Etude spectrale à basse temperature chez Euglena gracilis Z en culture synchrone sur milieu lactate: Variation de teneur en cytochrome 556

Summary Low temperature spectra are described for whole Euglena cells. Euglena growing in synchronous culture with lactate medium show a cyclic variation of cytochrome 556 content during each cellular generation. The greatest quantity of cytochrome 556 seems to coincide with the non-dividing phase of the cells, the phase in which the mitochondrial network is observed. On the other hand after treatment of the Euglena with antimycin A, a correlation exists between the formation of giant mitochondria and an increase in the quantity of cytochrome 556.These results demonstrate the existence of a cyclic variation of cytochrome 556 synthesis in Euglena during synchronous growth on lactate medium.     

Cytological Characterization of a Giant Strain of Euglena gracilis Obtained from Dark-starved Cultures

Euglena gracilis green cells were dark-starved for four months. After this period almost the entire population died, while a few giant, viable cells appeared in the culture. The giantism was maintained after repeated subcultures in growth medium in light or dark conditions. However, the phenomenon was not permanent, and the morphological characteristics of the wild-type Euglena were gradually restored. In giant cells nuclei enlarged greatly, DNA content increased and the Golgi apparatus greatly proliferated. Chloroplasts and mitochondria increased in number and size and often presented structural modifications when compared with normal Euglena. Importantly, in the giant cells that were maintained in darkness in resting or growth conditions chloroplasts persisted as structured organelles which appeared red-fluorescent under UV illumination. Whether giantism is a phenotypic or a genotypic change is still debated. In our case, the evolution of this phenomenon, chiefly the enhanced DNA content, suggests that teratism is a multiploid mutation with the possibility of a return to the normoploid condition. Constitutive chloroplasts are present in most algae, except for a few species, among which is Euglena gracilis. The persistence of differentiated plastids in darkness in giant Euglena is considered to be a return to an ancestral condition and may, therefore, be phylogenetically important.     

Tubulin Genes in the Algal Protist Euglena gracilis

ABSTRACT Alpha- and beta-tubulin cDNA were selected from a Euglenaλgt11 expression library, recloned and either sequenced (α-tubulin cDNA) or hybridized to Euglena RNA and DNA (α- and β-tubulin cDNA). RNA for hybridization was extracted at 30 minute intervals after flagellar amputation and quantitated for cDNA binding. Unlike previous reports on most other flagellates, no net increase in either α- or β-tubulin RNA could be detected during regeneration—suggesting steady state or constitutive tubulin RNA synthesis. Incubation of the cDNA with genomic DNA after restriction digestion produced patterns of hybridization consistent with the presence of one to two kinds each of the α- and β-tubulin genes. The deduced amino acid sequence of the α-tubulin cDNA was more than 90% identical to the α-tubulins of Trypanosoma, Chlamydomonas, Naegleria, Tetrahymena and higher plants. The carboxy terminus of the α-tubulin cDNA and the previously sequenced β-tubulin of Euglena showed greatest identity to the carboxy terminus of the tubulins from Trypanosoma brucei. The sequence data for α and β-tubulins of Euglena provides direct evidence for the similarity of two gene products from euglenas and trypanosomes and adds support to earlier suggestions that these organisms are phylogenetically related.     

Monoxenic cultivation of the rotifer Brachionus calyciflorus in a defined medium

Summary Techniques are described for the initiation and maintenance of axenic cultures of Euglena gracilis strain Z and monoxenic cultures of Brachionus calyciflorus variety pala with the Euglena, using in both the same defined, buffered medium. The medium, which is inorganic—except for the citrate chelating agent, the buffer, and vitamins B₁ and B₁₂ — has been used for the axenic cultuvation of the Euglena for more than 13 months. The monoxenic Brachionus cultures, established by inoculating rotifers into Euglena cultures, have been maintained for more than 8 months. Contamination tests on the rotifer cultures were performed frequently in three different test media.Mictic females, males, and resting eggs of Brachionus were observed in the monoxenic cultures, and considerable variation in the length of the posterolateral spines was noted.The compatibility of a rotifer to a defined medium which sustains the axenic culture of its food organism is a feature of this system which is convenient, useful, and unique to date in synxenic rotifer culture work.Supported by National Science Foundation Grant No. GB 7717.     

Chemosensory Responses Toward Oxygen in Euglena gracilis*

SYNOPSIS Euglena gracilis strain Z, at a concentration of 10⁶ cells/ml and in containers of ∽ 0.1-mm thickness, spontaneously forms dynamic ring patterns in the dark. These patterns are modified differentially by illumination with red and with blue light. The red light effect is abolished by treatment with an inhibitor of photosynthesis. Pattern formation is apparently the result of chemophobic responses to oxygen dissolved in the medium. Euglena can respond to both negative and positive concentration gradients, depending upon the absolute magnitude of oxygen concentration. The photo- and chemosensory transduction systems of Euglena interact at a stage which precedes the overt expression of motor responses.     

Isolation, fractionation and template activity of the continuously-condensed chromatin of Euglena gracilis

A technique for isolating whole chromatin from nuclei of the lower eukaryote Euglena gracilis is presented. This chromatin, which appears under the electron microscope as uniformly condensed fibers, can, nevertheless, be subfractionated into distinct heterochromatic and euchromatic fractions. The euchromatin, comprising about 14% of the total DNA of the nucleus, contains over 80 % of the total endogenous RNA polymerase activity measured. The K_m for this enzyme is higher than that found for prokaryotes, but falls in the range found for other eukaryotes. Stability constants, calculated from cation-chromatin binding data, suggest that internal carboxyl groups of chromosomal proteins, at least, are involved in the condensation of Euglena chromatin. The relationship between Euglena chromatin and that of higher eukaryotes is discussed.     

Growth of Euglena gracilis on whey

Summary To extend the use of industrial wastes, we have studied the growth of Euglena cells on demineralized whey powder, an industrial dairy waste from cheese making. The demineralized whey powder was solubilized (15 g/l) in 0.04 N HCl and autoclaved for two hours at 120°C. The solution was then brought to pH 3.5 with NH₄OH and tested for its ability to support Euglena growth. In the dark, cell densities of 4.5 to 5.5×10⁶ cells/ml were obtained when vitamin B₁₂, thiamine and minerals were added to the hydrolyzed whey solution. Although growth of Euglena is possible on whey, the industrial application may be limited due to the need to hydrolyze the whey and to the low utilization of carbon (20%) as the glucose, but not the galactose, released during hydrolysis is used.     

Partial purification of a chloroplast DNA polymerase from Euglena gracilis

A single DNA polymerase has been purified 965 fold from isolated chloroplasts of $\text{Euglena}$$\text{gracilis}$ with a yield of 53%. The isolation methods include solubilization of the enzyme with 1M NaCl, ammonium sulfate precipitation, DNA affinity and DEAE-cellulose chromatography. The enzyme requires all four deoxynucleotide triphosphates, magnesium and denatured DNA for maximal activity. The chloroplast DNA polymerase is free of contaminating nucleases and phosphatases, has a sharp pH optimum at pH 7.2 and magnesium optimum of 6mM.     

Comparaison, à la lumière et à l'obscurité, des synthèses spécifiques d'ARN chez Euglena gracilis Z en cultures synchrones sur milieu lactate et étude du Chondriome

Summary Synchronization of Euglena gracilis (Z) on lactate medium is shown to be independent of illumination. The existence of a mitochondrial cycle in lightgrown as well as in dark-grown Euglena is demonstrated. When RNA synthesis is studied by pulse labeling with tritiated uracil in synchronously growing cells, a discontinuous RNA synthesis is found. Two peaks of preferential RNA synthesis in dark-grown cells and three peaks in light-grown cells are seen; the significance of the third peak of RNA synthesis in light-grown Euglena is discussed.     

Effects of Photosynthetic Intermediates on the Activation State of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from Euglena gracilis Z

The half-saturating concentration of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) from Euglena gracilis Z for CO₂ in its activation by CO₂ in the presence of a saturating concentration of MgCl₂ (KJ was measured by analyzing the partial reversible inactivation of the fully activated enzyme in the medium with dilute CO₂. The K_d of the Euglena enzyme was 12.5 μm. The K,_d values were 6.3/im for the enzyme from soybean, 10.8 fiM from maize, 23.3 jiM from Scenedesmus obliquus, and 20.8 μm from Anabaena 7120. The activated state of Euglena RuBisCO was stabilized by 6-phosphogluconate, fructose 1,6-bisphosphate, and 3-phosphoglycerate in the medium containing low concentrations of CO₂. Both fructose 6-phosphate and ATP stimulated inactivation in the medium. NADPH not only stabilized the activated state of the enzyme, but also enhanced the enzyme activity over the full activity measured in the absence of NADPH. NADP⁺ did not nullify the effects of NADPH on the activation at all. The physiological significance of the effects of these photosynthetic metabolites on the activated state of Euglena RuBisCO is discussed.     

Comparative role of polyamines division and plastid differentiation of Euglena gracilis

Regulation of polyamine biosynthesis during growth and differentation of Euglena gracilis was investigated. Increased activity of l-ornithine decarboxylase (EC 4.1.1.17), the enzyme which catalyzes the initial step in polyamine synthesis in Euglena, and accumulation of polyamines were observed prior to DNA replication in synchronous cultures of heterotropically or photoautotrophically grown cells. In photoatotrophic cells three maxima of polyamine synthesis were observed during the light period of the cell cycle. The transition from quiescence of active growth was accompanied in heterotrophic Euglena by a very large stimulation of ornithine decaboxylase activity and polyamine synthesis; the decrease in growth potential of these cells was correlated with a decrease in polyamine levels. In contrast, differentiation of Euglena, i.e., a shift from heterotrophic to photoautotrophic mode of living in the absence of division, led only to a minor stimulation of polyamine biosynthesis. α-Methylornithine, an inhibitor of ornithine decarboxylase, blocked the growth of heterotrophic Euglena, and depletion of intracellular polyamines decreased the differentiation rate. Both events could be reversed only by addition of putrescine to the growth medium. This study suggests that Euglena requires a minimal intracellular level of polyamines to grow and differentiate under optimal conditions. This requirement seems to be more stringent for cell division.     

Chloroplast ribosomal RNA genes in the chloroplast DNA of Euglena gracilis

Euglena chloroplast DNA has a buoyant density in CsCI of 1.686. Shearing this DNA produces a satellite band at density 1.700. The satellite, easily lost during preparative CsCI gradient centrifugation of chloroplast DNA, contains the genes for chloroplast ribosomal RNA. Pure Euglena chloroplast DNA is shown to contain one set of ribosomal RNA genes for each 90 × 10⁶ daltons of DNA.     

The presequence of the precursor to the nucleus-encoded 30 kDa protein of photosystem II in Euglena gracilis Z includes two hydrophobic domains

A cDNA clone for the extrinsic 30 kDa protein (OEC30) of photosystem II in Euglena gracilis Z was isolated and characterized. The open reading frame of the cDNA encoded a polypeptide of 338 amino acids, which consisted of a long presequence of 93 amino acids and a mature polypeptide of 245 amino acids. Two hydrophobic domains were identified in the presequence, in contrast to the presence of a single hydrophobic domain in the presequence of the corresponding proteins from higher plants. At the N- and C-terminal regions, respectively, of the presequence, a signal-peptide-like sequence and a thylakoid-transfer domain were identified. The presence of a long and unique presequence in the precursor to OEC30 is probably related to the complexity of the intracellular processes required for the synthesis and/or transport of the protein in Euglena.Abbreviations ER endoplasmic reticulum - cDNA complementary DNA - SSU small subunit; Rubisco, ribulose 1,5-bisphosphate carboxylase/oxygenase - Rubico, ribulose 1,5 bisphosphate carboxylase/oxygenase - LHC II light-harvesting chlorophyll protein of photosystem II - PS II photosystem II - OEC30 the extrinsic 30 kDa protein of photosystem II in Euglena - PCR polymerase chain reaction - SDS sodium dodecyl sulfate - TE a solution containing 10 mM Tris-HCl and 1 mM EDTA pH 8.0 - SSPE a solution containing 0.15 M NaCl, 10 mM NaH₂PO₄ and 1 mM EDTA pH 7.4 - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - PVDF poly(vinylidene difluoride)     

Analysis of the RNA of Euglena gracilis on polyacrylamide gels

Summary Five peaks of RNA from bleached Euglena gracilis are resolved by polyacrylamide-gel electrophoresis. The extraction of these RNA's and their subsequent resolution on gels is dependent upon pH and the presence of an RNase inhibitor (e.g., macaloid). Careful control of ionic strength also appears necessary. Inorganic phosphate is incorporated first by low molecular weight RNA, then by a high molecular weight RNA (hRNA) and a peak with a sedimentation coefficient of 13S, and then by rRNA.The electrophoretic pattern of RNA from Astasia longa is similar to that of bleached Euglena whereas that from wild-type Euglena is more complex and presumably reflects the presence of chloroplast RNA's in these latter cells.     

Purification and Some Properties of Extracellular Protease of Euglena gracilis Z

The extracellular protease of Euglena gracilis z was purified to a single protein. It was an endopeptidase as found by the Nunokawa’s method, and showed optimum pH for the proteinase, esterase and amidase activities at 7.3, 7.0 and 6.3, respectively. It had a molecular weight of 41,000 and isoelectric point of 8.3. The bleached mutant of E. gracilis produced higher activity of extracellular protease than the wild strain, and supplementation of peptone to the growth medium augmented the enzyme production in both green and bleached cells. The Euglena extracellular protease was markedly inhibited by diisopropylfluorophosphate and Streptomyces subtilicin inhibitor, and to lesser extents by EDTA and p-chloromercuribenzoate. The enzyme was potentiated by some sulfhydryl compounds, activated greatly by Fe²⁺ and stabilized by Ca²⁺ and K⁺.     

INTRON REVEALED BY NUCLEOTIDE SEQUENCE OF LARGE SUBUNIT OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE FROM CODIUM FRAGILE (CHLOROPHYTA): PHYLOGENETIC ANALYSIS1

The coding sequence for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) from Codium fragile (Suringar) Hariot chloroplast DNA is 1428 bp in length and contains a 1813-bp group II intron. The only other organisms in which introns have been found in the rbcL gene are Euglena and Astasia. The Codium intron likely had a separate origin from the Euglena and Astasia introns, based on comparisons of intron sizes and sequences. Phylogenetic analyses of rbcL nucleotide and amino acid sequences place Codium between Chlorella and two Chlamydomonas spp., indicating that the Chlorophyceae may be polyphyletic.     

Precursor of the nuclear-encoded extrinsic 30 kDa protein in photosystem II of Euglena gracilis Z is not a polyprotein

Polyprotein-type precursors have been reported for the nuclear-encoded proteins such as the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and the apoproteins of light-harvesting chlorophyll-protein (LHC) in Euglena. We report here that the precursor of the extrinsic 30 kDa protein of photosystem II (PS II) encoded by nuclear DNA is not a polyprotein. The precursor was identified as a 45 kDa protein by immunoprecipitation of in vitro translation products of mRNA and by a pulse-chase experiment. It is probable that the structure of the precursor of the nuclear-encoded protein in Euglena chloroplast is closely related to the feature of assembly, as well as of transport, of the protein in chloroplast.     

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     

PURIFICATION AND PROPERTIES OF THE MUCUS OF EUGLENA GRACILIS (EUGLENOPHYCEAE)1

Anionic groups were demonstrated in the mucus of Euglena gracilis Klebs var. bacillaris Cori by histochemical staining with alcian blue or diaminobenzidine tetrahydrochloride and were quantified during the growth cycle with an alcian blue dye-binding assay. Mucus in the culture increased during growth and became high when the culture entered the stationary phase. Cultures were grown under conditions which uniformly labeled all sulfur containing compounds with ³⁵S. A purification scheme was devised using 0.15 M NaCl and 0.10 M EDTA at pH 8 (Marmur's solution) to separate the mucus from the cells without cell breakage. The isolated purified mucus was fractionated with sodium dodecyl sulfate (SDS) at 100° C into soluble and insoluble components. The soluble fraction was separated by SDS polyacrylamide gel electrophoresis into 18 polypeptide bands ranging from 22 to 320 kdaltons that stained with Coomassie blue; 16 of these bands also stained for carbohydrates using periodic acid-Schiffs reagent, indicating their glycoprotein nature. On hydrolysis, the SDS soluble fraction yielded xylose, fucose, rhamnose, and hexose. The SDS insoluble fraction contained no ³⁵S label, and, therefore, presumably no protein or bound sulfate; this gelatinous material does not contain the same sugar residues as the glycoproteins in the SDS soluble fraction. Its staining properties with alcian blue and its resistance to hydrolysis suggested the presence of uronic acids. Comparison with other Euglena fractions showed that bands comigrating with the mucus glycoproteins were not detectable in the fractions containing the whole cells or the culture medium. Although the mucus of Euglena yielded appreciable sulfate during mild acid treatment, most if not all of this sulfate appears to have come from the oxidation of reduced sulfur rather than from the hydrolysis of covalently bound sulfate. An infrared spectrum of the mucus showed only minor peaks in the correct regions for the S-O linkage. Thus, the mucus of Euglena is composed of glycoproteins and polysaccharides which contain little or no ester sulfate.