Deletion mapping delimits DNA sequences capable of selfreplication in plastids

In cereals, most plastid genes are dispensable allowing the isolation of deletion mutants lacking most of the plastid genome (see Section 3.1 above). This natural deletion mapping identifies a region of plastid DNA located around the trnE(UUC) gene (Fig. 6c) that is self-replicating as linear DNA molecules (Day and Ellis 1985; Ellis and Day 1986; Harada et al. 1992; Kawata et al 1997; Zubko and Day 2002; Cahoon et al. 2003). The retained region does not contain any known plastid replication origins mapped by electron microscopy, gel electropho-resis or biochemical methods (Fig. 6b, 6c). Maintenance of these linear DNA molecules might involve a replication origin that is not highly active in WT shoots and leaves. The relationship between the replication mechanisms maintaining small linear molecules and WT plastid DNA is not known but hairpin ends have been found at low frequency in WT H. vulgare plastids (Collin and Ellis 1990; Section 3.1).

Recombination events between sequences in the large single copy region and those located in either the large inverted repeat or small single copy region give rise to deleted plastid DNA molecules with circular maps (Day and Ellis 1984) and circular structures (Day and Ellis 1985) that have been found in albino T. aes-tivum plants from anther culture. The region present in the smallest circles (39 kbp, Day and Ellis 1985), containing only 30% of the plastid genome, stretches from the trnE gene region to the end of the adjacent large inverted repeat (Fig. 6c). These molecules contain only one large inverted repeat sequence and lack the replication origin mapped near rpl16 in Z. mays (Fig. 6c; Gold et al. 1987). Deleted circular plastid DNA molecules containing only one large inverted repeat have also been found in WT N. tabacum chloroplasts by DNA fibre-based FISH (Lilly et al. 2001). These results show that sub-genomic plastid DNA molecules lacking the small single copy region, most of the large single copy region and one large inverted repeat can be maintained as circular DNA molecules in plastids.

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