Complete sequence of Euglena gracilis chloroplast DNA.

We report the complete DNA sequence of the Euglena gracilis, Pringsheim strain Z chloroplast genome. This circular DNA is 143,170 bp, counting only one copy of a 54 bp tandem repeat sequence that is present in variable copy number within a single culture. The overall organization of the genome involves a tandem array of three complete and one partial ribosomal RNA operons, and a large single copy region. There are genes for the 16S, 5S, and 23S rRNAs of the 70S chloroplast ribosomes, 27 different tRNA species, 21 ribosomal proteins plus the gene for elongation factor EF-Tu, three RNA polymerase subunits, and 27 known photosynthesis-related polypeptides. Several putative genes of unknown function have also been identified, including five within large introns, and five with amino acid sequence similarity to genes in other organisms. This genome contains at least 149 introns. There are 72 individual group II introns, 46 individual group III introns, 10 group II introns and 18 group III introns that are components of twintrons (introns-within-introns), and three additional introns suspected to be twintrons composed of multiple group II and/or group III introns, but not yet characterized. At least 54,804 bp, or 38.3% of the total DNA content is represented by introns.     

Dark-induced chloroplast dedifferentiation in Euglena gracilis

Transfer of light-grown autotrophic Euglena gracilis cells to darkness and carbon (glucose) containing heterotrophic media causes structural and functional decomposition of the photosynthetic apparatus. The process can be ascribed to a strict diluting-out mechanism of stroma constituents among the progeny, as shown for ribulose-1,5-bisphosphate carboxylase (RuBPCase, EC 4.1.1.39), and aminoacyl-tRNA synthetases (Aa-RS; especially Leu-RS, EC 6.1.1.4) activities. The diluting-out effect of thylakoid membranes and chlorophyll seems to be superimposed by additional degradations, beginning soon after the transfer of cells to darkness. Cultivation of cells in darkness in 0.03 M KCl or without utilizable organic carbon (resting media) preserves chloroplast structure and function over a long period, indicating negligible turnover in these cells. Thus, under both growing and resting conditions, darkness induces the arrest of synthesis of plastid constituents. Experiments with the inhibitors cycloheximide, chloramphenicol, and nalidixic acid demonstrate that chloroplast dedifferentiation does not require organelle gene expression, but it is more strictly dependent on biosynthetic events in the nucleo-cytoplasmic compartment than the reverse process, light-induced chloroplast formation. Since cycloheximide at low concentrations in growth medium causes a marked suppression of precursor uptake or re-utilization similar to that in cells of resting media, intracellular precursor deficiency is suggested to control the observed blockade in cytoplasmic synthesis of plastid proteins. On the other hand, darkness might signalize the stop of gene expression in the organelles.Abbreviations Aa aminoacid - CH cycloheximide - CM chloramphenicol - Leu-RS leucyl-tRNA synthetase - RuBP ribulose-1,5-bisphosphate - TCA trichloroacetic acid     

Base composition heterogeneity of Euglena gracilis chloroplast DNA.

Euglena gracilis chloroplast DNA has an average buoyant density of 1.685 gm/cm3, corresponding to 25 mol% G . C base pairs. To test for base compositional heterogeneity within this 130 kilobase pairs (kbp) genome, previously mapped restriction endonuclease fragments were isolated, and characterized by equilibrium buoyant density centrifugation. The chloroplast DNA can be characterized as containing two major buoyant density components. A segment of 17 kbp, representing 13% of the genome and containing the rRNA genes is 43--44 mol% G . C. The remaining 113 kbp, accounting for 87% of the genome, has an average 20--21 mol% G . C content.     

Maturation of chloroplast rRNA in Euglena gracilis

RNA and protein synthesis in the myocardium were stimulated after a short preincubation period with calcium. This elevation of macromolecular synthesis persisted in the absence of the ion for at least four hours. It appears that the uptake and/or the concentration of intracellular calcium induced a persistent and optimum enhancement of RNA and protein synthesis.     

Restriction endonuclease map of Euglena gracilis chloroplast DNA.

A physical map of the Euglena gracilis chloroplast genome has been constructed, based on cleavage sites of Euglena gracilis chloroplast DNA treated with bacterial restriction endonucleases. Covalently close, circular chloroplast DNA is cleaved by restriction endonuclease SalI into three fragments and by restriction endonuclease BamHI into six fragments. These nine cleavage sites have been ordered by fragment molecular weight analysis, double digestions, partial digestions, and by digestion studies of isolated DNA fragments. A fragment pattern of the products of EcoRI restriction endonuclease digestion of Euglena chloroplast DNA is also described. One of these fragments has been located on the cleavage site map.     

Purification of chloroplast DNA from Euglena gracilis

A procedure is described for purification of chloroplast DNA from Euglena gracilis. This procedure differs from previous procedures in utilization of differential detergent extraction. Deoxycholate preferentially solubilizes most of the cellular DNA but leaves the chloroplast DNA bound to particulate material. Final purification of the chloroplast DNA is accomplished by equilibrium density gradient ultracentrifugation.     

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.     

Phenolic acids inhibit chloroplast mutagenesis in Euglena gracilis

The mutagenicity (bleaching activity) of ofloxacin (43 microM) and acridine orange (AO) (13.5 microM) in Euglena gracilis is inhibited by plant phenolics. Caffeic acid (CA), p-coumaric acid (PCA), ferulic acid (FA) and gentisic acid (GA) (25, 50, 100 and 250 microM) exhibited a significant concentration-dependent inhibitory effect against ofloxacin-induced mutagenicity, which was very effectively eliminated by the highest concentration of all four of those phenolic acids. The mutagenicity of AO was also significantly reduced in the presence of CA, PCA and FA (25, 50, 100 and 250 microM). However, GA exhibited no significant activity, even at the concentration of 250 microM. Based on the UV spectrophotometric measurements, we suggest that the antimutagenic effect of CA, PCA, FA and GA resulted from the scavenging of reactive oxygen species (ROS) produced by ofloxacin. On the other hand, the reduction of AO-induced mutagenicity correlates with the binding capabilities of CA, PCA and FA, with the exception of GA.     

Euglena gracilis chloroplast elongation factor Tu. Purification and initial characterization

The chloroplast protein synthesis elongation factor Tu (EF-Tuchl) has been purified to near homogeneity from Euglena gracilis. Chromatography of the postribosomal supernatant of light-induced Euglena on DEAE-Sephadex reveals two forms of EF-Tuchl. Further purification has shown that one species consists of a complex between EF-Tuchl and a factor that stimulates its activity. The other species consists of free EF-TUchl. The factor has been purified from both chromatographic forms by taking advantage of the molecular weight shift that occurs upon disruption of the complex between EF-Tuchl and the stimulatory factor. EF-Tuchl consists of a single polypeptide chain with a molecular weight of about 50,000. EF-Tuchl is as active on Escherichia coli ribosomes as it is on its homologous ribosomes but displays no detectable activity on eukaryotic cytoplasmic ribosomes. It is stimulated in polymerization by E. coli EF-Ts and will form a complex with the prokaryotic factor that can be isolated by gel filtration chromatography. Like E. coli EF-Tu, it is sensitive to modification by N-ethylmaleimide and is inhibited by the antibiotic kirromycin. Thus, the chloroplast factor has many features that reflect the close relationship between prokaryotic and chloroplast translational systems.     

Physical mapping of the ribosomal DNA region of Euglena gracilis chloroplast DNA.

1. The relative positions of endo R . EcoRI and endo R . Bg/II cleavage sites are mapped within the linked DNA fragments Bam-E-E-D of the Euglena gracilis chloroplast DNA. 2. The DNA segment Bam-E-E-D contains three contiguous repeated segments of approximately 5600 base pairs. 3. Each repeated segment can code for an rRNA gene (16-S and 23-S).     

The Euglena gracilis chloroplast genome: analysis by restriction enzymes.

1. Chloroplast DNA was isolated from autotrophically and mixotrophically grown Euglena gracilis cells. 2. Aliquots of chloroplast DNA were mechanically degraded to an average molecular weight of 4-7 X 10(6) and G+C-rich DNA fragments (density 1.701 g/cm3) were separated from the bulk DNA (density 1.685 g/cm3) using preparative CsCl density gradients. 3. Total chloroplast DNA and its DNA subfractions, which first were characterized with respect to average G+C content and hybridization capacity for chloroplast rRNA, were hydrolysed with restriction endonucleases (endo R-EcoRI, end R-HindII, endoR-HindIII, endo R-HindII+III, endoR-Hpal, endo R-HpaII and endoR-HaeIII). The fragments were separated on gels under a variety of electrophoretic conditions. 4. With each enzyme tested, a rather large number of bands was obtained. In all cases, different banding patterns were obtained for total DNA, and the DNA subfractions. 5. Chloroplast DNA from autotrophically and mixotrophically grown cells gave identical banding patterns. 6. Digestion of total DNA with the endoR-HaeIII yielded 51-52 fragments separated in the gels in a total of 36 bands of which 11-12 bands were composed of 2-3 fragments as estimated by densitometry. The molecular weights of all fragments combined was 87 X 10(6) or 95% of the genome (92 X 10(6)). 7. Chloroplast RNA hybridized to 5.1% with total chloroplast DNA, equal to three RNA cistrons per genome (Mr92 X 10(6)). These cistrons are located on seven different types of endo R-HaeIII fragments. The hybridising fragments are preferentially found in the G+C-rich subfraction and in bands which are composed of 2-3 fragments.     

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.