Analysis of soybean chloroplast DNA replication by two-dimensional gel electrophoresis

Abstract

Chloroplast DNA replication was studied in the green, autotrophic suspension culture line SB-1 of Glycine max. Three regions (restriction fragments Sac I 14.5, Pvu II 4.1 and Pvu II 14.8) on the plastome were identified that displayed significantly higher template activity in in vitro DNA replication assays than all other cloned restriction fragments of the organelle genome, suggesting that these clones contain sequences that are able to direct initiation of DNA replication in vitro. In order to confirm that the potential in vitro origin sites are functional in vivo as well, replication intermediates were analyzed by two-dimensional gel electrophoresis using cloned restriction fragments as probes. The two Pvu II fragments that supported deoxynucleotide incorporation in vitro apparently do not contain a functional in vivo replication origin since replication intermediates from these areas of the plastome represent only fork structures. The Sac I 14.5 chloroplast DNA fragment, on the other hand, showed intermediates consistent with a replication bubble originating within its borders, which is indicative of an active in vivo origin. Closer examination of cloned Sac I 14.5 sub-fragments confirmed high template activity in vitro for two, S/B 5 and S/B 3, which also seem to contain origin sites utilized in vivo as determined by two-dimensional gel electrophoresis. The types of replication intermediate patterns obtained for these sub-fragments are consistent with the double D-loop model for chloroplast DNA replication with both origins being located in the large unique region of the plastome [17,18]. This is the first report of a chloroplast DNA replication origin in higher plants that has been directly tested for in vivo function.

Characterization of replication origins flanking the 23S rRNA gene in tobacco chloroplast DNA

“…The presence of D-loop regions associated with the rRNA genes has also been observed in Oenothera, where EM analysis has identified four D-loops, two in each IR flanking the 16S rRNA genes [8]. However, ctDNA replication origins have been mapped at different locations in Chlamydomonas reinhardii [26,47,48], soybean [ 18], petunia [10] and maize [3]. A single replication origin was mapped near the additional 16S rRNA gene in Euglena gracilis [21,37].…”

“…However, Takeda et al enriched nucleic acids by BND cellulose from cultured tobacco cell plastids and labeled the DNA by nick translation, resulting in a difference between the signal and background of only 10-20%. However, this technique has only recently been applied to the analysis of ctDNA replication [ 18,32]. The presence of a second origin in each inverted repeat as reported here but not by Takeda et al [42] may be due to the low specificity of probes labeled by nick translation of replication intermediates after BND cellulose purification and inherent difficulties in accurately measuring DNA For fragments with D-loops near the ends, breakage may occur to generate a simple Y type pattern at each end but with one side of the Y being single-stranded (C).…”

Stable transformation of the cotton plastid genome and maternal inheritance of transgenes

“…Several methods have been used to study ctDNA replication origins in higher plants including electron microscopy, two-dimensional (2D) agarose gel electrophoresis, primer extension mapping of nascent 5¢ ends from total plastid DNA (Kolodner and Tewari, 1975;Meeker et al, 1988;Chiu and Sears, 1992;Hedrick et al, 1993; Nielsen et al, 1993;Kunnimalayaan et al, 1997), and in vitro replication analysis of ctDNA ori subclones (Kunnimalayaan and Nielsen, 1997 a, b). All these studies have identified two replication origins (oriA and oriB) that flank the 23S rRNA gene in tobacco ctDNA (Kunnimalayaan and Nielsen, 1997a,b).…”

Linear molecules of tobacco ptDNA end at known replication origins and additional loci

“…There is no report of an origin of replication in this area in the inverted repeats. But in G. max (soybean) two ORIs are reported to be in the large single copy region near one inverted repeat and the rpl16 gene (Hedrick et al 1993). Also in Z. mays (Carrillo and Bogorad 1988) and C. reinhardtii (Lou et al 1987) a putative origin of replication is near rpl16.…”