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.     

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.     

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.     

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.     

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.     

DNA polymerases of Euglena gracilis: heterogeneity of molecular weight and subunit structure.

Sedimentation analysis of glycerol-density gradients has shown that freshly purified DNA polymerases A and B (pol A and pol B) of Euglena gracilis have molecular weights of 185,000 (8.7S) and 240,000 (10.3S) respectively. They can aggregate in fresh preparations to give forms of higher molecular weight as shown by gel filtration through Sepharose 6B, but on ageing pol B progressively generates species with sedimentation coefficients of 7.4-7.7S, 6.3-6.5S, 4.8S and finally 3.0S. Pol A apparently behaves in a similar fashion though it is unstable. Exposure of pol A and pol B to high ionic strengths can also cause their breakdown to species with lower sedimentation coefficients. The mitochondrial DNA polymerase is distinct, having a molecular weight of 170,000. It is proposed that pol A and pol B are oligomers of the 3.0S subunit and possibly other dissimilar subunits, with pol B having additional factors conferring upon it its extra catalytic functions.     

Isolation of Euglena gracilis chloroplast 5S ribosomal RNA and mapping the 5S rRNA gene on chloroplast DNA.

Ribosomal RNA (5S) from Euglena gracilis chloroplasts was isolated by preparative electrophoresis, labeled in vitro with 125I, and hybridized to restriction nuclease fragments from chloroplast DNA or cloned chloroplast DNA segments. Euglena chloroplast 5S rRNA is encoded in the chloroplast genome. The coding region of 5S rRNA has been positioned within the 5.6 kilobase pair (kbp) repeat which also codes for 16S and 23S rRNA. There are three 5S rRNA genes on the 130-kbp genome. The order of RNAs within a single repeat is 16S-23S-5S. The organization and size of the Euglena chloroplast ribosomal repeat is very similar to the ribosomal RNA operons of Escherichia coli.     

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     

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.     

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).     

Cellular content of chloroplast DNA and chloroplast ribosomal RNA genes in Euglena gracilis during chloroplast development.

The cellular content of chloroplast DNA in Euglena gracilis has been quantitatively determined. DNA was extracted from Euglena cells at various stages of chloroplast development and renatured in the presence of trace amounts of 3H-labeled chloroplast DNA. From the kinetics of renaturation of the 3H-labeled chloroplast DNA, compared with the kinetics of renaturation of excess nonradioactive chloroplast DNA, the fraction of cellular DNA represented by chloroplast DNA was calculated. The content of chloroplast DNA was found to increase from 4.9 to 14.6% of cellular DNA during light-induced chloroplast development. Correcting for the change in DNA mass per cell, the number of copies of chloroplast DNA is found to vary from 1400 to 2900 per cell. During this developmental transition, the cellular content of the chloroplast ribosomal RNA genes varies from 1900 to 5200 copies per cell. The ratio of the number of copies of rRNA genes to chloroplast genomes per cell remains in the range of 1-2 throughout chloroplast development, ruling out selective amplification of chloroplast rRNA genes as a means of regulation of rRNA gene expression. Direct measurement of the number of rRNA cistrons per 9.2 X 10(7) dalton genome yields a value of 1 or 2.     

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.     

Isolation of covalently closed circular DNA of high molecular weight from bacteria.

A simple method is described in detail for the efficient isolation of high molecular weight covalently closed circular DNA (ccc-DNA) from Agrobacterium. Although this method was developed for isolating ccc-DNA of molecular weights greater than 10⁸ daltons in Agrobacterium, the technique also proves to be useful in isolating ccc-DNA of varying sizes from a variety of other bacteria. The technique involves the shearing and alkali denaturation of the chromosomal DNA, followed by the preferential removal of the single-stranded DNA by phenol extraction. The DNA which remains is largely ccc-DNA. The DNA is then concentrated, and the ccc-DNA is separated from the chromosomal DNA by centrifugation in a cesium chloride-ethidium bromide density gradient. By this technique, ccc-DNA of varying sizes has also been isolated from species of Escherichia, Rhizobium, Citrobacter, and Lactobacillus.