Replication origins mapped to the large inverted repeat

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Electron-microscopy combined with restriction enzyme digestion enables D-loops and replication bubbles to be mapped onto restriction fragments of plastid DNA. The P. sativum D-loops (OriA and OriB) flank the 23S ribosomal RNA gene (Fig. 6a; Meeker et al. 1988). Unlike most angiosperm plastid genomes P. sativum lacks a large inverted repeat (Chapter 3). Restriction fragments of proplastid DNA with a high frequency of D-loops from N. tabacum BY2 suspension culture cells

Loop Model Dna Replication

Fig. 5. Plastid DNA replication models. a) Displacement-loop (D-loop) model of plastid DNA replication. Two D-loops converge to give rise to bidirectional replication (Kolodner and Tewari 1975). b) Rolling circle replication arising from strand displacement at a nick. Movement of the replication fork is shown by anti-clockwise rotation of the circle marked by "a" c) Recombination-dependent DNA replication (Kowalczykowski 2000) on a circular template with D-loop gives rise to a bubble-containing circle with tail. d) Recombination-dependent DNA replication on a linear DNA template gives rise to a branched molecule.

Fig. 5. Plastid DNA replication models. a) Displacement-loop (D-loop) model of plastid DNA replication. Two D-loops converge to give rise to bidirectional replication (Kolodner and Tewari 1975). b) Rolling circle replication arising from strand displacement at a nick. Movement of the replication fork is shown by anti-clockwise rotation of the circle marked by "a" c) Recombination-dependent DNA replication (Kowalczykowski 2000) on a circular template with D-loop gives rise to a bubble-containing circle with tail. d) Recombination-dependent DNA replication on a linear DNA template gives rise to a branched molecule.

mapped close to the end of the 23S rRNA gene in the large inverted repeat (Fig. 6b Nt (pro)) and a less active D-loop mapped to a 2.3 kbp Stu I fragment containing part of the psaA and psaB genes in the large single copy region (Takeda et al. 1992). Later work, using two dimensional agarose gel electrophoresis to map bubbles in cloned plastid DNA templates replicated in chloroplast fractions, in vitro DNA replication assays and primer extension on nascent DNA strands, suggested different positions for two D-loops (named OriA and OriB) in plastid DNA from N. tabacum leaves. N. tabacum OriA mapped to the intron of the trnI (GAU) gene located between the 16S and 23S rRNA genes (Lu et al. 1996; Kunnimalaiyaan and Nielsen 1997a). OriB mapped to the large inverted repeat close to the border of the small single copy region in orf350 or ycfl (Kunnimalaiyaan and Nielsen 1997a; Kunnimalaiyaan et al. 1997b).

Phase Generator Windings

Fig. 6. Schematic diagram showing the locations of potential replication origins. Origin locations are shown outside the circular maps as triangles or bar-ended lines. a) P. sativum (Meeker et al. 1988) lacks a large inverted repeat, b) Dicots containing large inverted repeats. N. tabacum Nt (Ori A) and Nt (Ori B) (Kunnimalaiyaan and Nielsen 1997; Kunni-malaiyaan et al. 1997), N. tabacum D-loops Nt (pro) in proplastids (Takeda et al. 1992), Oenothera hookeri Oe (Ori A) and Oe (Ori B) (Chiu and Sears 1992; Sears et al. 1996), Glycine max bubbles Gm (Hedrick et al. 1993). c) Oryza sativa (Os) replication origins in suspension culture cells (Os!), leaf blades (Os2), and coleoptiles (Os3) mapped by Wang et al. 2003, Z mays (Zm, Gold et al. 1987), linear DNA replicons (Ellis and Day 1986; Ha-rada et al. 1992; Zubko and Day 2002), Hordeum vulgare (Hv), Oryza sativa (Os). d) C. reinhardtii Ori A and Ori B (Waddell et al. 1984; Chang and Wu 2000) and initiation of novobiocin-resistant replication (Woelfle et al. 1993). The large inverted repeats are shown as converging box arrows on maps b-d; arrow orientation according to rrn operon transcription direction.

Fig. 6. Schematic diagram showing the locations of potential replication origins. Origin locations are shown outside the circular maps as triangles or bar-ended lines. a) P. sativum (Meeker et al. 1988) lacks a large inverted repeat, b) Dicots containing large inverted repeats. N. tabacum Nt (Ori A) and Nt (Ori B) (Kunnimalaiyaan and Nielsen 1997; Kunni-malaiyaan et al. 1997), N. tabacum D-loops Nt (pro) in proplastids (Takeda et al. 1992), Oenothera hookeri Oe (Ori A) and Oe (Ori B) (Chiu and Sears 1992; Sears et al. 1996), Glycine max bubbles Gm (Hedrick et al. 1993). c) Oryza sativa (Os) replication origins in suspension culture cells (Os!), leaf blades (Os2), and coleoptiles (Os3) mapped by Wang et al. 2003, Z mays (Zm, Gold et al. 1987), linear DNA replicons (Ellis and Day 1986; Ha-rada et al. 1992; Zubko and Day 2002), Hordeum vulgare (Hv), Oryza sativa (Os). d) C. reinhardtii Ori A and Ori B (Waddell et al. 1984; Chang and Wu 2000) and initiation of novobiocin-resistant replication (Woelfle et al. 1993). The large inverted repeats are shown as converging box arrows on maps b-d; arrow orientation according to rrn operon transcription direction.

Two D-loops separated by 4 kbp (Chiu and Sears 1992; Sears et al. 1996) were found in the large inverted repeat of Oenothera hookeri plastid DNA, where they flank the 16S ribosomal genes (Fig. 6b). The locations of two origins in the large inverted repeat suggest four potential replication origins in N. tabacum and O. hookeri plastid DNA; two in each inverted repeat. The complexity of mapping plastid origins is illustrated by the observation that the locations of origins appear to vary in different cells, tissues and organs from the same species. Differences in the mechanism of DNA replication and locations of plastid origins have been observed in suspension culture cells, coleoptiles, and leaves of Oryza sativa (rice,

Wang et al. 2003). O. sativa plastid replication origins were mapped to the small single copy region and to two positions in the large inverted repeat (Fig. 6c; Wang et al. 2003). Comparisons of mapped origins show that the location of only one is conserved in N. tabacum (OriA), O. hookeri (OriB), and P. sativum (OriA). This is located in the intergenic region between the 16S and 23S rRNA genes (Fig. 6a, 6b; Lu et al. 1996). Conservation in location might suggest this region is important for plastid genome maintenance. However, deletion of OriA in N. tabacum using plastid transformation has revealed that it is not essential for plastid DNA replication and maintenance (Mühlbauer et al. 2002). Of the two copies of OriB present in the large inverted repeat the one located in orf350 (OriB2) could be deleted. The other copy of OriB (OriB1) cannot be removed without mutating the essential ycf1 gene and hence OriB1 dispensability cannot be addressed by a deletion that removes ycf1 function (Mühlbauer et al. 2002). In a recent study, a stem-loop in OriB1 was mutated that left ycf1 intact (Scharff and Koop 2007). This allowed the isolation of plants in which OriB1 was mutated and OriB2 was deleted indicating that neither OriB sequence was essential. The copy number of plastid DNA appeared to be the same in shoot tips but lower in young and older leaves of deleted OriA lines (down ~1.5-fold) and lines lacking both OriA and OriB2 (down ~2-fold) compared to WT. The plastid DNA copy number in young and older leaves of OriB mutated lines (OriB2 deleted, OriB1 mutated) was higher (up ~1.7-fold) than WT plants (Scharff and Koop 2007).

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