Conformation of chloroplast DNA in wheat leaves - Semantic Scholar

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plasts were previously treated with DNase (deoxyribonuclease), but the tertiary configuration was some- what disorganized by RNase (ribonuclease).
Conformation of chloroplast DNA in wheat leaves REGISMAC HE^

AND

E. R. WAYGOOD

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Department of Botany, University of Manitoba, Winnipeg, Manitoba Received August 22, 1969 MACHE,R., and E. R. WAYGOOD. 1970. Conformation of chloroplast DNA in wheat leaves. Can. J. Bot. 48: 173-175. Sedimentation and electron microscopic studies have provided no evidence for the presence of circular DNA (deoxyribonucleic acid) in chloroplasts from wheat leaves. Electron microscopy of osmotically disrupted chloroplasts has revealed the presence of linear duplex filaments, some with a tertiary helicoidal configuration and possibly monostranded filaments. None of these structures was observed if the chloroplasts were previously treated with DNase (deoxyribonuclease), but the tertiary configuration was somewhat disorganized by RNase (ribonuclease).

Introduction Whereas linear filaments of DNA are known to be the predominant components in the nucleus, circular DNA has been found in tumor viruses, bacteriophages, and also mitochondria of several animal species as well as in the colicinogenic factor E (10). Mitochondria1 DNA and chloroplast DNA feature, in common, the property of renaturing under conditions in which nuclear DNA remains denatured (4), suggesting that the DNA structure of both organelles may be of circular conformation. This was of interest to the authors since NAD, which has been show11 to be a non-oxidative coenzyme of the "DNA-joining enzyme" in E. coli by Zimmerman et al. (16), brings about a rapid degeneration of Elodea and wheat leaf chloroplasts (7, 11, 15). While circular DNA was not observed in the electron microphotographs of chloroplasts (14) from Acetabularia by Wertz and Kellner (13) or from spinach by Woodcock and Fernandez-Moran (14), an investigation was undertaken to determine whether circular DNA was a component of wheat leaf chloroplasts. Mache and Waygood (6) have recently characterized DNA in wheat leaf chloroplasts isolated by a 'laceration technique' which provides a source of chloroplasts with outer membranes intact and free from nuclear contamination. Electron microscopic examination of such chloroplasts exploded by osmotic shock revealed only the presence of linear DNA filaments having at least two conformations, a linear duplex type and another of tertiary 1Present address: FacultC des Sciences E.N.S.A.T. Physiologic VCgCtale, Toulouse, France.

helicoidal conformation and possibly monostranded filaments. The tertiary structure is probably attached to other macromolecules. The absence of circular DNA was supported also by buoyant density studies in the presence of ethidium bromide. Materials and Methods Chloroplast Preparation and Extraction of D N A Primary leaves of wheat (Triticum aestivum L. var Selkirk) were harvested 10 days after they were sown in the greenhouse. Chloroplasts were isolated by a modified procedure of Jensen and Bassham (5, 6) and by the 'laceration technique' of Mache and Waygood (6). DNA was extracted by a modified procedure of Chiang and Sueoka (2) as previously described (6). When chloroplasts were prepared by the procedure of Jensen and Bassham (5, 6), chloroplast DNA was purified from nuclear DNA by sedimentation in CsC1. Chloroplast DNA was centrifuged in CsCl with 100 pg/ml or 300 pg/ml of ethidium bromide according to the procedure described by Radloff et al. (9) and Bauer and Vinograd (1). After they were centrifuged for 60h at 39 000 r.p.m. (IEC, rotor SB405), the tubes were irradiated with ultraviolet to locate the bands. Fractions were collected with a microsiphon and mixed with a measured volume of a slurry of Dowex 50 X 8 Na+ to remove ethidium cations and analyzed at 260 mp. Electron Microscopy Only the chloroplasts isolated by the 'laceration technique' (6) free from nuclear contamination were used in this study. DNA was released from the chloroplasts by osmotic shock treatment according to the procedure of Woodcock and Fernandez-Moran (14). The extracted DNA was mixed with 0.01% cytochrome c (Sigma, type 11) and spread on a 0.1 M ammonium acetate solution, deposited on a copper grid, and stained with 1% uranyl acetate. Control samples were treated for 15 min with 100 pg/ml DNase I1 (Calbiochem). Some samples of isolated chloroplasts were treated for 15 min with 100 pg/ml pancreatic RNase (Calbiochem). An AEI-EM6B electron microscope equipped with an anticontamination device was used.

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Experimental Results Chloroplast DNA centrifuged at equilibrium in the presence of ethidium bromide, either at coilcentrations of 100 pg/ml or 300 pg /ml, sedimented in one discrete band as observed by ultraviolet radiation (Fig. 1, A). This was confirmed by spectrophotometric analysis, indicating that chloroplast DNA could be either of the linear or circular type. If closed circular DNA were present in the chloroplast a "DNA-joining enzyme" would be essential to repair single strand breaks (3, 8). Since NAD+ (12), ATP (16), and Mg2+ ions are essential cofactors for the enzyme, these were added in appropriate concentrations to the chloroplast suspensions from either the Jensen and Bassham (5) procedure or the 'laceration technique' (6) before extraction and sedimentation of DNA with ethidium bromide. Again only one discrete band resulted at the same position as in Fig. 1, A.

Electron Microscopy The DNA of chloroplasts isolated by the 'laceration technique' of Mache and Waygood (6) appeared in at least two or perhaps three conformations, having diameters of 30 to 60 A, (Figs. 1, 2, 3) as follows. (1) Linear double-stranded filaments-These were never over 10 to 15 p in length attached to fragments of chloroplasts, possibly the internal membrane (Fig. 1). Such a filament with numerous particles attached is shown in Fig. 1, B. Possibly, this could be a DNA filament still entangled with or attached to other macromolecules or similar to the short linear filament or association of filaments shown in Fig. 1, C. (2) Linear tertiary Izelicoidal filaments-The structures shown in Fig. 1, E and F are interpreted as tertiary helicoidal arrangements attached to the internal membrane structure or perhaps the grana of the chloroplast. A detached fragment of this tertiary helicoidal structure is clearly visible in Fig. 2, A and attached to a membrane or grana in Fig. 2, B. (3) Linear monostrandedfilaments-A tangled filament of deproteinized DNA (previously treated with heated 'pronase') (6) is shown in Fig. 2, C. The part in the top left hand corner is thicker (55 A) than that in the lower right hand corner (22 A). Possibly, the latter could be monostranded DNA. This, as well as thicker

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filaments (Fig. 2, D), is also shown in Fig. 2, E. None of these electron micrographs show the presence of circular DNA. Efect of DNnse and RNnse When the extracted DNA was treated with either DNase or RNase as described in Methods, none of the three configurations was observed. After DNase treatment there was little or no fine structure remaining. With RNase treatment there were some fine structures, but these appeared completely disorganized (Fig. 3). Discussion

The sedimentation and electron microscopic studies indicate that chloroplast DNA is linear rather than circular. The 'laceration technique' (6) and the use of ethidium bromide preclude the action of shearing forces and DNase action making it unlikely that a possible circular DNA could have been split into linear fragments. Furthermore, the addition of NAD+, ATP, and Mg2+ ions to the chloroplast suspension, which are essential cofactors for the 'DNA-joining enzyme' (12, 16), did not reveal any circular DNA in the sedimentation studies. There appear to be at least two, but possibly three conformation types of DNA in wheat leaf chloroplasts. The double stranded and tertiary helicoidal configurations appear to be attached to internal membranes, as has been shown for linear DNA in chloroplasts from spinach by Woodcock and Fernandez-Moran (14). However, we have not detected DNA filaments of different diameters unless they have been deproteinized. After deproteinization, thin filaments, perhaps monostranded, could be observed but this may be an artefact and is questionable since Wertz and Kellner (13) found only duplex DNA in chloroplasts of Acetabularia. The extended, linear, double-stranded, and compact helicoidal fragments are both of short length. None of these structures was observed after treatment with DNase. However, with RNase treatment structures were observed, but these appeared quite disorganized (Fig. 3) as compared to their counterparts in Figs. 1 and 2. If RNase disorganizes the tertiary structure, it would be interesting to determine whether this macromolecule contains RNA or not.

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FIG. 1. A. Photograph of the centrifuge tube irradiated with ultraviolet showing chloroplast DNA with ethidium bromide in CsCl sedinienting in one band. B, C, D, E, F. Electron micrographs of DNA from osmotically disrupted chloroplasts. B, X 63 000; C, X 95 000; D, X 10 000; E, X 82 000; F, x 80 000.

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FIG.2. A, B. Electron nlicrographs of chloroplast DNA from osn~oticallydisrupted chloroplasts, in tertiary helicoidal conforn~ation.A, X 80 000) B,, X 100 000. C. Tanglcd filaments of DNA after deproteinization X 96 000. D, E. DNA after deprotelnlzatlon. D, X 235 000; E, X 125 000.

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FIG. 3. Elcctron micrographs of D N A from osmotically disrupted chloroplasts previously treated with RNase. X 63 000.

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MACHE AND WAYGOOID: C1HLOROPLAST DNA

Acknowledgments This work was supported by a grant-in-aid (A2698) from the National Research Council of Canada. The senior author gratefully acknowledges the award of a Canada Council and University of Manitoba Postdoctorate Fellowship. We thank Dr. B. D. Sanwal of the Department of Microbiology for reading the manuscript and his advice, and Dr. P. K. Isaac and Miss M. DeGeus of the Department of Botany for their assistance in electron microscopy. The assistance of Dr. R. Haselkorn of the Department of Biophysics, University of Chicago, in interpreting the electron micrographs is also gratefully appreciated. 1. BAUER,W., and J. VINOGRAD. 1968. The interaction of closed circular DNA with intercalative dyes. I. The superhelix density of SV40 DNA in the presence and absence of dye. J. Mol. Biol. 33: 141-171. 2. CHIANG,K. S., and N. SUEOKA. 1967. Replication of chloroplast DNA in Chlan~ydomonas reinhardi during vegetative cell cycle: its mode and regulation. Proc. Nat. Acad. Sci. U.S.A. 57: 148-155. 3. GELLERT, M. 1967. Formation of covalent circles of Lambda bv E. coli extracts. Proc. Nat. Acad. - ~ .DNA . s;. U.S.A. 57: