The chloroplast DNA of a wild type photosynthetic variant of Euglena gracifs (ATCC n° 10616) with five ribosomal cistrons has been analyzed by restriction mapping. The results complete the electron microscope study of Koller and Delius (MGG 188, 305, 1982); they support a model of formation of the variant DNA by rearrangement of the wild type ribosomal cistrons through unequal crossing-over. The recombination sites have been determined. The recombination model proposed also explains the formation of the "Z-S" variant with a single ribosomal cistron (Wurtz and Buetow 1981).

Chloroplast DNA was isolated from total cellular DNA of a bleached mutant of Euglena gracilis (Y3BUD) by enrichment of the light component (p = 1.686) by repeated CsCl equilibrium centrifugations. Electron microscope visualization of this DNA showed minicircular DNA molecules in addition to large circular molecules (42 pm) identical to wild type chloroplast DNA. They were heterogenous in size and their contour lengths ranged from 0.8 to 8.5 μm. Fractionnation by agarose gel electrophoresis gave several discrete bands. Some of them hybridized with pure chloroplast DNA and with several cloned chloroplast DNA fragments, particularly to ribosomal fragments, while others did not show homology with chloroplast DNA being probably of extrachloroplatic origin.

The evolution of chloroplast DNA was analysed during streptomycin induced mutagenesis in Euglena gracilis strain bacillaris and strain Z. In addition to a massive reduction of the cellular level of chloroplast DNA, several structural modifications have been observed in early stages of mutagenesis but they are generally eliminated during the later stages. The ribosomal cistrons are regularly rearranged: two of the three tandemly arranged cistrons occuring in wild type chloroplast DNA decrease while the third one is relatively more conserved and amplified during mutagenesis and in bleached mutants.

The level of chloroplast DNA has been estimated in bleached mutants of Euglena by the increase of the renaturation rate of a radioactive chloroplast DNA probe in response to the addition of total mutant DNA.

Ultraviolet-irradiation of Euglena gracilis drastically increases the proportion of green colonies resistant to chloroplast-inhibiting antibiotics. Some green colonies persist at high UV doses. These results might indicate the occurrence in the chloroplastic DNA of repair processes, of which one could be error-prone.

Studies of nuclear and chloroplastic-DNA repair after ultraviolet irradiation of Euglena gracilis show that photoreactivation is very efficient at both the nuclear and chloroplastic level. Liquid-holding or split-dose experiments and treatment with caffeine reveal, furthermore, that dark-repair is very efficient in nuclear DNA but not in chloroplastic DNA (ctDNA). The possibility of a chloroplastic dark-repair of restricted efficiency is discussed.

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.

During multiplication of irradiated cells, a segregation may take place between bleached cells, whose progeny is unable to green, and green ones. Some of the green cells give progenies exclusively made of green cells; the progeny of others is partly composed of bleached cells.

1.1. Addition to greening Euglena of levulinic acid, a competitive inhibitor of δ-aminolevulinate dehydratase, temporarily stops chlorophyll synthesis and produces accumulation of δ-aminolevulinate. The amount of δ-aminolevulinate accumulated 30 min after inhibitor is stoichiometrically equivalent (within ± 10%) to the chlorophyll produced in control cells during the same time.2.2. In the presence of inhibitor, a small quantity of δ-aminolevulinate is produced by etiolated Euglena during growth in the dark. In greening Euglena returned to dark, the synthesis of δ-aminolevulinate is progressively abolished after 20 min. Thus, the synthesis of δ-aminolevulinate in Euglena shows an indirect dependence towards light.3.3. The observations lead to the recognition of at least three ways of control for the synthesis of δ-aminolevulinate and its precursors: one way is related to cellular growth in the dark, the second depends on active photosynthesis and may be blocked by 3(3,4-dichlorphenyl)-1,1-dimethylurea (DCMU), and the last, unimpaired by DCMU, depends on light but not on photosynthesis. This last way is effective at the beginning of greening, and is turned off before the end of the process. Besides, δ-aminolevulinate synthesis can be limited by factors related with the abundance and mobility of cellular reserves.4.4. Addition of δ-aminolevulinate does not increase the production of chlorophyll, except during the terminal phase of greening, when cellular reserves are nearly consumed. Thus, the synthesis of chlorophyll pigments seems to be regulated either by a limitation of δ-aminolevulinate precursors or by a limitation in the last stages of the synthesis, according to the stage of greening. In the latter case, a feedback control limits δ-aminovulinate production.

1. 1. Addition to greening Euglena of levulinic acid, a competitive inhibitor of δ-aminolevulinate dehydratase, temporarily stops chlorophyll synthesis and produces accumulation of δ-aminolevulinate. The amount of δ-aminolevulinate accumulated 30 min after inhibitor is stoichiometrically equivalent (within ± 10%) to the chlorophyll produced in control cells during the same time. 2. 2. In the presence of inhibitor, a small quantity of δ-aminolevulinate is produced by etiolated Euglena during growth in the dark. In greening Euglena returned to dark, the synthesis of δ-aminolevulinate is progressively abolished after 20 min. Thus, the synthesis of δ-aminolevulinate in Euglena shows an indirect dependence towards light. 3. 3. The observations lead to the recognition of at least three ways of control for the synthesis of δ-aminolevulinate and its precursors: one way is related to cellular growth in the dark, the second depends on active photosynthesis and may be blocked by 3(3,4-dichlorphenyl)-1,1-dimethylurea (DCMU), and the last, unimpaired by DCMU, depends on light but not on photosynthesis. This last way is effective at the beginning of greening, and is turned off before the end of the process. Besides, δ-aminolevulinate synthesis can be limited by factors related with the abundance and mobility of cellular reserves. 4. 4. Addition of δ-aminolevulinate does not increase the production of chlorophyll, except during the terminal phase of greening, when cellular reserves are nearly consumed. Thus, the synthesis of chlorophyll pigments seems to be regulated either by a limitation of δ-aminolevulinate precursors or by a limitation in the last stages of the synthesis, according to the stage of greening. In the latter case, a feedback control limits δ-aminovulinate production.

1.1. Illumination of dark-grown Euglena results in a rapid formation of polysomes, even in conditions limiting rRNA synthesis.2.2. After return to dark of dark-grown cultures submitted to limited illumination, kinetic analysis of polysome degradation suggests the existence of two polysome classes: “photoactive” polysomes which undergo rapid degradation and “photoinduced” polysomes which are synthesized for some time in the dark. In the light of results obtained in different conditions, possible cellular activities of these polysomes are discussed.3.3. Polysome formation seems to result from synthesis of different mRNAs. Photoactive polysomes could correspond to continuously light-dependent mRNA synthesis.