Hybridization studies with RNA and DNA isolated from Euglena gracilis chloroplasts and mitochondria

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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 lo6 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, endo R . HindII, endo R . HindIII, endo R . Hind11 +III, endo R . HpaI, endo R . HpaII and endo R . 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 endo R . 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 lo6 or 95% of the genome (92 x lo6).7. Chloroplast RNA hybridized to 5.1% with total chloroplast DNA, equal to three RNA cistrons per genome ( M , 92 x lo6). 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.

Section: Discussionmentioning

confidence: 99%

The Euglena gracilis Chloroplast Genome:

Kopecká

Crouse

Stutz

1977

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“…of 0.05 M Tris-HC1, pH 7.9, 0.01 M EDTA, 0.25 M sucrose. Cells were opened by grinding with glass beads [ l l ] and mitochondria separated by differential centrifugation and banding in Renographin [5].…”

Section: Isolation Of Mitochondriamentioning

confidence: 99%

“…Gel Filtration DNA was isolated from purified mitochondria as reported [5]. The crude DNA samples were treated with ribonuclease B (bovine pancreas, type VII, Sigma) and chromatographed through a methylated serum albumin -kieselguhr column.…”

Section: Dna Isolationmentioning

confidence: 99%

“…Manning et al [3] have found that Euglena mitochondrial DNA shows a very wide distribution of lengths ranging from 1 to 19 pm with a mean length of 1.3 pm; Nass and Ben-Shad [4], in contrast, found lengths of 0.6-0.9 pm; Crouse e f al. [5] have calculated from the hybridization plateau of mitochondrial rRNA and its molecular weight, a genome complexity of 40 . lo6, a value which agrees with the size of the longest molecules seen by Manning et al [3].…”

mentioning

confidence: 99%

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The Mitochondrial Genome of Euglena gracilis

Fonty

Bernardi

Crouse

et al. 1975

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Mitochondria1 DNA from Euglena gracilis has been investigated in its chemical and physical properties. Its G + C content is equal to 25 %; its buoyant density in a CsCl density gradient (1.690 g/ cm3) is higher, by 5 mg/cm3, than expected for a bacterial DNA having the same base composition. The buoyant densities of denatured and renatured DNA are higher than that of native DNA by 10-12 mg/cm3 and 6 mg/cm3, respectively. The melting temperature, T,, is 77 "C in standard saline citrate; the first derivative of the melting curve shows a striking multimodality. Degradation of the DNA by micrococcal nuclease indicates that about 40% of the DNA is formed by stretches lower than 10% in G + C. In all its properties the mitochondrial DNA from Euglena gracilis is strikingly similar to that of Saccharomyces cerevisiae.The information currently available on the mitochondrial genome of Euglena gracilis is rather limited. Since Ray and Hanawalt [I] and Edelman et al. [2] independently reported that Euglena mitochondria contained a DNA having a buoyant density in CsCl gradients of 1.690-1.691 g/cm3, only four papers have dealt with this subject. Manning et al. [3] have found that Euglena mitochondrial DNA shows a very wide distribution of lengths ranging from 1 to 19 pm with a mean length of 1.3 pm; Nass and Ben-Shad [4], in contrast, found lengths of 0.6-0.9 pm; Crouse e f al. [5] have calculated from the hybridization plateau of mitochondrial rRNA and its molecular weight, a genome complexity of 40 . lo6, a value which agrees with the size of the longest molecules seen by Manning et al. [3]. Finally Stutz and Bernardi [6] showed that Euglena mitochondrial DNA could be separated from nuclear DNA by chromatography on hydroxyapatite.In the present work, we have characterized in some of its chemical and physical properties the mitochondrial DNA from Euglena. In addition we have asked ourselves whether the mitochondrial genome of Euglena contains A +T-rich spacers. Such a question has been prompted by recent investigations on the organization of the mitochondrial genome of wildDefinition. A260 unit, the quantity of material contained in 1 ml of a solution which has an absorbance of 1 at 260 nm, when measured in a 1-cm path-length cell.Enzyme. Micrococcal nuclease (EC 3.1.4.7).type Saccharomyces cerevisiae cells [7] which have shown that this genome is made up, in equal parts, by "spacers" having a G + C content lower than 5 % and by "genes" having an average G + C content of 32 %.It was considered to be of interest to know whether the existence of spacers in mitochondrial DNA is unique to yeast, an organism which does not have an absolute requirement for functional mitochondria, or is of more general significance. In this case, spacers do not need to be of the A + T-rich type found in yeast.The methodology developed by Prune11 and Bernardi [7] for the analysis of A + T-rich spacers led us, however, to consider first mitochondrial DNAs having very low G + C levels and which were, therefore, good candidates for containing A + T-r...

“…The melting transition indicates that about 30% of the chloroplast DNA is lower than 22 "/, dG + dC (Fig.4 and Table 3) and that the remaining DNA is strongly heterogeneous in base composition (see also [27,28]. The minor component melting at 88 -89 "C can tentatively be assigned to ribosomal cistrons, since it corresponds to the expected dG + dC level (47%) and amount (10%).…”

Section: Sall Cleavagementioning

confidence: 99%

Compositional Heterogeneity of the Chloroplast DNAs from Euglena gracilis and Spinacia oleracea

Schmitt

Bohnert

Gordon

et al. 1981

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The chloroplast genomes of Euglena gracilis and Spinacia oleracea were investigated in their compositional heterogeneity, by using several different experimental approaches.Euglena chloroplast DNA has a dG +dC content of 28 %. Preparations averaging 20 x 1 O6 in molecular weight exhibit a gross heterogeneity in their elution profiles from hydroxyapatite and in their buoyant densities because the rRNA genes have a high rG + rC content. Finer analysis by melting, buoyant density of restriction fragments and micrococcal nuclease degradation have revealed an extended compositional heterogeneity. From micrococcal nuclease digestion data, approximately 30% of the chloroplast genome is as low as 12% in its dG + dC content, whereas 10% is higher than 60% dG + dC. Since the average dG + dC content of large restriction endonuclease fragments varied to a lesser extent, most of dA + dT-rich sequences must occur in short stretches interspersed with dG + dC-rich stretches.Spinach chloroplast DNA (dG + dC = 36.5%) did not exhibit any gross compositional heterogeneity in its hydroxyapatite elution or in its buoyant density profile. But the higher resolution methods of melting, buoyant densities of restriction fragments and micrococcal nuclease degradation revealed a high degree of heterogeneity which appears to be due to interspersion of short DNA stretches of different base composition. About 30%of genome is as low as 22 in dG + dC, while 10 % is higher than 60 % in dG + dC.Studies on the sequence organization of large mitochondrial genomes from a variety of unicellular organisms, including a number of yeasts, Euglena gracilis and Acanthamoeba castellanii have disclosed a marked compositional heterogeneity [l -41. In several cases, it has been possible to demonstrate that these genomes share not only a c-value paradox (namely the fact that they contain much more DNA than needed for coding purposes), and a strong compositional heterogeneity, but also the property of containing internally repetitive sequences. In the case of Saccharomyces cerevisiae, for example, the 'excess DNA' property is largely due to the presence of 'AT spacers' which contain repeated sequences of dA and dT [I]. At present, it is not known whether the sequence organization of the mitochondrial genomes just mentioned is of more general occurrence.In view of providing such an answer we have studied h'ere two chloroplast genomes, those of Euglena and of spinach. The chloroplast genomes investigated so far (see [5 -71 for recent reviews) consist of large circular units varying in contour length from 37 pm to 62 Fm. Physical maps are available for a variety of chloroplast DNAs, including those of Euglena and spinach [8 -141. Our choice was dictated in one case by the very low d G + d C content of the Euglena chloroplast genome, a property allowing easy comparisons with dA + dTrich mitochondrial genomes. In the other case, our choice was due to the fact that spinach chloroplast DNA is a similar to all other chloroplast DNAs from higher plants and has, in contras...