Ura uracil - Urd uridine - CHI cycloheximide - -AIB -aminoisobutyric acid Dedicated to Prof. Dr. Dr. h.c. mult. K. Mothes on the occasion of his 75th birthday     

The effect of cycloheximide on Euglena gracilis phenylalanyl-tRNA synthetases

The effect of cycloheximide on the chloroplastic, cytoplasmic and mitochondrial phenylalanyltransferRNA synthetases of Euglena gracilis was studied by growing both logarithmic and stationary phase cultures in the presence of the antibiotic. Enzyme activity was measured relative to untreated control cultures. At very low concentrations of cycloheximide (1 g/ml), all three log phase enzymes showed an increase in activity of 40–50%. At slightly higher concentrations (2.5 g/ml), the phenylalanyl-tRNA synthetase activities were comparable to those of the control cultures. At a cycloheximide concentration of 5g/ml the enzyme activities from stationary phase cultures showed only very slight decreases (5–20%). The cytoplasmic and mitochondrial enzymes behaved similarly in log phase cultures at this concentration. However, the chloroplastic phenylalanyl-tRNA synthetase from log phase cultures treated with 5g/ml cycloheximide showed a marked decrease in activity (70%). A further increase in antibiotic concentration to 10g/ml resulted in significant losses of activity of all three enzymes, from both growth stages. The implications of the data with regard to identification of the site(s) of chloroplast enzyme synthesis are discussed.     

A consideration of Euglena gracilis W3BUL as a cytoplasmic control for the wild-type phenylalanyl-tRNA synthetase system

The studies described indicate that the UV bleached mutant, Euglena gracilis W₃BUL does not serve as a suitable cytoplasmic control for the phenylalanyl-tRNA synthetase system. Chromatography of wild-type E. gracilis on Sephadex G100 revealed three peaks of activity identified as the chloroplastic, cytoplasmic and mitochondrial enzymes. The chloroplastic activity was greater in log than in stationary phase cells and was the only activity recovered from purified chloroplasts. Cell-free extracts of the achloroplastic mutant, E. gracilis W₃BUL, contained wild-type levels of the cytoplasmic and mitochondrial phenylalanyl-tRNA synthetases. However, no chloroplastic synthetase was detected in the mutant extracts. Anomalies in the aminoacylation behavior of the W₃BUL system were observed which suggest the possibility of a mutation affecting non-chloroplastic tRNAs in this UV-induced mutant. These anomalies significantly reduce the ability of the E. gracilis W₃BUL mutant to serve as a cytoplasmic control in the phenylalanyl-tRNA synthetase system.     

Effects of increased salinity on gravitaxis in Euglena gracilis

The unicellular freshwater flagellate Euglena gracilis regulates its position in the water column by means of phototactic and gravitactic behavior. Recent experiments have revealed that the cells switch between negative and positive gravitaxis depending upon environmental stimuli such as solar radiation. In this study, the effect of increased salinity on gravitaxis in Euglena gracilis was investigated. In some experiments it was found that salt concentrations up to 5 gL-1 (in some experiments 10 gL-1) increased the motility, velocity and precision of negative gravitactic orientation. Higher salt concentrations decreased all these parameters. At concentrations of about 15 gL-1, cells which did not become immobile, switched from negative to positive gravitaxis. Positive gravitaxis persisted for several hours or even days when the cells were transferred back to standard culture medium. Most of the cells in cultures exposed to salt concentrations above 20 gL-1 lost their motility (partial formation of palmella stages) but recovered when transferred back to standard medium or de-ionised water. Post recovery, the cells showed pronounced positive gravitaxis. Additional investigations on the pigmentation, revealed that the cells showed a complete loss of a carotenoid shoulder in the spectrum, which reappeared when the cells were brought back to standard medium.     

Transfer RNA genes of Euglena gracilis chloroplast DNA

Summary Transfer RNA genes have been mapped to at least nine different loci on the physical map of the Euglena gracilis chloroplast genome. One of these loci in the ribosomal RNA operons is present three times per genome. The DNA sequences of six of the nine different loci, containing 21 different tRNA genes, have been determined. Genes corresponding to the amino acids Ala, Arg, Asn, Cys, Gln, Gly (2), Glu, His, Ile, Leu (2), Met (2), Phe, Ser, Thr, Trp, Tyr, Val, and one unassigned species have been identified. All genes except one are found in clusters of 2–6 genes. None of the known genes contains introns, nor codes for the 3-CCA terminus. In addition to these genes, two pseudo tRNA genes are present in the rDNA leader region.     

Fatty acid content in wild type and WZSL mutant of Euglena gracilis

The effects of growth conditions on fatty acid profilewere examined in the photosynthetic wild type and inthe spontaneous non-photosynthetic WZSL mutant of theunicellular flagellate Euglena gracilis. Inthe light, the amount of polyunsaturated fatty acids(PUFAs) is higher in the wild type than in the mutant,independent of the carbon source. Among importantPUFAs, linolenic acid (18:3 3) is present inhigh amount only in wild type cells grown in the lightwith any of the tested carbon sources. The content ofother PUFAs, such as arachidonic acid (20:46), EPA (20:5 3) and DHA (22:63), is not correlated with the presence oflight or chloroplasts.The main effect of the dark in both strains is tolower the content of PUFAs and mono-unsaturated fattyacids and to increase the content of saturated fattyacids with all the carbon sources.     

Photosensory transduction in Euglena gracilis: Effect of some metabolic drugs on the photophobic response

We have investigated the effect of some metabolic drugs, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2,4-dinitrophenol (DNP), sodium azide (NaN₃), on the photobehavior of single cells of Euglena gracilis, in order to clarify the relevance of different metabolic pathways in the process of photoperception and sensory transduction in this alga. The results obtained show that the photophobic response of Euglena is not affected by the action of these drugs. This suggests that neither the photosynthetic process nor oxidative phosphorylation play a significant role in the phenomenon of photosensory transduction in Euglena.List of Abbreviations DNP 2,4-dinitrophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PSI Photosystem I - PSII Photosystem II     

Simulation of phototaxis in the flagellateEuglena gracilis

A three-dimensional model of the flagellateEuglena gracilis was developed to simulate phototaxis and movement in space. The simulation of the phototactic behavior was compared with thein vivo behavior in order to determine the mechanism of orientation with respect to light. Phototactic behavior with respect to one light source, can be explained by the shading hypothesis as well as by a dichroic orientation of the absorbing vectors of the photoreceptor pigments. In contrast, the behavior of the cells when exposed to two perpendicular light beams is not compatible with the shading hypothesis. Likewise, the phototactic orientation of stigmaless cells cannot be accounted for on the basis of the shading hypothesis. In contrast, simulations andin vivo observations of the behavior under polarized light strongly indicate the validity of the dichroic orientation of the photoreceptor pigments.     

Nucleotide sequence of cytoplasmic 5S ribosomal RNA fromEuglena gracilis

Summary The nucleotide sequence of cytoplasmic 5S ribosomal RNA fromEuglena gracilis has been determined to be: G- _A ^C -G-U-A-C-G-G-C-C-A-U-A-C-U-A-C-C-G-G-G-A-A-U-A-C-A-C-C-U-G-A-A-C-C-C-G--U-C-G-A-U-U-U-C-A-G-A-A-G-U-U-A-A-G-C-C-G-G-G-U-U-A-G-G-C-C-C-A-G-U-U-A-G-U-A-C-U-G-A-G-U-G-G-G-C-G-A-C-C-A-C-U-U-G-G-G-A-A-C-A-C-U-G-G-G-U-G-C-U-G-U-A-C-G-C-U-Up. This RNA is 119 nucleotides long and the sequence of a probable tRNA-binding site is GAUU (position 41–44 from the 5-terminus), which is the same as that of a trypanosoma species,Crithidia fasci-culata. TheEuglena 5S rRNA has a pseudouridine residue at position 38 and 3-terminus is phosphorylated. The 5S rRNA sequence ofEuglena resembles those of several other protozoa and higher animals rather than plants.On leave from Department of Zoology, Hiroshima University, Hiroshima, Japan     

Anti-u.v. activity of lignin biopolymers on Euglena gracilis

Sulphur-free lignin biopolymer and its oxidized and reduced derivatives have been prepared and their inhibitory activity against u.v.-induced mutagenesis in Euglena gracilis was evaluated. The structure- and dose-dependent anti-u.v. activity of lignins was observed at concentrations higher than 250gml^–1. The oxidized lignin showed the most antimutagenic activity, followed by the reduced lignin and the unmodified lignin had the least antimutagenic activity.     

Structure and rearrangements of rRNA genes in chloroplast DNA in two strains of Euglena gracilis

Summary The organisation of the rRNA genes in the chloroplast genomes of two strains of Euglena gracilis were analyzed and compared. It was previously shown that the bacillaris strain contains three complete rrn (rRNA) operons (7) and that the Z-S strain contains one operon (21). Using heteroduplex analysis it was found that the bacillaris strain contains, apart from the three complete rrn operons, an extra 16S rRNA gene, an extra partial 23S rRNA gene sequence and an inverted duplication of a stretch within the 5S–16S spacer. In addition a short (<100 bp) inverted repeat sequence (13) which forms a stem/loop structure in single-stranded cpDNA was located between the 3-end of the extra 16S rRNA gene and the partial 23 S rRNA sequence.The Z-S strain differs from the bacillaris strain by a deletion of two units of the complete rrn operons. The region upstream of the single complete rrn operon, including the inverted repeats, the partial 23S and the extra 16S rRNA sequences is identical with the bacillaris strain.The only non-homology found in heteroduplexes between the SalI fragments of B of the two strains is the deletion-insertion loop which represents the two rrn operons. A small deletion loop was found occasionally in hetero-and in homoduplexes of both strands in the region of variable size. Apart from the deletion/insertion of two rrn operons the two genomes appear to be colinear as can be seen from partial denaturation mapping. The organisation of the rRNA genes of the two strains is compared with those of the Z strain and the bacillaris-ATCC strain.     

Cytochromec oxidase ofEuglena gracilis: Purification,characterization, and identification of mitochondrially synthesized subunits

Cytochromec oxidase was purified from mitochondria ofEuglena gracilis and separated into 15 different polypeptide subunits by polyacrylamide gel electrophoresis. All 15 subunits copurify through various purification procedures, and the subunit composition of the isolated enzyme is identical to that of the immunoprecipitated one. Therefore, the 15 protein subunits represent integral components of theEuglena oxidase. In anin vitro protein-synthesizing system using isolated mitochondria, polypeptides 1–3 were radioactive labeled in the presence of [³⁵S]methionine. This further identifies these polypeptides with the three largest subunits of cytochromec oxidse encoded by mitochondrial DNA in other eukaryotic organisms. By subtraction, the other 12 subunits can be assigned to nuclear genes. The isolatedEuglena oxidase was highly active withEuglena cytochromec ₅₅₈ and has monophasic kinetics. Using horse cytochromec ₅₅₀ as a substrate, activity of the isolated oxidase was rather low. These findings correlate with the oxidase activity of mitochondrial membranes. Again, reactivity was low with cytochromec ₅₅₀ and 35-fold higher with theEuglena cytochromec ₅₅₈. The data show that the cytochromec oxidase of the protistEuglena is different from other eukaryotic cytochromec oxidases in number and size of subunits, and also with regard to kinetic properties and substrate specificity.Abbreviations kDa kilodalton - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TN turnover number     

Photosensory transduction in the flagellated alga,Euglena gracilis

Euglena were cultured under 3 W m^-2 constant white light. In culture medium, cells show immediate and long lasting step-down photophobic responses and photoaccumulation behavior to blue light if dim red light-adapted for 30 min. However, if cells are suspended in buffered, saltcontaining solutions (adaptation buffers), strong step-down photobehavior and photoaccumulation responses are not observed for several hours. These behaviors gradually increase in strength to reach a maximum after 6–12 h; after which a stable response is maintained. The relative rates of appearance and the relative strengths of the responses are influenced by the concentrations of Ca²⁺ and K⁺, but not H⁺ or Na⁺ ions, in the adaptation buffers. Expression of the stepdown photobehavior thus requires that the cells adapt to the chemical environment in which they are suspended.Abbreviations Hepes N-2-hydroxypiperazine-N-2-ethanesulfonic acid - Mes 2(N-morpholino)-ethanesulfonic acid - Pipes piperazine-N,N-bis (2-ethanesulfonic acid) - Taps tris(hydroxymethyl) methylaminopropanesulfonic acid This work was supported, in part, by grant No. PCM-79-05320 from the U.S. National Science Foundation to B.D.     

High-salt solutions prevent reactivation of euglenoid movement in detergent-treated cell models ofEuglena gracilis

Summary Detergent-treated cell models ofEuglena gracilis showed rounded-up movement of the cell body upon addition of ATP and Ca²⁺. Reactivation of the cell models was inhibited when the cell models had been treated with solutions containing >150 mM NaCl or >300 mM KC1. When the supernatant of salt-extracted cell models was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two distinctive bands of 120 and 160 kDa were found to be enriched. The cell membrane and associated cytoskeleton (pellicular complex) were isolated after treatment with salt solutions, and examined by electron microscopy to identify essential cortical structures required for reactivating rounding-up cell movement. Among three regularly arranged microtubules, only one and its associated structures were selectively dissolved from the pellicular strips, while the other pellicular elements remained intact. These structures were located in the groove region where sliding between strips is believed to occur during cell shape change. These results suggest a possible involvement of microtubule 2 and its associated bridges in active sliding between adjacent pellicular strips during euglenoid movement.