Potential replication origins have also been located well away from the ribosomal genes. In C. reinhardtii two D-loops (OriA and OriB) spaced 7 kb apart (Waddell et al. 1984) map to the single copy region of plastid DNA (Fig. 6d). C. reinhardtii OriA was localised to a 224 bp region containing the rpl16 gene (Chang and Wu 2000) whereas "OriB is located in or adjacent to chl L" (Wu personal communication). Replication at OriA is influenced by transcription across rpl16 (Chang and Wu 2000). Novobiocin inhibition of D-loop replication led to novobiocin-resistant replication starting close to a 'hot spot' of recombination near the 3' end of the psbA gene in the large inverted repeat of C. reinhardtii plastid DNA (Woelfle et al. 1993). In Z. mays, preferential DNA synthesis from a 1368 bp plastid DNA sequence amongst cloned templates representing 94% of the Z. mays plastid genome by a partially purified P. sativum plastid DNA polymerase suggested this region promoted replication (Gold et al. 1987). The Z. mays region promoting DNA synthesis was found to promote bidirectional replication using a partially purified ~90 kDa Z. mays DNA polymerase. This was localised to a 455 bp sequence containing the 3' end of the rpl16 gene in the large single copy region of plastid DNA (Fig. 6c; Carrillo and Bogorad 1988). A one kbp region including the 3' end of the psbA gene also promoted DNA synthesis using the Z. mays DNA polymerase fraction (Fig. 6c; Carrillo and Bogorad 1988). Whilst the sequence organizations of Z. mays and C. reinhardtii plastid DNA are not conserved it is interesting that a po tential replication origin overlaps with the rpl16 genes in both species (Gold et al. 1987; Chang and Wu 2000).
Two replication bubbles were mapped to 1.8 kbp Sac I-Bam HI and 2.5 kbp Bam HI fragments in the large single copy region of Glycine max (soybean) chloroplast DNA by two-dimensional gel electrophoresis (Fig. 6b; Hedrick et al. 1993). The recently published G. max plastid genome (Saski et al. 2005) locates these bubble-containing fragments to regions containing the rps12-clp P1 and pet B-petD genes in the large single copy region. Petunia hybrida and N. tabacum plastid DNA sequences located in the small copy region near the location of N. ta-bacum OriB promote autonomous replication in yeast nuclei (Ohtani et al. 1984; Dehaas et al. 1986). Although interesting, the finding that some A + T rich plastid sequences resembling yeast autonomously replicating elements (ARS) promote replication in S. cerevisiae nuclei would appear to provide weak evidence for locating plastid origins of replication. The locations of these ARS regions are not shown in Figure 6. The lack of agreement between different experimental approaches in locating origins of replication (Fig. 6) has hindered progress in this difficult area of research. It might indicate a lack of conservation in the location of plastid replication origins between different species, the presence of multiple origins, differences in the accuracy of locating origins or limitations of the plastid dual D-loop replication model.
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