I

Monomer Head to tall dimer

~ 15% Unclassified molecules -10% Lasso-like - 5-10% With bubbles, D - Loop

C. Oldenburg and Bendich, 2004b n=1 to 8

Monomer Head to tall dimer

~ 15% Unclassified molecules -10% Lasso-like - 5-10% With bubbles, D - Loop

C. Oldenburg and Bendich, 2004b

Simple forms

High MW branched linear structures

Number of molecules Mass of DNA

Simple forms

High MW branched linear structures

Fig. 3. a) Topological forms of plastid DNA revealed by electron microscopy (Kolodner and Tewari 1972). Only head-to-tail dimers were found in P. sativum while head-to-head dimers were predominant in L. sativa and S. oleracea (Kolodner and Tewari 1979). Breakage of circles during extraction will give rise to a variable percentage of circles and sub-genomic linear forms. b) Topological forms of plastid DNA in A. thaliana and N. tabacum revealed by DNA fibre-based FISH with plastid DNA probes (Lilly et al. 2001). Only the monomer and dimer are shown in the circular and linear multimeric series. Arrows indicate sequence orientation. c) Structures of plastid DNA molecules in fluorescent images of ethidium-stained DNA from purified Z. mays chloroplasts lysed in agarose plugs. DNA fibres in compact high MW structures were extended by an electric field or flowing liquid (Oldenburg and Bendich 2004b).

were unclassified (15%), lasso-like (~10%) or contained bubbles or D-loops (510%). In N. tabacum chloroplasts (Lilly et al. 2001), monomelic circles are the most abundant (55%) followed by dimers (17%), trimers (10%), tetramers (7%), pentamers (5%), and hexameric circles (1%). Rare higher-order multimers of unit genome sized (genome size = 156 kbp) linear DNA molecules were found up to the octomer. In A. thaliana and N. tabacum multimers were comprised of monomers arranged head-to-tail (Lilly et al. 2001) in contrast to the earlier results of predominantly head-to-head dimers in L. sativa and S. oleracea (Kolodner and Tewari 1979). The ends of the linear DNA molecules were not mapped and they could represent real linear plastid DNA species or the breakage products of large circles (Lilly et al. 2001). A number of lines of evidence indicate that linear DNA molecules found in plastids are not simply breakage products of large circles (see Section 3.1, 3.2, and 7 below). Using a similar DNA fibre-based FISH method, C. reinhardtii plastid DNA was found to be mainly comprised of monomeric and dimeric linear and circular forms (Maul et al. 2002). The multimeric series of linear and circular DNA molecules found in plastids (Fig. 3b) must result from the action of plastid DNA-RRR pathways.

Embedding cells in agarose plugs prior to cell and chloroplast lysis reduces DNA breakage and allows the isolation of large DNA molecules. DNA in agarose plugs can then be analysed by pulsed-field gel electrophoresis or microscopy after staining with ethidium bromide. Circular DNA does not enter pulsed field gels and remains within the agarose plugs at the origin at the relatively short pulse times used to fractionate DNA in the 100-1000 kbp range (Bendich and Smith 1990; Backert et al. 1995). Linear chloroplast DNA molecules enter the agarose gel and can be identified by blot hybridization with chloroplast DNA probes. Some of these linear DNA molecules might result from breakage of circular DNA molecules (Backert et al. 1995; Bendich 2004). A multimeric series comprised of monomer (most abundant) and higher molecular weight (MW) linear plastid DNA forms can be visualised on pulsed-field gels. The largest multimers found were tetramers for S. oleracea (Deng et al. 1989) and N. tabacum (Lilly et al. 2001), dimers (Lilly et al. 2001) or trimers for P. sativum, and up to the octomer for Citrullus vulgaris plastid DNA (watermelon, Bendich and Smith 1990). The banding pattern can be disrupted by altering the activities of plastid DNA-RRR proteins. Inhibition of plastid-targeted gyrase (see Section 13.4 below), which is required to decatenate newly replicated DNA, reduces the levels of discrete bands corresponding to the monomer and dimer, and gives rise to a heterogeneous mixture of plastid DNA molecules, some of which are greater than 1000 kbp in size on pulsed field gels (Cho et al. 2004).

Whilst the bands seen on pulsed-field gels were useful for visualising multimers of plastid DNA they represent a minor proportion of plastid DNA and give a distorted view of the topological forms of plastid DNA molecules (Bendich 2004). The bulk of plastid DNA molecules including circles, high MW linear branched forms (Bendich 2004; Oldenburg and Bendich 2004b), tangled DNA fibres and any DNA in unlysed plastids remains immobile in the agarose plugs at the origin and does not enter pulsed field gels. N. tabacum leaf chloroplast DNA remained at the origin (migration into the gel was not detected) whereas about 35% of Chenopidium album plastid DNA from a non-green suspension-culture entered the gel revealing monomer and dimer bands (pulse times of 30-60 seconds, Backert et al. 1995). The presence of electrophoretically-mobile linear plastid DNA molecules in C. album non-pigmented plastids but not in N. tabacum chloroplasts might reflect changes in plastid DNA topologies in different plastid types, also indicated from other studies on Z. mays (Oldenburg and Bendich 2004a), or result from breakage during extraction. Mild DNase I treatment of high MW N. tabacum chloroplast DNA and blot hybridization with a plastid probe revealed a smear of DNA (representing molecules of different lengths) within which discrete monomer to tetramer bands were clearly visualised (Backert et al. 1995). These discrete linear bands are likely to be derived from circular DNA because a single double strand break mediated by DNase I will convert a circle to its linear form.

The structures of plastid DNA molecules in agarose plugs prepared from 10-14 day old Z. mays seedlings have been studied by fluorescence microscopy following ethidium bromide staining (Oldenburg and Bendich 2004b). The DNA was present as simple DNA molecules and high MW DNA complexes with a central core and attached DNA fibres (Fig. 3 c). In the presence of an electric field or liquid flow the simple molecules migrate whereas linear fibres extend from the immobile cores of the high MW complexes. Simple DNA molecules are comprised of circles and linear molecules and represent 94% of the DNA molecules but only 7% of the mass of DNA in plastids due to their small sizes relative to the high MW DNA complexes. The high MW complexes contained on average a minimum of eight plastid genomes (not including bright fluorescent cores) and were suggested to be largely comprised of linear and complex-branched molecules (Oldenburg and Bendich 2004b). A reduction in high MW complexes and an increase in simple forms were correlated with chloroplast maturation during leaf development in Z. mays (Oldenburg and Bendich 2004a). The multigenome complexes were reported to represent 93% of plastid DNA by mass. Following removal of linear DNA molecules from multigenome complexes by pulsed-field gel electrophoresis the immobile high MW core was suggested to be comprised of complex-branched DNA structures representing 50% of the mass of DNA in plastids (Oldenburg and Bendich 2004b). The complex high MW branched forms have been suggested to represent replication intermediates and their analysis is particularly important (Oldenburg and Bendich 2004b; Scharff and Koop 2006). The ~15% of tangled DNA fibres that were unclassified (Fig. 3b) by Lilly et al. (2001) might correspond to these high MW DNA complexes identified by Oldenburg and Bendich (2004b). DNA fibre-based FISH using plastid DNA probes would confirm the presence of plastid DNA in these high MW complexes and might be a useful tool to study their sequence organisation.

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