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Characterization of replication origins flanking the 23S rRNA gene in tobacco chloroplast DNA
Abstract
Using 5' end-labeled nascent strands of tobacco chloroplast DNA (ctDNA) as a probe, replication displacement loop (D-loop) regions were identified. The strongest hybridization was observed with restriction fragments containing the rRNA genes from the inverted repeat region. Two-dimensional gel analysis of various digests of tobacco ctDNA suggested that a replication origin is located near each end of the 7.1 kb BamHI fragment containing part of the rRNA operon. Analysis of in vitro replication products indicated that templates from either of the origin regions supported replication, while the vector alone or ctDNA clones from other regions of the genome did not support in vitro replication. Sequences from both sides of the BamHI site in the rRNA spacer region were required for optimal in vitro DNA replication activity. Primer extension was used for the first time to identify the start site of DNA synthesis for the D-loop in the rRNA spacer region. The major 5' end of the D-loop was localized to the base of a stem-loop structure which contains the rRNA spacer BamHI site. Primer extension products were insensitive to both alkali and RNase treatment, suggesting that RNA primers had already been removed from the 5' end of nascent DNA. Location of an origin in the rRNA spacer region of ctDNA from tobacco, pea and Oenothera suggests that ctDNA replication origins may be conserved in higher plants.
... 2014; Gowrishankar, 2015). However, the cpDNA region with the highest copy number in cptk1 mutants does not coincide with replication origins that have been described for maize and other plant species (Chiu and Sears, 1992;Lu et al., 1996;Kunnimalaiyaan and Nielsen, 1997;Bendich, 2004, 2016;Day and Madesis, 2007). We explored the possibility that there is a relationship between the copy number gradient and GC skew (Fig. 3A), which is an asymmetry in G and C content between the two DNA strands. ...
... A gradient in copy number along the genome was more pronounced in the double mutant but was also visible in TK1A/tk1A tk1B (Fig. 7B). This gradient had a maximum in the IR region, consistent with the idea that replication origins map close to the rRNA genes, as described for other eudicots (Chiu and Sears, 1992;Lu et al., 1996;Kunnimalaiyaan and Nielsen, 1997;Bendich, 2004, 2016;Day and Madesis, 2007). A minimum in the GC skew plot of the Arabidopsis cpDNA was also found upstream of the rpoB operon, but contrarily to maize cptk1, there was no evidence that replication initiated from that locus in the Arabidopsis mutants. ...
... then map to the locus where replication is reinitiated. Surprisingly, in maize cptk1, that locus differs from the proposed replication origins mapped in several flowering plants in the IRs of the cpDNA (IR A and IR B ), close to rRNA gene sequences (Chiu and Sears, 1992;Lu et al., 1996;Kunnimalaiyaan and Nielsen, 1997;Oldenburg and Bendich, 2004;Day and Madesis, 2007). These putative promoter regions often coincide with the extremities of linear cpDNA molecules that were mapped both in maize and tobacco (Nicotiana tabacum; Bendich, 2004, 2016;Scharff and Koop, 2006). ...
... 227 Gowrishankar, 2015;Nordman et al., 2014). However, the cpDNA region with the highest 228 copy number in cptk1 mutants does not coincide with replication origins that have been 229 described for maize and other plant species ( Chiu et Sears, 1992;Day et Madesis, 2007;230 Kunnimalaiyaan et Nielsen, 1997;Lu et al., 1996;Oldenburg et Bendich, 2004. We 231 explored the possibility that there is a relationship between the copy number gradient and 232 GC skew ( Figure 3A), which is an asymmetry in G and C content between the two DNA 233 strands. ...
... A gradient in 335 copy number along the genome was more pronounced in the double mutant but was also 336 visible in TK1A/tk1A tk1B ( Figure 7B). This gradient had a maximum in the IR region, 337 consistent with the idea that replication origins map close to the rRNA genes, as described 338 for other eudicots ( Chiu et Sears, 1992;Day et Madesis, 2007;Kunnimalaiyaan et Nielsen, 339 1997;Lu et al., 1996;Oldenburg et Bendich, 2004. A minimum in the GC skew plot of 340 the Arabidopsis cpDNA was also found upstream of the rpoB operon, but contrarily to maize 341 cptk1 there was no evidence that replication initiated from that locus in the Arabidopsis 342 mutants. ...
... Maximum sequence copy number 411 should then map to the locus where replication is re-initiated. Surprisingly, in maize cptk1 412 that locus differs from the proposed replication origins mapped in several flowering plants in 413 the IRs of the cpDNA (IR A and IR B ), close to rRNA gene sequences ( Chiu et Sears, 1992;414 Day et Madesis, 2007;Kunnimalaiyaan et Nielsen, 1997;Lu et al., 1996;Oldenburg et 415 Bendich, 2004). These putative promoter regions often coincide with the extremities of linear 416 cpDNA molecules that were mapped both in maize and tobacco (Nicotiana tabacum) 417 ( Oldenburg et Bendich, 2004Scharff et Koop, 2006). ...
Thymidine kinase (TK) is a key enzyme of the salvage pathway that recycles thymidine nucleosides to produce deoxythymidine triphosphate. Here, we identified the single TK of maize (Zea mays), denoted CPTK1, as necessary in the replication of the plastidial genome (cpDNA), demonstrating the essential function of the salvage pathway during chloroplast biogenesis. CPTK1 localized to both plastids and mitochondria, and its absence resulted in an albino phenotype, reduced cpDNA copy number and a severe deficiency in plastidial ribosomes. Mitochondria were not affected, indicating they are less reliant on the salvage pathway. Arabidopsis (Arabidopsis thaliana) TKs, TK1A and TK1B, apparently resulted from a gene duplication after the divergence of monocots and dicots. Similar but less-severe effects were observed for Arabidopsis tk1a tk1b double mutants in comparison to those in maize cptk1 TK1B was important for cpDNA replication and repair in conditions of replicative stress but had little impact on the mitochondrial phenotype. In the maize cptk1 mutant, the DNA from the small single-copy region of the plastidial genome was reduced to a greater extent than other regions, suggesting preferential abortion of replication in this region. This was accompanied by the accumulation of truncated genomes that resulted, at least in part, from unfaithful microhomology-mediated repair. These and other results suggest that the loss of normal cpDNA replication elicits the mobilization of new replication origins around the rpoB (beta subunit of plastid-encoded RNA polymerase) transcription unit and imply that increased transcription at rpoB is associated with the initiation of cpDNA replication. © 2018 American Society of Plant Biologists. All rights reserved.
... Considering the highly conserved structure and gene content of ctDNA, the observed differences in number and location of plastome D-loops are somewhat surprising. Two-dimensional gel electrophoresis of in vivo and in vitro ctDNA replication intermediates and other experimental approaches to further define plastome ori regions (Hedrick et al., 1993;Kunnimalaiyaan and Nielsen, 1997b;Kunnimalaiyaan et al., 1997;Lu et al., 1996;Nielsen et al., 1993;Wang, Y. et al., 2002) for the most part support the presence of two D-loops but also failed to establish a consensus ori location on the plastome of higher plants. In keeping with these results is the general lack of primary structure homology between those ori regions that have been sequenced (Kunnimalaiyaan and Nielsen, 1997b;Lu et al., 1996;Nielsen et al., 1993;Takeda et al., 1992;Wu, M. et al., 1986); rev in (Heinhorst and Cannon, 1993;Kunnimalaiyaan and Nielsen, 1997a). ...
... Two-dimensional gel electrophoresis of in vivo and in vitro ctDNA replication intermediates and other experimental approaches to further define plastome ori regions (Hedrick et al., 1993;Kunnimalaiyaan and Nielsen, 1997b;Kunnimalaiyaan et al., 1997;Lu et al., 1996;Nielsen et al., 1993;Wang, Y. et al., 2002) for the most part support the presence of two D-loops but also failed to establish a consensus ori location on the plastome of higher plants. In keeping with these results is the general lack of primary structure homology between those ori regions that have been sequenced (Kunnimalaiyaan and Nielsen, 1997b;Lu et al., 1996;Nielsen et al., 1993;Takeda et al., 1992;Wu, M. et al., 1986); rev in (Heinhorst and Cannon, 1993;Kunnimalaiyaan and Nielsen, 1997a). ...
... Plastome origins do, however, share certain structural features, such as an abundance of (A+T)-rich regions flanked by (G+C)-rich motifs, and several short inverted repeats that are predicted to form stable stem/loop structures (Kunnimalaiyaan and Nielsen, 1997b;Nielsen et al., 1993;Wu, M. et al., 1986). Such secondary structure elements may be important for initiation site selection, like the in vitro D-loop extension site that was mapped to the base of a (G+C)-rich potential stem/loop in the cloned oriA region of the tobacco plastome (Lu et al., 1996). Hairpin structures might also mediate specific interactions with the organellar DNA replication machinery, since gel mobility shift analysis revealed that the cloned oriB from tobacco (Kunnimalaiyaan et al., 1997) and oriA from Chlamydomonas (Nie, Z.Q. ...
Plastids and mitochondria fulfill important metabolic functions that greatly affect plant growth and productivity. One can
therefore easily envision that division of the organelles themselves, as well as replication, maintenance and partitioning
of their genomes must be carefully controlled processes that ensure even organelle distribution during cell division and coordinate
the organellar metabolic processes with the needs of the cell, tissues and the entire plant. This chapter reviews the combined
cytological, biochemical, genetic and genomics approaches that have led to novel insights into key players that mediate or
regulate these processes.
... Each ori contains imperfect indirect repeats that have the potential to form stem-loop structures, with additional short direct repeat sequence elements 5 (oriA) or both 5 and 3 (oriB) of the stem-loop regions. We have previously shown that deletions that remove the repeat sequence elements 5 of the start site of unidirectional DNA synthesis for single-ori clones result in complete loss of replication activity in vitro [10,13]. ...
... Fractions were assayed for DNA synthesis activity as described [13]. Active fractions (40,000-100,000 cpm each) were pooled and dialyzed against three changes of Buffer B (50 mM Tris-HCl, 20% glycerol, 0.1% Triton X-100, 100 mM NaCl, 0.2 mM PMSF, 5 mM 2-mercaptoethanol, 1 mM benzamidine, 1 mM sodium metabisulfite). ...
... Z00044). Direct repeats in each origin are underlined and italicized, restriction sites are in bold, and the location and direction of DNA synthesis initiation for each origin is indicated by the open arrows [9,13]. For oriB, two separate repeats are indicated as R1 and R2 (R2 is an imperfect repeat). ...
Two replication origins (oris) were previously mapped in each inverted repeat of tobacco chloroplast DNA (ctDNA) and each contains a potential stem-loop forming region. Here, we show that specific 45–285 bp deletions within or near the stem-loop regions in single-ori clones abolish replication activity in vitro. In addition, a double-ori clone with ends within the stem-loop region of both oris but with the original spacing lacks replication activity in vitro. These results provide support for the involvement of both the stem-loop and flanking sequences in ctDNA replication. Alteration of spacing in double-ori clones affects the mode of replication used in vitro. A clone with a 2.95 kbp deletion between the two oris replicates by the normal theta mechanism, while a clone with a 6.1 kbp deletion replicates by a rolling circle mechanism, similar to clones that contain a single ori. Similar results were obtained with single- and double-ori constructs after electroporation into isolated intact chloroplasts.
... In pea ctDNA the two D-loops (termed oriA and oriB) have been mapped¯anking the 23 S rRNA gene (Meeker et al., 1988), and oriA has been localized within a 0.45 kb fragment downstream of the 16 S rRNA gene in the rRNA spacer region (Nielsen et al., 1993). We have recently mapped the oriA region in tobacco ctDNA to the same location as in pea ctDNA (Lu et al., 1996). ctDNA replication origins have been found closely associated with the rRNA genes in some plant and algal species (Koller & Delius, 1982;Ravel-Chapuis et al., 1982;Chiu & Sears, 1992;Takeda et al., 1992). ...
... We have recently reported the presence of a pair of replication origins in each IR of tobacco ctDNA (Lu et al., 1996). We report here the localization and characterization of the oriB replication origin downstream of the 23 S rRNA genes in tobacco ctDNA using 2-D gel analysis of in vivo and in vitro tobacco ctDNA replication intermediates. ...
... Localization of ori B near the ends of the IRs by in vivo 2-D gel electrophoresis Previously, oriB was tentatively localized to the 4.29 kb SspI tobacco ctDNA fragment by 2-D gel analysis (Lu et al., 1996). To further localize this ori, speci®c DNA fragments from the IR region were used as probes for Southern hybridization with restricted tobacco ctDNA separated by 2-D gel electrophoresis. ...
We have mapped the origin of DNA replication (oriB) downstream of the 23 S rRNA gene in each copy of the inverted repeat (IR) of tobacco chloroplast DNA between positions 130,502 and 131,924 (IR(A)) by a combination of approaches. In vivo chloroplast DNA replication intermediates were examined by two-dimensional agarose gel electrophoresis. Extended arc patterns suggestive of replication intermediates containing extended single-stranded regions were observed with the 4.29 kb SspI fragment and an overlapping EcoRI fragment from one end of the inverted repeat, while only simple Y patterns were observed with a 3.92 kb BamHI-KpnI fragment internal to the SspI fragment. Other restriction fragments of tobacco chloroplast DNA besides those at the oriA region also generated only simple Y patterns in two-dimensional agarose gels. Several chloroplast DNA clones from this region were tested for their ability to support in vitro DNA replication using a partially purified chloroplast protein fraction. Templates with a deletion of 154 bp from the SspI to the BamHI sites near the end of the inverted repeat resulted in a considerable loss of in vitro DNA replication activity. These results support the presence of a replication origin at the end of the inverted repeat. The 5' end of nascent DNA from the replication displacement loop was identified at position 130,697 for IR(A) (111,832 for IR(B)) by primer extension. A single major product insensitive to alkali and RNase treatment was observed and mapped to the base of a stem-loop structure which contains one of two neighboring BamHI sites near the end of each inverted repeat. This provides the first precise determination of the start site of DNA synthesis from oriB. Adjacent DNA fragments containing the stem-loop structure and the 5' region exhibit sequence-specific gel mobility shift activity when incubated with the replication protein fraction, suggesting the presence of multiple binding sites.
... Using this approach partially purified chloroplast extracts from several plant sources have been shown to prefer specific cloned regions of ctDNA as in vitro DNA synthesis templates, yet to date it has been difficult to distinguish between true DNA replication and random repair synthesis (2,9). Using 2D gel electrophoresis (5) and other techniques we have recently reported the identification and localization of ctDNA replication origins (oriA and oriB) in each inverted repeat (IR) of tobacco (10,11). Four oris have been reported for Oenothera due to their location as identical pairs in each IR (12). ...
... DNA polymerase activity was assayed for each fraction. Active fractions were tested further for replication activity with specific ctDNA clones as described (10). Fractions which showed high DNA polymerase activity and low nuclease activity were used for further studies. ...
... We have earlier reported the identification and localization of ori regions in the IR of tobacco ctDNA (10,11). In this study we describe detailed in vitro 2D gel analysis of ori regions of tobacco ctDNA using a partially purified tobacco chloroplast DNA replication complex. ...
Using a partially purified replication complex from tobacco chloroplasts, replication origins have been localized to minimal sequences of 82 (pKN8, positions 137 683-137 764) and 243 bp (pKN3, positions 130 513-130 755) for ori A and ori B respectively. Analysis of in vitro replication products by two-dimensional agarose gel electrophoresis showed simple Y patterns for single ori sequence-containing clones, indicative of rolling circle replication. Double Y patterns were observed when a chloroplast DNA template containing both ori s (pKN9) was tested. Dpn I analysis and control assays with Escherichia coli DNA polymerase provide a clear method to distinguish between true replication and DNA repair synthesis. These controls also support the reliability of this in vitro chloroplast DNA replication system. EM analysis of in vitro replicated products showed rolling circle replication intermediates for single ori clones (ori A or ori B), whereas D loops were observed for a clone (pKN9) containing both ori s. The minimal ori regions contain sequences which are capable of forming stem-loop structures with relatively high free energy and other sequences which interact with specific protein(s) from the chloroplast replication fraction. Apparently the minimal ori sequences reported here contain all the necessary elements for support of chloroplast DNA replication in vitro.
... 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 trn I(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 ycf 1 (Kunnimalaiyaan and Nielsen 1997a;Kunnimalaiyaan et al. 1997b). ...
... Comparisons of these 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. ...
Plastid DNA is conserved, highly polyploid and uniform within aplant reflecting efficient
plastid DNA replication/recombination/repair (DNA-RRR) pathways. We will review the current understanding
of the DNA sequences, proteins, and mechanisms involved in plastid genome maintenance. This includes
analysis of the topological forms of plastid DNA, models of plastid DNA replication, homologous recombination,
replication slippage, DNA repair, and plastid DNA-RRR-proteins. We will focus on flowering plants
but include information from algae when relevant. Plastid DNA is comprised of amultimeric series
of circular, linear, and branched forms. Variant plastid DNA molecules include small linear palindromes
with hairpin ends. Plastid transformation has demonstrated an efficient homologous recombination
pathway, acting on short ∼200 bp sequences, that is active throughout shoot development.
These functional studies involving plastid transformation to manipulate DNA sequences, combined with
genomics and reverse genetics to isolate mutants in plastid DNA-RRR proteins, will be particularly
important for making progress in this field.
... In line with this model, two replication origins -oriA and oriBwere found in N. tabacum, on both of which, independently, r replication could be initiated in vitro and in isolated chloroplasts. h replication could be started in vitro and in isolated chloroplasts involving both oriA and oriB (Kunnimalaiyaan and Nielsen, 1997b;Kunnimalaiyaan et al., 1997;Lu et al., 1996;Lugo et al., 2004). Both oris are located in the inverted repeats, therefore there are two copies of both on one monomeric plastid DNA molecule. ...
... According to the Kolodner and Tewari (1975) model, plastid DNA replication of N. tabacum initiates on a pair of replication origins: oriA and oriB (Kunnimalaiyaan and Nielsen, 1997b;Kunnimalaiyaan et al., 1997;Lu et al., 1996;Lugo et al., 2004). Pairs of oris were also detected in other species: (Kunnimalaiyaan and Nielsen, 1997b), the suspension culture ori (Takeda et al., 1992;Wang et al., 2002), the putative homologue of oriA of Oenothera elata (Chiu and Sears, 1992), the putative homologue of an ori near psbA or rpl16, respectively, of Zea mays (Carrillo and Bogorad, 1988) and near rpl16 of Chlamydomonas reinhardtii (Lou et al., 1987) and Glycine max (Hedrick et al., 1993) are given. ...
According to the Kolodner and Tewari model [Kolodner, R.D. and Tewari, K.K. (1975) Nature, 256, 708.], plastid DNA replication involves displacement-loop and rolling-circle modes of replication, which are initiated on a pair of origins of replication (ori). In accordance with the model, such a pair of oris -oriA and oriB- was described in Nicotiana tabacum [Kunnimalaiyaan, M. and Nielsen B.L. (1997b) Nucl. Acids Res. 25, 3681.]. However, as reported previously, both copies of oriA can be deleted without abolishing replication. Deletion of both oriBs was not found [Mühlbauer, S.K. et al. (2002) Plant J. 32, 175.]. Here we describe new ori inactivation lines, in which one oriB is deleted and the other copy is strongly mutated. In addition, lines oriA and oriB were deleted from the same inverted repeat. In contrast to the expectations of the model, neither oriA nor oriB is essential. Some of the deletions led to reduced growth of plants and reduced plastid DNA copy number in later stages of leaf development. The gross structure of plastid DNA was unchanged; however, the location of the ends of branched plastid DNA complexes was different in the inactivation mutants. Taken together, the results indicate that there are additional mechanisms of plastid DNA replication and/or additional origins of replication. These mechanisms seem to be different from those found in eubacteria, which, according to the endosymbiont theory, are the progenitors of plastids.
... Proposed models include displacement-loop and rolling circle (θ and σ; [186]) replication of circular templates, and recombination dependent mechanisms acting on circular or linear templates [94, 187,188]. There is strong experimental evidence for multiple replication origins mapping to both the IR and single copy regions [189][190][191][192][193][194][195][196]. Several different groups using distinct approaches have demonstrated that ptDNA is present in vivo in many forms. ...
The plastid genome (plastome) has proved a valuable source of data for evaluating evolutionary relationships among angiosperms. Through basic and applied approaches, plastid transformation technology offers the potential to understand and improve plant productivity, providing food, fiber, energy, and medicines to meet the needs of a burgeoning global population. The growing genomic resources available to both phylogenetic and biotechnological investigations is allowing novel insights and expanding the scope of plastome research to encompass new species. In this chapter, we present an overview of some of the seminal and contemporary research that has contributed to our current understanding of plastome evolution and attempt to highlight the relationship between evolutionary mechanisms and the tools of plastid genetic engineering.
... In the flowering plants studied so far, it has been shown that flip-flop recombination and inversions predominantly occur around the origin of replication (ori). In some angiosperms, the ori B maps to the rDNA-ycf1 region within the IR, which is located more closely to the IR-SSC-boundary than to the IR-LSC junction (Thompson et al. 1986; Lu et al. 1996; Kunnimalaiyaan and Nielsen 1997; Eisen et al. 2000; Mackiewicz et al. 2001). ...
This review bridges functional and evolutionary aspects of plastid chromosome architecture in land plants and their putative ancestors. We provide an overview on the structure and composition of the plastid genome of land plants as well as the functions of its genes in an explicit phylogenetic and evolutionary context. We will discuss the architecture of land plant plastid chromosomes, including gene content and synteny across land plants. Moreover, we will explore the functions and roles of plastid encoded genes in metabolism and their evolutionary importance regarding gene retention and conservation. We suggest that the slow mode at which the plastome typically evolves is likely to be influenced by a combination of different molecular mechanisms. These include the organization of plastid genes in operons, the usually uniparental mode of plastid inheritance, the activity of highly effective repair mechanisms as well as the rarity of plastid fusion. Nevertheless, structurally rearranged plastomes can be found in several unrelated lineages (e.g. ferns, Pinaceae, multiple angiosperm families). Rearrangements and gene losses seem to correlate with an unusual mode of plastid transmission, abundance of repeats, or a heterotrophic lifestyle (parasites or myco-heterotrophs). While only a few functional gene gains and more frequent gene losses have been inferred for land plants, the plastid Ndh complex is one example of multiple independent gene losses and will be discussed in detail. Patterns of ndh-gene loss and functional analyses indicate that these losses are usually found in plant groups with a certain degree of heterotrophy, might rendering plastid encoded Ndh1 subunits dispensable.
... The only previous sequence-specific ctDNA origin binding protein that has been identified is from Chlamydomonas (Wu et al. 1989). Two replication oris (oriA and oriB) have been identified in chloroplasts of higher plants (Lu et al. 1996;Kunnimalaiyaan and Nielsen 1997a, b). We report here the purification and characterization of an ori-binding protein from total soybean chloroplast protein extracts via sequence-specific DNA affinity chromatography and MALDI-ToF mass spectrometry. ...
Replication of chloroplast DNA (ctDNA) in several plants and in Chlamydomonas reinhardii has been shown to occur by a double displacement loop (D-loop) mechanism and potentially also by a rolling circle mechanism. D-loop replication origins have been mapped in several species. Minimal replication origin sequences used as probes identified two potential binding proteins by southwestern blot analysis. A 28 kDa (apparent molecular weight by SDS-PAGE analysis) soybean protein has been isolated by origin sequence-specific DNA affinity chromatography from total chloroplast proteins. Mass spectrometry analysis identified this protein as the product of the soybean C6SY33 gene (accession number ACU14156), which is annotated as encoding a putative uncharacterized protein with a molecular weight of 25,897 Da, very near the observed molecular weight of the purified protein based on gel electrophoresis. Western blot analysis using an antibody against a homologous Arabidopsis protein indicates that this soybean protein is localized specifically in chloroplasts. The soybean protein shares some homology within a single-stranded DNA binding (SSB) domain of E. coli SSB and an Arabidopsis thaliana mitochondrial-localized SSB of about 21 kDa (mtSSB). However, the soybean protein induces a specific electrophoretic mobility shift only when incubated with a double-stranded fragment containing the previously mapped ctDNA replication oriA region. This protein has no electrophoretic mobility shift activity when incubated with single-stranded DNA. In contrast, the Arabidopsis mtSSB causes a mobility shift only with single-stranded DNA but not with the oriA fragment or with control dsDNA of unrelated sequence. These results suggest that the 26 kDa soybean protein is a specific origin binding protein that may be involved in initiation of ctDNA replication.
... The pLD-LH-CTB (Figure 1(a)) vector integrates the genes of interest into the inverted repeat regions through homologous recombination events between the trnI and trnA chloroplast border sequences of the transformation vector and the corresponding homologous sequences of the chloroplast genome. Unique advantages of integration of foreign genes at this site include accomplishment of homoplasmy in the first round of selection due to the presence of a complete chloroplast origin of replication within the flanking sequence 31,32 and doubling the copy number of foreign genes per cell. The chimeric aminoglycoside 3′ adenylyltransferase (aadA) gene, conferring resistance to spectinomycin and streptomycin, and the CTB gene downstream of it are driven by the constitutive promoter of the rRNA operon (Prrn), each with individual ribosome-binding sites (GGAGG), and the foreign transcripts are stabilized by the psbA3′ untranslated region. ...
The B subunits of enterotoxigenic Escherichia coli (LTB) and cholera toxin of Vibrio cholerae (CTB) are candidate vaccine antigens. Integration of an unmodified CTB-coding sequence into chloroplast genomes (up to 10,000 copies per cell), resulted in the accumulation of up to 4.1 % of total soluble tobacco leaf protein as functional oligomers (410-fold higher expression levels than that of the unmodified LTB gene expressed via the nuclear genome). However, expression levels reported are an underestimation of actual accumulation of CTB in transgenic chloroplasts, due to aggregation of the oligomeric forms in unboiled samples similar to the aggregation observed for purified bacterial antigen. PCR and Southern blot analyses confirmed stable integration of the CTB gene into the chloroplast genome. Western blot analysis showed that the chloroplast- synthesized CTB assembled into oligomers and were antigenically identical with purified native CTB. Also, binding assays confirmed that chloroplast-synthesized CTB binds to the intestinal membrane GM1-ganglioside receptor, indicating correct folding and disulfide bond formation of CTB pentamers within transgenic chloroplasts. In contrast to stunted nuclear transgenic plants, chloroplast transgenic plants were morphologically indistinguishable from untransformed plants, when CTB was constitutively expressed in chloroplasts. Introduced genes were inherited stably in subsequent generations, as confirmed by PCR and Southern blot analyses. Increased production of an efficient transmucosal carrier molecule and delivery system, like CTB, in transgenic chloroplasts makes plant-based oral vaccines and fusion proteins with CTB needing oral administration commercially feasible. Successful expression of foreign genes in transgenic chromoplasts and availability of marker-free chloroplast transformation techniques augurs well for development of vaccines in edible parts of transgenic plants. Furthermore, since the quaternary structure of many proteins is essential for their function, this investigation demonstrates the potential for other foreign multimeric proteins to be properly expressed and assembled in transgenic chloroplasts.
... In vivo chromosomal replication has been successfully mapped by 2-D gel analysis in a wide variety of eukaryotes (9)(10)(11)(12)(13)(14), but relatively few prokaryotes (15)(16)(17). Prokaryotes are often poor subjects for 2-D gel analysis as the majority are difficult to culture with synchronized chromosomal DNA replication. ...
The α-proteobacterium Caulobacter crescentus possesses a developmental cell cycle that restricts chromosome replication to a stalked cell type. The proposed C.crescentus chromosome replication origin (Cori) lies between hemE and RP001, an unusual intergenic region not previously associated with bacterial replication origins, although a similar
genomic arrangement is also present at the putative replication origin in the related bacterium Rickettsia prowazekii. The cloned Cori supports autonomous plasmid replication selectively in the stalked cell type implying that replication of the entire chromosome
also initiates between hemE and RP001. To confirm this location, we applied the 2-D (N/N) agarose gel electrophoresis technique to resolve and identify
chromosome replication intermediates throughout a 30 kb region spanning Cori. Replication initiation in Cori was uniquely characterized by an ‘origin bubble and Y-arc’ pattern and this observation was supported by simple replication
fork ‘Y-arc’ patterns that characterized the regions flanking Cori. These replication forks originated bi-directionally from within Cori as determined by the fork direction assay. Therefore, chromosomal replication initiates from the unusual hemE/RP001 intergenic region that we propose represents a new class of replication origins.
... Fine mapping of the sequences of both tobacco replication origins has been made in in vitro replication experiments (Kunnimalaiyaan and Nielsen, 1997b), which do not necessarily reproduce the situation in the plastid. In vivo analysis of replication origins has been restricted to the analysis of displacement loops up to now, which does not allow precise localisation (Kunnimalaiyaan et al., 1997;Lu et al., 1996). In an earlier study with proplastids of cultured tobacco cells, displacement loops were mapped to the ends of the Inverted Repeat near to, but not identical to the described oriB locus, and to the large single copy region of the plastome, but not to the region of oriA (Takeda et al., 1992). ...
Sequences described as chloroplast DNA replication origins were analysed in vivo by creating deletion and insertion mutants via plastid transformation in tobacco. Deletion of the described oriA sequence, which is located within the intron of the trnI gene, resulted in heteroplastomic transformants, when the selection marker was inserted within the intron. Removal of the complete intron sequence together with the oriA sequence, however, yielded homoplastomic transformants of normal phenotype, in which wild-type signals were no longer detectable through Southern analysis, thus bringing the role of the described oriA sequence for plastome replication into question. Similarly, deletion of sequence elements upstream of trnI, which have a possible ori function in Oenothera, did not show any effect in tobacco. The two copies of oriB, which are located at the very end of the plastome Inverted Repeats, were targeted with two different transformation vectors in a cotransformation approach. While in initial transformants integration of the selection marker could be detected at both sites, the transgene was found exclusively at one site or the other after additional rounds of regeneration. Whereas the copy of oriB in Inverted Repeat B could be completely deleted, targeting of the copy in Inverted Repeat A resulted in heteroplastomic lines, as the essential ycf1 gene was also affected. Due to the strong selection against cotransformants we conclude that at least one copy of the oriB sequence is essential for plastome replication, whereas replication appears possible without oriA elements.
... In vitro replication analysis has revealed that the minimal sequences required for activity of single-ori ctDNA templates in tobacco is 82 bp for oriA and 243 bp for oriB (Kunnimalayaan and Nielsen, 1997b). There are conflicting reports on the effect of deletions of chloroplast oris (Muhlbauer et al., 2002; Kunnimalayaan and Nielsen, 1997b; Lu et al., 1996; Lugo et al., 2004). It is quite possible that disruption of an ori in vivo is not lethal if another mechanism can take over. ...
Chloroplast genetic engineering overcomes concerns of gene containment, low levels of transgene expression, gene silencing, positional and pleiotropic effects or presence of vector sequences in transformed genomes. Several therapeutic proteins and agronomic traits have been highly expressed via the tobacco chloroplast genome but extending this concept to important crops has been a major challenge; lack of 100 homologous species-specific chloroplast transformation vectors containing suitable selectable markers, ability to regulate transgene expression in developing plastids and inadequate tissue culture systems via somatic embryogenesis are major challenges. We employed a 'Double Gene/Single Selection (DGSS)' plastid transformation vector that harbors two selectable marker genes (aph A-6 and npt II) to detoxify the same antibiotic by two enzymes, irrespective of the type of tissues or plastids; by combining this with an efficient regeneration system via somatic embryogenesis, cotton plastid transformation was achieved for the first time. The DGSS transformation vector is at least 8-fold (1 event/2.4 bombarded plates) more efficient than 'Single Gene/Single Selection (SGSS)' vector (aph A-6; 1 event per 20 bombarded plates). Chloroplast transgenic lines were fertile, flowered and set seeds similar to untransformed plants. Transgenes stably integrated into the cotton chloroplast genome were maternally inherited and were not transmitted via pollen when out-crossed with untransformed female plants. Cotton is one of the most important genetically modified crops (120 billion US dollars US annual economy). Successful transformation of the chloroplast genome should address concerns about transgene escape, insects developing resistance, inadequate insect control and promote public acceptance of genetically modified cotton.
... Replication origins have been reported in the ribosomal RNA genes of a number of organisms, including human, [47][48][49][50] mouse, 51,52 rat, 49 schizosaccharomyces pombe, 53 saccharomyces cerevisiae, 54,55 tetrahymena thermophila, 56,57 pisum sativum 58 and tobacco chloroplast. 59 Within human ribosomal RNA genes, analyses of replication intermediate structures suggested a broad initiation zone covering the entire 30 kb non-transcribed spacer region (NTS), while other approaches suggest a highly preferred initiation region exists within a 5-10 kb NTS sequence upstream of the 18S transcribed sequence. [47][48][49][50] This latter region contains Alu elements, A:T-rich regions, a DNA bending region and purine/pyrimidine tracts, 60,61 all sequence elements that have been found within other mammalian replication origins. ...
Numerous studies have demonstrated that DNA replication initiates within the 30 kB non-transcribed spacer (NTS) region of the human ribosomal RNA gene (rDNA). Using a series of closely spaced primer pairs to measure nascent leading strand abundance in mid and late S phase cells isolated by centrifugal elutriation, we find evidence for one highly preferred initiation site and two less utilized sites within a 6 kb region of the NTS. The initiation sites colocalize with significant DNA unwinding elements (DUEs), matrix attachment regions (MARs), and ARS-like sequences. An intrinsic DNA bending site was localized by circular permutation analysis to within several hundred base pairs of one initiation site. While DUE and MAR elements occur elsewhere throughout the 43 kb rDNA sequence, the close association of DUE and MAR elements occurs only near replication initiation sites, a juxtaposition also seen in other well-studied mammalian replication initiation sites. The utilization of rDNA initiation sites close to DUE and MAR elements in mid and late S phase, but not in very early S phase as previously shown, suggests that in rRNA genes, contributions from these sequence-associated properties may be more significant to initiation sites associated with transcriptionally inactive genes, than to initiation sites associated with transcriptionally active genes.
Plastids, like mitochondria, result from an ancient endosymbiosis event and contain a distinct genome. Though many plastid genes have since been transferred to the nuclear genome, the small plastid genome still encodes between 90 and 100 genes, which are notably involved in translation, transcription, and energy metabolism in the plastid. The many roles of this organelle, the most familiar being photosynthesis in chloroplasts, make it essential for the development of higher plants. As such, the ability of the plastid to maintain the stability of its genome represents a crucial element of plant life. The physical organization of the genome itself can have an influence on DNA metabolism, with its large inverted repeats acting as templates for recombination. Furthermore, the localization of chloroplast DNA near elements of the electron transport chain increases the importance of DNA repair mechanisms, as reactive oxygen species (ROS) appear as by-products of photosynthesis. These ROS, along with UV radiation and DNA double-strand breaks, create a genotoxic stress through their respective ability to oxidize nucleotides, link DNA bases, or rearrange the structure of the genome. To minimize the deleterious effects of these events, different mechanisms present in the nucleus such as homologous recombination exist in plastids. Some less conservative mechanisms based on sequences of microhomology are also found, and sometimes lead to copy-number variation in certain areas of the plastid genome. While some of these changes can remain silent, others can be linked to phenotypes such as variegation.
DNA replication, transcription of the circular plastid genome and plastid division represent fundamental processes that take place in all types of the multifunctional, multiple-form plastids. The components of the plastid genetic system are highly expressed during early phases of chloroplast development corresponding to the establishment of 'housekeeping' functions before the onset of photosynthesis. A survey of fundamental and recent work is presented concerning plastid division, the organization of the plastid genome and the characterization of enzymes implicated in replication and transcription.
To maintain and to differentiate into various plastid lineages, replication of the plastid DNA (ptDNA) and division of the plastid must take place. However, replication initiation of the ptDNA has been less understood. The present study describes identification of the initiation region (origin) of ptDNA replication in the rice cultured cells. RNA-primed newly replicated DNA strands pulse-labeled with bromodeoxyuridine were isolated and size-fractionated. Locations of these nascent strands on the ptDNA determined the two major origin regions around the 3′ region of each 23S rDNA in the inverted repeats (IRA and IRB). Two-dimensional agarose gel electrophoresis of the replication intermediates suggested that replication from each origin proceeds bidirectionally. This contrasted to replication by the double D - loop mechanism.
The plastid genome (plastome) has proved a valuable source of data for evaluating evolutionary relationships among angiosperms. Through basic and applied approaches, plastid transformation technology offers the potential to understand and improve plant productivity, providing food, fiber, energy and medicines to meet the needs of a burgeoning global population. The growing genomic resources available to both phylogenetic and biotechnological investigations are allowing novel insights and expanding the scope of plastome research to encompass new species. In this chapter we present an overview of some of the seminal and contemporary research that has contributed to our current understanding of plastome evolution and attempt to highlight the relationship between evolutionary mechanisms and tools of plastid genetic engineering.
Chloroplasts contain circular DNA molecules which are found in low copy number in proplastids but are amplified to very high copy number in actively dividing leaf cells. A double displacement loop (D-loop) mechanism for chloroplast DNA (ctDNA) replication has been proposed, and pairs of replication origins which fit this model have been identified in some species. It appears that ctDNA replication is under the control of at least some nuclear gene products, as genes for DNA polymerase, topoisomerases, DNA primase and other accessory replication proteins have not been reported in the sequenced chloroplast genomes, and ctDNA replication remains active in the absence of active chloroplast transcription or translation. Only a few chloroplast replication proteins have been isolated, and to date most have not been characterized in detail. The mechanism by which ctDNA copy number is regulated during plant development is not known. In this review we summarize the current understanding of ctDNA replication.
The methylation status of carrot (Daucus carota L.) mitochondrial DNA (mtDNA) was studied using isoschizomeric restriction enzymes MspI/HpaII (CCGG) and MvaI/EcoRII [CC(A/T)GG]. Southern hybridisations with probes for mitochondrial genes coxII and atpA were performed. MtDNAs isolated from non-embryogenic cell suspensions and roots were analysed. No differences were found
using MspI/HpaII but after digesting the mtDNA with MvaI and EcoRII, some qualitative and quantitative differences between the restriction patterns appeared. Distinction was also revealed
after Southern hybridisation with the coxII probe. These data indicate that the mtDNA of carrot is methylated in CNG trinucleotides and unmethylated in CG dinucleotides
in CCGG sequences. The results were reproducible for cell suspensions of various genotypes and even cultivars but the extent
of methylation was different in the root. The possible role of methylation in the mitochondrial genome of higher plants is
discussed.
To test the possibility of producing the novel vaccine in plants against diarrhea normally found in neonatal and newly weaned
piglets, the faeG gene, encoding a major F4ac fimbrial subunit protein, was introduced into the tobacco chloroplast genome. After two rounds
of selection under spectinomycin, we obtained the transgenic plants nearly homoplasmic. RNA gel blot analysis indicated that
faeG and the antibiotic selective gene aminoglycoside 3′ adenylyltransferase (aadA) were highly transcribed as a dicistron, while the translational level of recombinant FaeG in transplastomic tobacco was
about 0.15% of total soluble protein. The immunogenicity of recombinant FaeG produced in tobacco chloroplasts was confirmed
by the observation that FaeG-specific antibodies were elicited in mice immunized with total soluble protein of transgenic
plants, as well as the result that mouse sera stimulated by chloroplast-derived recombinant FaeG could neutralize F4ac enterotoxigenic
Escherichia coli (ETEC) in vivo. This study provides a new alternative for producing the ETEC vaccine using the chloroplast expression system.
In common with other apicomplexan parasites, Plasmodium falciparum, a causative organism of human malaria, harbours a residual plastid derived from an ancient secondary endosymbiotic acquisition of an alga. The function of the 35 kb plastid genome is unknown, but its evolutionary origin and genetic content make it a likely target for chemotherapy. Pulsed field gel electrophoresis and ionizing radiation have shown that essentially all the plastid DNA comprises covalently closed circular monomers, together with a tiny minority of linear 35 kb molecules. Using two-dimensional gels and electron microscopy, two replication mechanisms have been revealed. One, sensitive to the topoisomerase inhibitor ciprofloxacin, appears to initiate at twin D-loops located in a large inverted repeat carrying duplicated rRNA and tRNA genes, whereas the second, less drug sensitive, probably involves rolling circles that initiate outside the inverted repeat.
The 35kb apicoplast genomes (plDNA) of Plasmodium falciparum and Toxoplasma gondii share close sequence similarity but differ in their in vivo topologies. Although sequence analysis of tandem repeats of T. gondii plDNA has suggested the presence of replication initiation sites within the inverted repeat region, the replication origins (ori) of the P. falciparum circular plDNA have not been identified. Using 5' end-labelled nascent DNA as probe, we demonstrate that the ori of P. falciparum plDNA is localised within the inverted repeat region. Our results also indicate the presence of two initiation sites within each inverted repeat segment of the circular plDNA of P. falciparum suggestive of a four D-loop/bi-directional ori mechanism of DNA replication.
In a previous study, we mapped replication origin regions of the plastid DNA around the 3' end of the 23S rRNA gene in rice suspension-cultured cells. Here, we examined initiation of the plastid DNA replication in different rice cells by two-dimensional agarose gel electrophoresis. We show for the first time, to our knowledge, that the replication origin region of the plastid DNA differs among cultured cells, coleoptiles and mature leaves. In addition, digestion of the replication intermediates from the rice cultured cells with mung bean nuclease, a single-strand-specific nuclease, revealed that both two single strands of the double-stranded parental DNA were simultaneously replicated in the origin region. This was further confirmed by two-dimensional agarose gel analysis with single-stranded RNA probes. Thus, the mode of plastid DNA replication presented here differs from the unidirectional replication started by forming displacement loops (D-loops), in which the two D-loops on the opposite strands expand toward each other and only one parental strand serves as a template.
Mitochondrial genomes in higher plants are much larger and more complex as compared to animal mitochondrial genomes. There is growing evidence that plant mitochondrial genomes exist predominantly as a collection of linear and highly branched DNA molecules and replicate by a recombination-dependent mechanism. However, biochemical evidence of mitochondrial DNA (mtDNA) recombination activity in plants has previously been lacking. We provide the first report of strand-invasion activity in plant mitochondria. Similar to bacterial RecA, this activity from soybean is dependent on the presence of ATP and Mg(2+). Western blot analysis using an antibody against the Arabidopsis mitochondrial RecA protein shows cross-reaction with a soybean protein of about 44 kDa, indicating conservation of this protein in at least these two plant species. mtDNA structure was analyzed by electron microscopy of total soybean mtDNA and molecules recovered after field-inversion gel electrophoresis (FIGE). While most molecules were found to be linear, some molecules contained highly branched DNA structures and a small but reproducible proportion consisted of circular molecules (many with tails) similar to recombination intermediates. The presence of recombination intermediates in plant mitochondria preparations is further supported by analysis of mtDNA molecules by 2-D agarose gel electrophoresis, which indicated the presence of complex recombination structures along with a considerable amount of single-stranded DNA. These data collectively provide convincing evidence for the occurrence of homologous DNA recombination in plant mitochondria.
Higher plant plastid DNA (ptDNA) is generally described as a double-stranded circular molecule of the size of the monomer of the plastid genome. Also, the substrates and products of ptDNA replication are generally assumed to be circular molecules. Linear or partly linear ptDNA molecules were detected in our present study using pulsed-field gel electrophoresis and Southern blotting of ptDNA restricted with 'single cutter' restriction enzymes. These linear DNA molecules show discrete end points which were mapped using appropriate probes. One possible explanation of discrete ends would be that they represent origins of replication. Indeed, some of the mapped ends correlate well with the known origins of replication of tobacco plastids, i.e. both of the oriA sequences and--less pronouncedly--with the oriB elements. Other ends correspond to replication origins that were described for Oenothera hookeri, Zea mays, Glycine max and Chlamydomonas reinhardtii, respectively, while some of the mapped ends were not described previously and might therefore represent additional origins of replication.
Chloroplasts contain multiple copies of a DNA molecule (the plastome) that encodes many of the gene products required to perform photosynthesis. The plastome is replicated by nuclear-encoded proteins and its copy number seems to be highly regulated by the cell in a tissue-specific and developmental manner. Our understanding of the biochemical mechanism by which the plastome is replicated and the molecular basis for its regulation is limited. In this commentary we review our present understanding of chloroplast DNA replication and examine current efforts to elucidate its mechanism at a molecular level.
We have compared the sequence organization of four previously uncharacterized legume chloroplast DNAs - from alfalfa, lupine, wisteria and subclover — to that of legume chloroplast DNAs that either retain a large, ribosomal RNA-encoding inverted repeat (mung bean) or have deleted one half of this repeat (broad bean). The circular, 126 kilobase pair (kb) alfalfa chloroplast genome, like those of broad bean and pea, lacks any detectable repeated sequences and contains only a single set of ribosomal RNA genes. However, in contrast to broad bean and pea, alfalfa chloroplast DNA is unrearranged (except for the deletion of one segment of the inverted repeat) relative to chloroplast DNA from mung bean. Together with other findings reported here, these results allow us to determine which of the four possible inverted repeat configurations was deleted in the alfalfa-pea-broad bean lineage, and to show how the present-day broad bean genome may have been derived from an alfalfa-like ancestral genome by two major sequence inversions. The 147 kb lupine chloroplast genome contains a 22 kb inverted repeat and has essentially complete colinearity with the mung bean genome. In contrast, the 130 kb wisteria genome has deleted one half of the inverted repeat and appears colinear with the alfalfa genome. The 140 kb subclover genome has been extensively rearranged and contains a family of at least five dispersed repetitive sequence elements, each several hundred by in size; this is the first report of dispersed repeats of this size in a land plant chloroplast genome. We conclude that the inverted repeat has been lost only once among legumes and that this loss occurred prior to all the other rearrangements observed in subclover, broad bean and pea. Of those lineages that lack the inverted repeat, some are stable and unrearranged, other have undergone a moderate amount of rearrangement, while still others have sustained a complex series of rearrangement either with or without major sequence duplications and transpositions. Peer Reviewed http://deepblue.lib.umich.edu/bitstream/2027.42/46960/1/294_2004_Article_BF00355401.pdf
We have examined the replication patterns of ribosomal DNA plasmids in vivo and in vitro using Xenopus eggs. Plasmids carrying different parts of the Xenopus ribosomal DNA sequence were allowed to replicate either in vitro in an egg extract or in vivo after microinjection into unfertilized eggs. The replication intermediates were analyzed by the 2D gel electrophoretic technique
of Brewer and Fangman (1), using original or modified electrophoresis conditions. With standard electrophoresis conditions,
the patterns obtained for restriction fragments larger than 5 kb were unreliable because of artefactually distorted Y arcs
and unrecognizable bubble arcs. Interpretable patterns could nevertheless be obtained using suitably modified electrophoresis
parameters. Under these conditions, replication was found to initiate and terminate at multiple, random locations on each
plasmid both in vivo and in vitro. However, only one or very few of these potential initiation sites are used during the replication of an individual plasmid
molecule. We discuss the possible artefacts and misinterpretations that can result when the 2D electrophoresis parameters
are not adapted to the size of the fragment examined. We also discuss the relevance of the random replication mode to the
mechanisms and the control of DNA replication in eukaryotes.
We have investigated the frequency of replication origin usage in bacterial plasmids containing more than one potential origin. Escherichia coli recA- cells were selectively transformed with pBR322 monomers, dimers, or trimers. Plasmid DNA was isolated and digested with a restriction enzyme that cut the monomer only once, and the replicative intermediates (RIs) were analyzed by neutral/neutral two-dimensional agarose gel electrophoresis. Evidence for initiation outside the linearized plasmid was found only for oligomers. Moreover, in dimers, the intensity of the signal indicative for external initiation was equivalent to that reflecting internal initiation, whereas it was approximately twice as strong in trimers. To determine whether initiation could occur simultaneously at two origins in a single plasmid, we studied the replication of a neodimer in which both units could be unambiguously distinguished. The results showed that although both origins were equally competent to initiate replication, only one was active per plasmid. These observations strongly suggest that in ColE1 plasmids, replication initiates at a single site even when there are several identical potential origins per plasmid. In addition to the conventional two-dimensional gel patterns, novel specific patterns were observed with intensities that varied from one DNA sample to another. These unique patterns were the result of breakage of the RIs at a replication fork. This type of breakage changes both the mass and shape of RIs. When the entire population of RIs is affected, a new population of molecules is formed that may generate a novel pattern in two-dimensional gels.
Studies of chloroplast DNA variations, and several direct experimental observations, indicate the existence of recombination ability in algal and higher plant plastids. However, no studies have been done of the biochemical pathways involved. Using a part of a cyanobacterial recA gene as a probe in Southern blots, we have found homologous sequences in total DNA from Pisum sativum and Arabidopsis thaliana and in a cDNA library from Arabidopsis. A cDNA was cloned and sequenced, and its predicted amino acid sequence is 60.7% identical to that of the cyanobacterial RecA protein. This finding is consistent with our other results showing both DNA strand transfer activity and the existence of a protein of the predicted molecular mass crossreactive with antibodies to Escherichia coli RecA in the stroma of pea chloroplasts.
Two complementary two-dimensional gel electrophoretic techniques have recently been developed that allow initiation sites
to be mapped with relative precision in eukaryotic genomes at least as complex as those of yeast and Drosophila melanogaster.
We reported the first application of these mapping methods to a mammalian genome in a study on the amplified dihydrofolate
reductase (DHFR) domain of the methotrexate-resistant CHO cell line CHOC 400 (J.P. Vaughn, P.A. Dijkwel, and J.L. Hamlin,
Cell 61:1075-1087, 1990). Our results suggested that in this 240-kb domain, initiation of nascent DNA strands occurs at many
sites within a 30- to 35-kb zone mapping immediately downstream from the DHFR gene. In the course of these studies, it was
necessary to develop methods to stabilize replication intermediates against branch migration and shear. This report describes
these stabilization methods in detail and presents a new enrichment protocol that extends the neutral/neutral two-dimensional
gel mapping method to single-copy loci in mammalian cells. Preliminary analysis of replication intermediates purified from
CHO cells by this method suggests that DNA synthesis may initiate at many sites within a broad zone in the single-copy DHFR
locus as well.
Recently, two 2-dimensional (2D) gel techniques, termed neutral/neutral and neutral/alkaline, have been developed and employed
to map replication origins in eukaryotic plasmids and chromosomal DNA (1–11). The neutral/neutral technique, which requires
less DNA for analysis, has been preferentially used in recent studies. We show here that the signal predicted for an origin
is not detected using the neutral/neutral technique if the origin is located near the end of the analyzed restriction fragment.
We also demonstrate that analysis of the same batch of DNA by the two different mapping techniques can generate apparently
contradictory results: in some situations where neutral/alkaline 2D analysis indicates that a certain origin is always used,
neutral/neutral 2D analysis suggests that the origin is not always used. Several possible explanations for this type of disagreement
between the two techniques are discussed, and we conclude that it is important to use both techniques in combination in order
to minimize possible misinterpretations.
The locations of the two replication origins in pea chloroplast DNA (ctDNA) have been mapped by electron microscopic analysis of restriction digests of supercoiled ctDNA cross-linked with trioxalen. Both origins of replication, identified as displacement loops (D-loops), were present in the 44-kilobase-pair (kbp) SalI A fragment. The first D-loop was located at 9.0 kbp from the closest SalI restriction site. The average size of this D-loop was about 0.7 kbp. The second D-loop started 14.2 kbp in from the same restriction site and ended at about 15.5 kbp, giving it a size of about 1.3 kbp. The orientation of these two D-loops on the restriction map of pea ctDNA was determined by analyzing SmaI, PstI, and SalI-SmaI restriction digests of pea ctDNA. One D-loop has been mapped in the spacer region between the 16S and 23S rRNA genes. The second D-loop was located downstream of the 23S rRNA gene. Denaturation mapping of recombinants pCP 12-7 and pCB 1-12, which contain both D-loops, confirmed the location of the D-loops in the restriction map of pea ctDNA. Denaturation-mapping studies also showed that the two D-loops had different base compositions; the one closest to a SalI restriction site denatured readily compared with the other D-loop. The recombinants pCP 12-7 and pCB 1-12 were found to be highly active in DNA synthesis when used as templates in a partially purified replication system from pea chloroplasts. Analysis of in vitro-synthesized DNA with either of these recombinants showed that full-length template DNA was synthesized. Recombinants from other regions of the pea chloroplast genome showed no significant DNA synthesis activity in vitro.
In previous studies, we utilized a neutral/neutral two-dimensional (2-D) gel replicon mapping method to analyze the pattern of DNA synthesis in the amplified dihydrofolate reductase (DHFR) domain of CHOC 400 cells. Replication forks appeared to initiate at any of a large number of sites scattered throughout the 55 kb region lysing between the DHFR and 2BE2121 genes, and subsequently to move outward through the two genes. In the present study, we have analyzed this locus in detail by a complementary, neutral/alkaline 2-D gel technique that determines the direction in which replication forks move through a region of interest. In the early S period, forks are observed to travel in both directions through the intergenic region, but only outward through the DHFR gene. Surprisingly, however, replication forks also move in both directions through the 2BE2121 gene. Furthermore, in early S phase, small numbers of replication bubbles can be detected in the 2BE2121 gene on neutral/neutral 2-D gels. In contrast, replication bubbles have never been detected in the DHFR gene. Thus, replication initiates not only in the intergenic region, but also at a lower frequency in the 2BE2121 gene. We further show that only a small fraction of DHFR amplicons sustains an active initiation event, with the rest being replicated passively by forks from distant amplicons. These findings are discussed in light of other experimental approaches that suggest the presence of a much more narrowly circumscribed initiation zone within the intergenic region.
Studies of chloroplast DNA variations, and several direct experimental observations, indicate the existence of recombination ability in algal and higher plant plastids. However, no studies have been done of the biochemical pathways involved. Using a part of a cyanobacterial recA gene as a probe in Southern blots, we have found homologous sequences in total DNA from Pisum sativum and Arabidopsis thaliana and in a cDNA library from Arabidopsis. A cDNA was cloned and sequenced, and its predicted amino acid sequence is 60.7% identical to that of the cyanobacterial RecA protein. This finding is consistent with our other results showing both DNA strand transfer activity and the existence of a protein of the predicted molecular mass crossreactive with antibodies to Escherichia coli RecA in the stroma of pea chloroplasts.
Chloroplast DNA (ctDNA) generally occurs as circular molecules with molecular weights (MWs) in the range 70–130 × 106 depending on the species1,2. In Euglena gracilis, ctDNA (44 µm, 92 × 106 MW3) replicates through Cairns-type intermediates4 having structural aspects suggesting bidirectional replication5. Pea and corn ctDNA were shown to contain two displacement loops (D-loops) located 7,100 base pairs (bp) apart6. The displacing strands of the two D-loops are located on opposite strands of the parental DNA; they expand towards each other and form a Cairns replicative intermediate when the two strands elongate past each other. Rolling circle intermediates7, apparently resulting from the continuation of Cairns rounds of replication, have also been observed8. The origins of replication were not located on physical maps for any of these studies. We present here the results of an electron microscopic (EM) study indicating that replication of ctDNA in Euglena is initiated near the 5′ end of the supplementary 16S ribosomal RNA gene9.
Publisher Summary This chapter describes a filter hybridization approach for mapping chloroplast DNA (cpDNA) restriction sites along with several strategies for generating restriction fragments for use as hybridization probes. Approaches for visualizing and mapping cpDNAs that are difficult or impossible to isolate in very pure form are also discussed. The chloroplast genome is densely packed with genes and many of them have been isolated and sequenced. Chloroplast gene products identified thus far function either in photosynthesis or as components of the chloroplast protein synthesizing system. The basic strategy for mapping by overlap hybridization is to hybridize each one of a set of restriction fragments that together cover an entire chloroplast genome to replica membranes containing various single and double digests of total cpDNA. Three classes of individually purified cpDNA restriction fragments that can be used as hybridization probes are homologous, uncloned fragments; homologous, cloned fragments; and heterologous, cloned fragments. Procedures to obtain each class of probe fragments and the relative merits of each class are discussed.
An in vitro chloroplast DNA synthesizing lysate system prepared from purified chloroplasts of Petunia hybrida leaves has been developed. Both co-isolated endogeneous chloroplast (cp)DNA and externally added DNA can be used as DNA templates in the system. The system contains a -like DNA polymerase as determined by using DNA polymerase-specific inhibitors and synthetic templates. The molecular weight of this enzyme is about 85 kd. Part of the DNA synthesizing activity is repair synthesis. When a chimaeric plasmid containing a fragment with a potential cpDNA replication origin is used as a template (pPCY62), specific initiation of DNA synthesis is observed on this fragment which strongly suggests that the in vitro chloroplast lysate system is also capable of replication initiation.
The origins of chloroplast DNA (cpDNA) replication were mapped in two plastome types of Oenothera in order to determine whether variation in the origin of cpDNA replication could account for the different transmission abilities associated with these plastomes. Two pairs of displacement loop (D-loop) initiation sites were observed on closed circular cpDNA molecules by electron microscopy. Each pair of D-loops was mapped to the inverted repeats of the Oenothera cpDNA by the analysis of restriction fragments. The starting points of the two adjacent D-loops are approximately 4 kb apart, bracketing the 16S rRNA gene. Although there are small DNA length variations near one of the D-loop initiation sites, no apparent differences in the number and the location of replication origins were observed between plastomes with the highest (type I) and lowest (type IV) transmission efficiencies.
Covalently closed circular chloroplast DNA (ctDNA) molecules have been isolated from pea, bean, spinach, lettuce, corn and oat plants by ethidium bromide/cesium chloride density-gradient centrifugation. As much as 30–40% of the total ctDNA could be isolated as closed circular DNA molecules and up to 80% of the total ctDNA was found in the form of circular molecules.The size of pea, spinach, lettuce, corn and oat ctDNA relative to an internal standard (ΦX174 replicative form II monomer DNA) was determined by electron microscopy. The ctDNAs showed significant differences in their sizes, and their molecular weights ranged from 85.4 · 106 for corn ctDNA to 96.7 · 106 for lettuce ctDNA. Each of these ctDNAs contained 3–4% of the circular molecules as circular dimers and 1–2% of the circular molecules as catenated dimers.The molecular complexity of these ctDNAs was studied by renaturation kinetics using T4 DNA as a standard. The molecular weights of the unique sequence of the ctDNAs ranged from 83.7 · 106 for oat ctDNA to 93.1 · 106 for lettuce ctDNA, which are in excellent agreement with the sizes of the circular ctDNA molecules. No repeating sequences were detected in any of the ctDNAs.The ctDNAs from pea, lettuce, corn, and spinach were studied by thermal denaturation using T4 DNA as a standard. All of the ctDNAs melted more broadly than T4 DNA and they all had a distinctly different . For example, the of pea ctDNA was 1°C below the of T4 DNA and the of corn was 1.9°C higher than the of T4 DNA.
A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.
The single-stranded mitochondrial DNA (mtDNA) displacement-loop initiation sequence (7S mtDNA) is hydrogen-bonded at the origin of replication in animal cell mtDNA. Analysis of 7S mtDNA from several cell sources indicates that this initiation sequence exists as a family of fragments of relatively discrete lengths. mtDNA from both mouse L cells and mouse liver has four major sizes of 7S mtDNA fragments, ranging from 500 to 580 nucleotides in length. The 5′-end region of each of these species is the same; thus, the size heterogeneity is due primarily to differences in length at the 3′-end of these molecules. By contrast, 7S mtDNA from both human KB cells and human liver exists in three major forms, ranging from 555 to 615 nucleotides in length, due to differences at both terminal regions. The mtDNA initiation sequence from Xenopus laevis oocytes also exists in at least two forms, 1350 and 1510 nucleotides in length. Thus, the maintenance of multiple forms of mtDNA initiation sequence appears to be a general phenomenon of animal cells, although the precise mechanism of synthesis or processing of these forms is variable.
The sequence of 42 nucleotides at the 5′-end of 7S mtDNA from mouse L cells has been determined and found to be rich in dGuo and dThd residues, with no apparent palindromes or potential secondary structures. We thus present sequence information on the replication origin of mtDNA, as defined by the naturally occurring 7S mtDNA.
The major form of mouse L-cell mitochondrial DNA contains a small displacement loop at the replication origin, created by synthesis of a 550 to 670-nucleotide portion of the heavy strand. These short heavy-strand segments remain hydrogen-bonded to the parental light strand and are collectively termed 7 S mitochondrial DNA. The unique location of these 7 S mitochondrial DNAs at the heavy-strand origin suggests that they may function as primers in the synthesis of full-length heavy strands. Ribonucleotides have been detected at the 5′-end of some of these molecules, which are most likely remnants of primer RNAs. Using 5′-end labeling in vitro, we have determined that these ribonucleotides occur at several discrete positions along the nucleotide sequence of the origin region, which suggests that there may be variability in the precise initiation point of RNA priming or in the location of the switchover from RNA priming to DNA synthesis. The length of 5′-end RNA was estimated by alkali treatment of mitochondrial DNA prior to end labeling. A range of one to ten ribonucleotides was hydrolyzed from the 5′-end of some 7 S mitochondrial DNA strands. This is the first evidence of RNA priming at a eukaryotic cell DNA replication origin.
The chloroplast DNA (ctDNA) from pea and corn plants contains both Cairns type and rolling circle replicative intermediates. Denaturation mapping studies with pea ctDNA molecules have shown that the rolling circles initiate replication at or near the site where the Cairns replicative intermediates terminate replication. These results suggest that the rolling circles are initiated by a Cairns round of replication. A model for the replication of the chloroplast DNA is based on these results.
Covalently closed circular choloroplast DNA (ctDNA) molecules have been isolated from pea, bean, spinach, lettuce, corn and oat plants by ethidium bromide/cesium choloride density-gradient entrifugation. As much as 30-40% of the total ctDNA could be isolated as closed circular DNA molecules and up to 80% of the total ctDNA was found in the form of circular molecules. The size of pea, spinach, lettuce, corn and oat ctDNA relative to an internal standard (phiX174 replicative form II monomer DNA) was determined by electron microscopy. The ctDNAs showed significant differences in their sizes, and their molecular weights ranged from 85.4 - 10(6) for corn ctDNA to 96.7 - 10(6) for lettuce ctDNA. Each of these ctDNAs contained 3-4% of the circular molecules as circular dimers and 1-2% of the circular molecules as catenated dimes. The molecular complexity of these ctDNAs was studied by renaturation kinetics using T4 DNA as a standard. The molecular weights of the unique sequences of the ctDNAs ranged from 83.7 - 10(6) for oat ctDNA to 93.1 - 10(6) for lettuce ctDNA, which are in excellent agreement with the sizes of the circular ctDNA molecules...
We have used a combination of one- and two-dimensional agarose gel electrophoresis, and solution hybridization to strand-specific probes, to map the replication origin of sea urchin mitochondrial DNA and to investigate the structure of replication intermediates. These assays are consistent with replication initiating unidirectionally from the D-loop region by D-loop expansion, as in vertebrates. A prominent site of initiation of lagging-strand synthesis lies at, or near to, the boundary between the genes for ATPase 6 and COIII, which is also close to a pause site for leading-strand synthesis. These findings suggest a role for pause sites in the regulation of mitochondrial transcription and replication, possibly involving template-binding proteins.
When tobacco suspension culture line BY2 cells in stationary phase are transferred into fresh medium, replication of proplastid DNA proceeds for 24 h in the absence of nuclear DNA replication. Replicative intermediates of the proplastid DNA concentrated by benzoylated, naphthoylated DEAE cellulose chromatography, were radioactively labelled and hybridized to several sets of restriction endonuclease fragments of tobacco chloroplast DNA. The intermediates hybridized preferentially to restriction fragments in the two large inverted repeats. Mapping of D-loops and of restriction fragment lengths by electron microscopy permitted the localization of the replication origin, which was close to the 23S rRNA gene in the inverted repeats. The replication origins in both segments of the inverted repeat in tobacco proplastid DNA were active in vivo.
A DNA primase activity was isolated from pea chloroplasts and examined for its role in replication. The DNA primase activity was separated from the majority of the chloroplast RNA polymerase activity by linear salt gradient elution from a DEAE-cellulose column, and the two enzyme activities were separately purified through heparin-Sepharose columns. The primase activity was not inhibited by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase, or by polyclonal antibodies prepared against purified pea chloroplast RNA polymerase, while the RNA polymerase activity was inhibited completely by either tagetitoxin or the polyclonal antibodies. The DNA primase activity was capable of priming DNA replication on single-stranded templates including poly(dT), poly(dC), M13mp19, and M13mp19 + 2.1, which contains the AT-rich pea chloroplast origin of replication. The RNA polymerase fraction was incapable of supporting incorporation of 3H-TTP in in vitro replication reactions using any of these single-stranded DNA templates. Glycerol gradient analysis indicated that the pea chloroplast DNA primase (115-120 kDa) separated from the pea chloroplast DNA polymerase (90 kDa), but is much smaller than chloroplast RNA polymerase. Because of these differences in size, template specificity, sensitivity to inhibitors, and elution characteristics, it is clear that the pea chloroplast DNA primase is an distinct enzyme form RNA polymerase. In vitro replication activity using the DNA primase fraction required all four rNTPs for optimum activity. The chloroplast DNA primase was capable of priming DNA replication activity on any single-stranded M13 template, but shows a strong preference for M13mp19 + 2.1. Primers synthesized using M13mp19 + 2.1 are resistant to DNase I, and range in size from 4 to about 60 nucleotides.
Two-dimensional (2D) agarose gel electrophoresis is progressively replacing electron microscopy as the technique of choice to map the initiation and termination sites for DNA replication. Two different versions were originally developed to analyze the replication of the yeast 2 microns plasmid. Neutral/Neutral (N/N) 2D agarose gel electrophoresis has subsequently been used to study the replication of other eukaryotic plasmids, viruses and chromosomal DNAs. In some cases, however, the results do not conform to the expected 2D gel patterns. In order to better understand this technique, we employed it to study the replication of the colE1-like plasmid, pBR322. This was the first time replicative intermediates from a unidirectionally replicated plasmid have been analyzed by means of N/N 2D agarose gel electrophoresis. The patterns obtained were significantly different from those obtained in the case of bidirectional replication. We showed that identification of a complete are corresponding to molecules containing an internal bubble is not sufficient to distinguish a symmetrically located bidirectional origin from an asymmetrically located unidirectional origin. We also showed that unidirectionally replicated fragments containing a stalled fork can produce a pattern with an inflection point. Finally, replication appeared to initiate at only some of the potential origins in each multimer of pBR322 DNA.
Replication of ribosomal DNA replicons in cells of Pisum sativum (cv. Alaska) occurs bidirectionally by displacement loops. Replication is initiated on opposite parental strands and nascent chains are elongated moving 5'----3' along each parental template. Replicative intermediates were analyzed by 2-dimensional agarose gel electrophoresis under neutral--neutral and neutral--alkaline conditions. Southern blots of ribosomal DNA fragments separated in the second dimension under neutral conditions show slowly migrating replicative fragments that hybridize with specific probes in a manner consistent with bidirectional replication. The replicative fragments are present in root meristems with cells in S phase; they are absent or few in number in meristems with cells in G2 phase. The following observations indicate that the replicative fragments are single stranded. The apparent length of the replicative fragments is not the same when separated under neutral and alkaline conditions. They contain rDNA without breaks and they do not exhibit the smaller nascent chains expected from replication bubbles and forks. They are not cleaved by restriction enzymes that require duplex DNA as substrate and they are digestible by S1 nuclease.
In the diploid vegetative plant cell, the nuclear DNA is present in two copies, whereas the chloroplast and mitochondria genomes are present in a higher and variable copy number. We have studied the replication of the nuclear, chloroplast and mitochondrial DNA in cultured Nicotiana tabacum cells using density and radioactive markers. Essentially all the 10,000 chloroplast genomes in a given cell replicate in one cell cycle as do all the mitochondrial DNA molecules. No measurable level of unreplicated organellar DNA molecules can be detected in these cells.
We have used two complementary two-dimensional gel electrophoretic methods to localize replication inititation sites and to determine replication fork direction in the amplified 240 kb dihydrofolate reductase domain of the methotrexate-resistant CHO cell line CHOC 400. Surprisingly, our analysis indicates that replication begins at many sites in several restriction fragments distributed throughout a previously defined 28 kb initiation locus, including a fragment containing a matrix attachment region. Initiation sites were not detected in regions lying upstream or downstream of this locus. Our results suggest that initiation reactions in mammalian chromosomal origins may be more complex than in the origins of simple microorganisms.
Mammalian mitochondrial DNA maintains a novel displacement-loop region containing the major sites of transcriptional initiation and the origin of heavy strand DNA replication. Because the exact map positions of the 5' termini of nascent mouse displacement-loop strands are known, it is possible to examine directly a potential relationship between replication priming and transcription. Analyses of in vivo nucleic acids complementary to the displacement-loop region reveal two species with identical 5' ends at map position 16 183. One is entirely RNA and the other is RNA covalently linked to DNA. In the latter the transition from RNA to DNA is sharp, occurring near or within a series of previously identified conserved sequences 74-163 nucleotides downstream from the transcriptional initiation site. These data suggest that the initial events in replication priming and transcription are the same and that the decision to synthesize DNA or RNA is a downstream event under the control of short, conserved displacement-loop template sequences.
An enzyme system prepared from maize chloroplasts catalyzes the synthesis of DNA from maize chloroplast DNA sequences cloned
in bacterial plasmids. Cloned maize chloroplast DNA fragments Bam HI 17′ (2470 bp) and Eco RI x (1368 bp) have been shown
to be preferred templates for in vitro DNA synthesis catalyzed by pea chloroplast DNA polymerase preparations [Gold et al. (1987) Proc. Natl. Acad. Sci. USA 84, 194–198]. Analysis of replicative intermediates indicates that although the template
activity of the recombinant plasmid pZmcBam 17′ is substantially greater than that of the pZmcEco ×, replication in both cases
originates from within a 455 bp region which overlaps the two plasmids. The remaining approximately 1500 basepair portion
of maize chloroplast BamHI fragment 17′ is not more active because it contains additional origins for replication. The overlapping
region shows sequence homology with a portion of the Chlamydomonas reinhardtii chloroplast chromosome that contains a replication origin. Replication is shown to proceed bidirectionally within the 455
bp origin region. Recombinant plasmid pZmc 427, which is also active in the in vitro DNA synthesis assay, promoted localized replication initiation within a 1 kbp Bg1II-Eco RI fragment of the chloroplast DNA
insert, a region that includes the 3′ terminal part of the psbA gene.
Individual promoters for transcription of each strand of human mtDNA are located near the origin of heavy-strand DNA replication in the displacement-loop region. Initiation of heavy-strand synthesis represents the first event in mtDNA replication. Analyses of the 5' and 3' map positions of displacement-loop nucleic acids from mitochondria of cultured human cells reveal a close correspondence between the 3' ends of RNA, whose 5' ends map at a unique site, and the 5' ends of DNA strands. The 5' ends of the RNA species all map at nucleotide position 407 in the genomic sequence, which corresponds exactly to the major 5' transcriptional start site, determined previously in vitro, that is contained within the light-strand promoter sequence. Displacement-loop heavy-strand DNAs map immediately adjacent to the 3' termini of these RNAs, and these transition points between RNA and DNA lie within short conserved sequence blocks in the template sequence. The simplest interpretation of these data is that replication is initiated at the major transcriptional promoter with subsequent precise cleavage of primary transcripts to provide the appropriate primer species.
Replication of the approximately 200 tandem copies of yeast ribosomal RNA genes (rDNA) is known to be initiated within a subset of the repeats, with transcription continuing during the replication process. To examine replication fork movement in this gene cluster, we used a two-dimensional (2D) agarose gel electrophoresis procedure that distinguishes molecules with different branched structures. Replication forks move through most of the rDNA in the same direction in which RNA polymerase I transcribes the 35S rRNA precursor: the 3' end of this transcription unit acts as a barrier to replication forks moving in the direction opposite to RNA polymerase I. The replication fork barrier (RFB) is observed as the accumulation of branched intermediates of specific size. We propose that the act of transcription may influence the movement of replication forks, creating barriers at the 3' ends of actively transcribed genes.
In our previous study of chloroplast (Cp) DNA replication in Chlamydomonas reinhardtii, one D-loop site with its flanking regions was cloned and sequenced. The D-loop site mapped by electron microscopy (EM) overlaps with an open reading frame (ORF) potentially coding for a polypeptide of 136 amino acids. In this report, the corresponding D-loop isolated from another species of Chlamydomonas was sequenced. An ORF was also detected. Sequence comparison indicated that most conserved sequences between these two cloned origins are located within the ORF. Amino acid sequences of these two ORFs are highly conserved. The corresponding sequence for this ORF in the tobacco Cp genome was located by a Southern blotting analysis. Since the complete sequence data of Cp DNAs from a liverwort and from tobacco have been determined in 2 Japanese laboratories recently, it has been possible for us to show that this ORF encodes a protein homologous to the Cp ribosomal protein (r-protein) L16, by sequence comparison.
Initiation sites for DNA synthesis were located at the resolution of single nucleotides in and about the genetically defined origin of replication (ori) in replicating SV40 DNA purified from virus-infected cells. About 50% of the DNA chains contained an oligoribonucleotide of six to nine residues covalently attached to their 5' ends. Although the RNA-DNA linkage varied, the putative RNA primer began predominantly with rA. The data reveal that initiation of DNA synthesis is promoted at a number of DNA sequences that are asymmetrically arranged with respect to ori: 5' ends of nascent DNA are located at several sites within ori, but only on the strand that also serves as the template for early mRNA, while 5' ends of nascent DNA with the opposite orientation are located only outside ori on its early gene side. This clear transition between discontinuous (initiation sites) and continuous (no initiation sites) DNA synthesis defines the origin of bidirectional replication at nucleotides 5210--5211 and demonstrates that discontinuous synthesis occurs predominantly on the retrograde arms of replication forks. Furthermore, it appears that the first nascent DNA chain is initiated within ori by the same mechanism used to initiate nascent DNA ("Okazaki fragments") throughout the genome.
Chloroplast DNA, isolated from a synchronized culture of Chlamydomonas reinhardii , was digested with restriction endonucleases and examined in the electron microscope. Restriction fragments containing displacement
loops (D-loop) were photographed and measured to determine the position of replicated sequences in relation to the restriction
enzyme sites. D-loops were located at two positions on the physical map of chloroplast DNA. One replication origin was mapped
at about 10 kb upstream of the 5′ end of a 16s rRNA gene. The second origin was spaced 6.5kb apart from the first origin and
was about 16.5 kb upstream of the same 16s rRNA. Initiations at those two sites were not always synchronized. Replication
initiated with the formation of a D-loop resulting from the synthesis of one daughter strand. After a short initial lag phase,
corresponding to the synthesis of 350±130 bp of one daughter strand, DNA synthesis then proceeded in both directions. Both
D-loop regions were preferred binding sites of undetermined protein complexes.
The entire mitochondrial genome of Schizosaccharomyces pombe ura4-294h- was analyzed by the 2D pulsed field gel electrophoresis technique developed by Brewer and Fangman. The genome consists of multimers with an average size of 100 kb and analysis of the overlapping restriction fragments of the complete mitochondrial DNA (mtDNA) genome resulted in simple Y 2D gel patterns. Large single-stranded DNA molecules or double-stranded DNA molecules containing large or numerous single-stranded regions were found in the S. pombe mtDNA preparation. The replication of mtDNA monomers was found to occur in either direction. On the basis of these results, a replication mechanism for S. pombe mtDNA that is most consistent with a rolling circle model is suggested.
A partially purified replicative system of pea chloroplast that replicates recombinant DNAs containing pea chloroplast origin sequences has been characterised. Polymerisation by this system is very fast and insensitive to chain terminators like dideoxynucleotides, arabinosylcytosine 5′-triphosphate, etc. Both strands of template DNA are synthesized and single-stranded DNA templates undergo more than one round of replication.
When sequences of either of the two chloroplast origins of replication (OriA or OriB) are used as templates, the replicative intermediates are found to have sigma structures. Electron microscopic analysis of the sigma structures restricted with various enzymes reveals that the initiation site of in vitro replication maps near the displacement-loop regions where replication initiates also in vivo. Although the observed replication initiation in the OriA recombinant template is chloroplast-DNA-specific, the mode of replication is different from that observed in vivo with intact ctDNA. However, when the template DNA contains both the OriA and OriB sequences, the in vitro replication proceeds in the theta mode, the mode of replication usually observed in vivo.
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.
One of the two origins of replication in pea chloroplast DNA (oriA) maps in the rRNA spacer region downstream of the 16S rRNA gene, and further characterization of this origin is presented here. End-labeling of nascent DNA strands from in vivo replicating ctDNA was used to generate probes for Southern hybridization. Hybridization data identified the same region that was previously mapped to contain D-loops by electron microscopy. Subclones of the oriA region were tested for their ability to support in vitro DNA replication using a partially purified pea ctDNA replication system. Two-dimensional agarose gel electrophoresis identified replication intermediates for clones from the region just downstream of the 16S rRNA gene, with a 450-bp SacI-EcoRI clone showing the strongest activity. The experiments presented in this paper identify the 940 base pair region in the rRNA spacer between the 3' end of the 16S rRNA gene and the EcoRI site as containing oriA. Previous studies by electron microscopy localized the D-loop in the spacer region just to the right of the BamHI site, but the experiments presented here show that sequences to the left of the BamHI site are required for replication initiation from oriA. DNA sequence analysis of this region of pea ctDNA shows the presence of characteristic elements of DNA replication origins, including several direct and inverted repeat sequences, an A + T rich region, and dnaA-like binding sites, most of which are unique to the pea ctDNA oriA region when compared with published rRNA spacer sequences from other chloroplast genomes.
This chapter focuses on the analysis of replication intermediates by two-dimensional (2D) agarose gel electrophoresis. The first step in the analysis of replication intermediates on 2-D gels involves the isolation of branched molecules in a way that preserves their structure. Branched molecules are delicate, and shearing is minimized by use of large-bore pipette tips and gentle hand mixing. Shearing can lead to loss of replication intermediates, as well as generation of novel branched artifacts. Exposure of the DNA to excessive heat is avoided in order to preserve the integrity of DNA at the fork. Restriction fragments of larger or smaller size can be analyzed on 2-D gels if some modifications are made to the electrophoresis conditions. Finding the correct electrophoresis conditions for larger or smaller fragments require some experimentation using fragments of known size and replication pattern. Fragments that are larger than 5.5 to 6 kb must be run under conditions of lower agarose concentration and lower voltage in both dimensions in order to separate successfully the bubble and simple Y arcs. If gels are run under the conditions intended for smaller fragments (2 to 5.5 kb), the bubble and simple Y patterns are not clearly separated, and the signals for each are distorted. After the second dimension of electrophoresis, nonreplicating molecules are seen as a shallow arc, whereas the rare replicating molecules run above this arc.
The occurrence of DNA recombination in plastids of higher plants is well documented. However, little is known at the enzymic level. To begin dissecting the biochemical mechanism(s) involved we focused on a key step: strand transfer between homologous parental DNAs. We detected a RecA-like strand transfer activity in stromal extracts from pea (Pisum sativum L.) chloroplasts. Formation of joint molecules requires Mg2+, ATP, and homologous substrates. This activity is inhibited by excess single-stranded DNA (ssDNA), suggesting a necessary stoichiometric relation between enzyme and ssDNA. In a novel assay with Triton X-100-permeabilized chloroplasts, we also detected strand invasion of the endogenous chloroplast DNA by 32P-labeled ssDNA complementary to the 16S rRNA gene. Joint molecules, analyzed by electron microscopy, contained the expected displacement loops.