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Electron microscopic localization of replication origins in Oenothera chloroplast DNA
Abstract
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.
... 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.
... 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. (Waddell et al., 1984), G. max (Hedrick et al., 1993), O. elata (Chiu and Sears, 1992) and Pisum sativum (Meeker et al., 1988). ...
... 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. (Waddell et al., 1984), G. max (Hedrick et al., 1993), O. elata (Chiu and Sears, 1992) and Pisum sativum (Meeker et al., 1988). The pair is located in the inverted repeats in N. tabacum and O. elata (in P. sativum, lacking an inverted repeat, the pair of origins is also located in the same gene-order context), whereas in G. max and C. reinhardtii it is found in the large single copy region. ...
... It is located in the intron of the trnI gene. An ori was also detected in this region in O. elata (Chiu and Sears, 1992) and P. sativum (Nielsen et al., 1993). Ends of linear molecules and/or branched complexes of plastid DNA were also found in this region in Z. mays (Oldenburg and Bendich, 2004a) and N. tabacum (Scharff and Koop, 2006). ...
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.
... Some species of the family also have medicinal value 57 and are widely used to make oil, spices, and nectar [31]. 58 Inheritance of the plastid genome in Onagraceae has attracted great attention from 59 botanists [32][33][34][35][36][37]. Both maternal and paternal (or possibly biparental) transmission 60 of plastid genomes has been reported in Onagraceae. ...
... Most SSRs in the newly 282 sequenced Onagraceae plastomes were found to be mononucleotides (A/T) ( Table 2), 283 which is similar to reports in other families of angiosperms. The genus Circaea contained 284 more types of SSRs and repeats than the other two genera (Figure 6, subsection are also known to have biparentally inherited plastid genomes [34,75], which 303 is rare in angiosperms [15]. ...
Convergent evolution, often viewed as the inevitable outcome of natural selection, has received special attention since the time of Darwin. Clematis is well known for its climbing habit, but it has some shrubby species, known as sect. Fruticella s.l. The shrubby Clematis species are distributed in the dry habitats of Central Asia and adjacent areas showing possible convergent evolution. In this study, we assembled the complete plastome and nuclear ribosomal DNA (nrDNA) sequences of 56 Clematis species, representing most sections and covering most of the shrubby species, to reconstruct their evolutionary histories. Using both maximum likelihood and Bayesian methods, the plastome and nrDNA datasets generated similar, but not identical, phylogenetic relationships, which are better resolved than in previous studies. Then, molecular dating, historical range reconstruction, and character optimization analyses were conducted based on this updated phylogenetic framework. All the morphological characters widely used for taxonomy were shown to have evolved multiple times. Molecular dating inferred that Clematis diverged from its sister in the mid Miocene, and all six major clades of Clematis originated during the late Miocene, with a species radiation during the Pliocene to Pleistocene. The results clearly showed that the shrubby habit evolved independently in four lineages of Clematis in Asia. We also revealed that the shrubby lineages have emerged since the very beginning of Pliocene. Asian monsoon variation in the Pliocene and glacial period fluctuation in the Pleistocene may be the driving forces for the origin and diversification of the shrubby Clematis in Central Asia and adjacent dry areas.
... This finding argues against plastid DNA (ptDNA) replication per se being responsible for differences in chloroplast competitiveness. This conclusion is in line with previous analyses of the Oenothera replication origins, which had suggested that their variability does not correlate with the competitive strength of the plastids [17,18] (also see Supplementary Text). We further confirmed this by determining the relative ptDNA amounts of chloroplasts with different inheritance strengths in a constant nuclear background. ...
... First, the number of D-loop initiation sites (i.e. oris) does not differ between weak and strong plastomes and their locations in the chloroplast genome is identical [18]. Second, in a previous association mapping study that investigated the hypervariable repeat region of oriB, a short repeat series was identified as the sole determinant that could explain the difference between the strong and intermediate plastomes I, III and II on one side, and the weak plastome IV on the other side [17]. ...
In most eukaryotes, organellar genomes are transmitted preferentially by the mother, but molecular mechanisms and evolutionary forces underlying this fundamental biological principle are far from understood. It is believed that biparental inheritance promotes competition between the cytoplasmic organelles and allows the spread of so-called selfish cytoplasmic elements. Those can be, for example, fast replicating or aggressive chloroplasts (plastids) that are incompatible with the hybrid nuclear genome and therefore maladaptive. Here we show that the ability of plastids to compete against each other is a metabolic phenotype determined by extremely rapidly evolving genes in the plastid genome of the evening primrose Oenothera . Repeats in the regulatory region of accD (the plastid-encoded subunit of the acetyl-CoA carboxylase, which catalyzes the first and rate limiting step of lipid biosynthesis), as well as in ycf2 (a giant reading frame of still unknown function), are responsible for the differences in competitive behavior of plastid genotypes. Polymorphisms in these genes influence lipid synthesis and most likely profiles of the plastid envelope membrane. These in turn determine plastid division and/or turn-over rates and hence competitiveness. This work uncovers cytoplasmic drive loci controlling the outcome of biparental chloroplast transmission. Here, they define the mode of chloroplast inheritance, since plastid competitiveness can result in uniparental inheritance (through elimination of the “weak” plastid) or biparental inheritance (when two similarly “strong” plastids are transmitted).
Significance statement
Plastids and mitochondria are usually uniparentally inherited, typically maternally. When the DNA-containing organelles are transmitted to the progeny by both parents, evolutionary theory predicts that the maternal and paternal organelles will compete in the hybrid. As their genomes do not undergo sexual recombination, one organelle will “try” to outcompete the other, thus favoring the evolution and spread of aggressive cytoplasms. The investigations described here in the evening primrose, a model species for biparental plastid transmission, have discovered that chloroplast competition is a metabolic phenotype. It is conferred by rapidly evolving genes that are encoded on the chloroplast genome and control lipid biosynthesis. Due to their high mutation rate these loci can evolve and become fixed in a population very quickly.
... Electron microscopic examination of ctDNA from other plant species has supported this model to various degrees. In Chlamydomonas and Oenothera, two D-loops 7 and 4 kbp apart, respectively, have been mapped on the plastome Chiu and Sears, 1992). However, conversion to discontinuous synthesis apparently occurs soon after D-loop initiation, since mainly forks with exclusively double-stranded arms have been observed in preparations of replication intermediates. ...
... One D-loop initiation site is located within the spacer region, and the other approximately 7 kbp away, downstream of the 23 S rRNA gene (I,II in Fig. 1). Chiu and Sears (1992) recently mapped two D-loop regions to the inverted repeats of the Oenothera plastome by electron microscopic analysis of replicative intermediates. oriA was located in the spacer region between the rRNA genes, close to the 16 S rRNA gene and oriB was found 4 kbp away, upstream of the 16 S rRNA gene (OA,OB in Fig. 1). ...
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.
... Electron microscopy has been used to map D-loops on the plastomes of several higher plants and algae. Although the predicted two D-loops (Kolodner and Tewari, 1975a; b) have rarely been observed on a single ctDNA fragment, between one and four D-loops have been reported for plastomes from various plant species and organelles at different developmental stages (Chiu and Sears, 1992; Koller and Delius, 1982; Meeker et al., 1988; Ravel-Chapuis et al., 1982; Takeda et al., 1992; Waddell et al., 1984). Considering the highly conserved structure and gene content of ctDNA, the observed differences in number and location of plastome D-loops are somewhat surprising. ...
... Since the initiation region of this alternative DNA replication mode was mapped to a recombination hotspot on the C. reinhardtii plastome, the possibility of a recombination-dependent replication mechanism was invoked. Of the collective experimental evidence that addresses the plastome replication mechanism only a fraction supports the expected unidirectional D-loop extension (Kunnimalaiyaan and Nielsen, 1997b; Kunnimalaiyaan et al., 1997; Lu et al., 1996; Nielsen et al., 1993), whereas results from several other studies are consistent with bi-directional fork movement (Chiu and Sears, 1992; Ravel-Chapuis et al., 1982; Takeda et al., 1992; Waddell et al., 1984; Wang, Y. et al., 2002). Some of these apparent discrepancies can probably be explained by the presence of a single versus two cloned ori regions in in vitro DNA replication templates (Kunnimalaiyaan and Nielsen, 1997b; Kunnimalaiyaan et al., 1997; Lu et al., 1996; Nielsen et al., 1993; Reddy et al., 1994) and per se may not contradict the accepted plastome replication model. ...
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.
... (Meeker et al. 1988) lacks a large inverted repeat, b) Dicots containing large inverted repeats. N. tabacum Nt (Ori A) and Nt (Ori B) (Kunnimalaiyaan and Nielsen 1997;Kunnimalaiyaan et al. 1997), N. tabacum D-loops Nt (pro) in proplastids (Takeda et al. 1992), Oenothera hookeri Oe (Ori A) and Oe (Ori B) (Chiu and Sears 1992;Sears et al. 1996), Glycine max bubbles Gm (Hedrick et al. 1993). c) Oryza sativa (Os) replication origins in suspension culture cells (Os 1 ), leaf blades (Os 2 ), and coleoptiles (Os 3 ) mapped by Wang et al. 2003, Z. mays (Zm, Gold et al. 1987, linear DNA replicons (Ellis and Day 1986;Harada et al. 1992;Zubko and Day 2002), Hordeum vulgare (Hv), Oryza sativa (Os). ...
... Two D-loops separated by 4 kbp (Chiu and Sears 1992;Sears et al. 1996) were found in the large inverted repeat of Oenothera hookeri plastid DNA, where they flank the 16S ribosomal genes (Fig. 6b). The locations of two origins in the large inverted repeat suggest four potential replication origins in N. tabacum and O. hookeri plastid DNA; two in each inverted repeat. ...
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.
... As the different plastid types show different replication rates when transferred to the same nuclear background, the region showing these sequence differences is assumed to be involved in the regulation of DNA replication. This is corroborated by the fact that a replication origin (confusingly named oriB in Oenothera) was mapped in this region by electron microscopic analysis of displacement loops (Chiu and Sears, 1992). The sequence differences are caused by repetition and variation of short DNA elements. ...
... The question of whether this region contains the replication origin in Oenothera can only be answered when plastid transformation of this species has been achieved. As electron microscopic mapping of a displacement loop within this region did not allow precise localisation (Chiu and Sears, 1992), the replication origin in Oenothera might also be within the trnI intron, analogous to tobacco oriA. As published sequences of the different Oenothera plastid types contain a sequence with high homology to tobacco oriA, one might speculate that this sequence acts as an origin of replication also in Oenothera, although our experiments question the function of the described oriA sequence. ...
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.
... Based on our physical mapping of M. truncatula cpDNA, the predominant ends of linear molecules lie near 20, 30, 60, 100, and 120 kb. Interestingly, two of the ends (Fig. 1C, maps c and e) lie near a sequence homologous to replication origins previously identified in Oenothera (Chiu and Sears, 1992); the other ends could be near previously unclassified origins. These results suggest that the ends may represent initiation sites for recombination-dependent DNA replication of linear molecules, as we inferred for maize (Oldenburg and Bendich, 2004b). ...
... The ends of linear cpDNA molecules have been mapped for the IR-containing maize (Oldenburg and Bendich, 2004b) and tobacco (Scharff and Koop, 2006) and now for M. truncatula that has no IR. For all three species, ends are located at positions near regions homologous to tobacco oriA (homologous to Oenothera oriB) and Oenothera oriA (Fig. 1C, maps c and e; Chiu and Sears, 1992;Kunnimalaiyaan and Nielsen, 1997b;Oldenburg and Bendich, 2004b), but for each species there are additional ends. For example, an end for tobacco, but not maize or M. truncatula, is located near tobacco oriB Koop, 2006, 2007). ...
We used pulsed-field gel electrophoresis and restriction fragment mapping to analyze the structure of Medicago truncatula chloroplast DNA (cpDNA). We find most cpDNA in genome-sized linear molecules, head-to-tail genomic concatemers, and complex branched forms with ends at defined sites rather than at random sites as expected from broken circles. Our data suggest that cpDNA replication is initiated predominantly on linear DNA molecules with one of five possible ends serving as putative origins of replication. We also used 4',6-diamidino-2-phenylindole staining of isolated plastids to determine the DNA content per plastid for seedlings grown in the dark for 3 d and then transferred to light before being returned to the dark. The cpDNA content in cotyledons increased after 3 h of light, decreased with 9 h of light, and decreased sharply with 24 h of light. In addition, we used real-time quantitative polymerase chain reaction to determine cpDNA levels of cotyledons in dark- and light-grown (low white, high white, blue, and red light) seedlings, as well as in cotyledons and leaves from plants grown in a greenhouse. In white, blue, and red light, cpDNA increased initially and then declined, but cpDNA declined further in white and blue light while remaining constant in red light. The initial decline in cpDNA occurred more rapidly with increased white light intensity, but the final DNA level was similar to that in less intense light. The patterns of increase and then decrease in cpDNA level during development were similar for cotyledons and leaves. We conclude that the absence in M. truncatula of the prominent inverted repeat cpDNA sequence found in most plant species does not lead to unusual properties with respect to the structure of plastid DNA molecules, cpDNA replication, or the loss of cpDNA during light-stimulated chloroplast development.
... Changes in light conditions can trigger plastid DNA replication as well as degradation in M. truncatula 42 . Flanking regions of the rrn16 rRNA gene that showed high coverage are not just high GC sites (see Fig. 1B) but also the putative origins of replication (oriA and oriB) 42,43 . Many steps in NGS can introduce GC bias, but PCR during library preparation is a principal source 44 . ...
Medicago truncatula is a model legume that has been extensively investigated in diverse subdisciplines of plant science. Medicago littoralis can interbreed with M. truncatula and M. italica; these three closely related species form a clade, i.e. TLI clade. Genetic studies have indicated that M. truncatula accessions are heterogeneous but their taxonomic identities have not been verified. To elucidate the phylogenetic position of diverse M. truncatula accessions within the genus, we assembled 54 plastid genomes (plastomes) using publicly available next-generation sequencing data and conducted phylogenetic analyses using maximum likelihood. Five accessions showed high levels of plastid DNA polymorphism. Three of these highly polymorphic accessions contained sequences from both M. truncatula and M. littoralis. Phylogenetic analyses of sequences placed some accessions closer to distantly related species suggesting misidentification of source material. Most accessions were placed within the TLI clade and maximally supported the interrelationships of three subclades. Two Medicago accessions were placed within a M. italica subclade of the TLI clade. Plastomes with a 45-kb (rpl20-ycf1) inversion were placed within the M. littoralis subclade. Our results suggest that the M. truncatula accession genome pool represents more than one species due to possible mistaken identities and gene flow among closely related species.
... Inheritance of the chloroplast genome in Onagraceae has attracted great attention of botanists (Cleland, 1972;Chiu et al., 1988;Chiu and Sears, 1992;Chiu and Sears, 1993;Sears et al., 1996;Massouh et al., 2016;Sobanski et al., 2019). Both maternal and biparental inheritance of chloroplast genomes has been reported in the family . ...
The evening primrose family, Onagraceae, is a well defined family of the order Myrtales, comprising 22 genera widely distributed from boreal to tropical areas. In this study, we report and characterize the complete chloroplast genome sequences of 13 species in Circaea, Chamaenerion, and Epilobium using a next-generation sequencing method. We also retrieved chloroplast sequences from two other Onagraceae genera to characterize the chloroplast genome of the family. The complete chloroplast genomes of Onagraceae encoded an identical set of 112 genes (with exclusion of duplication), including 78 protein-coding genes, 30 transfer RNAs, and four ribosomal RNAs. The chloroplast genomes are basically conserved in gene arrangement across the family. However, a large segment of inversion was detected in the large single copy region of all the samples of Oenothera subsect. Oenothera. Two kinds of inverted repeat (IR) region expansion were found in Oenothera, Chamaenerion, and Epilobium samples. We also compared chloroplast genomes across the Onagraceae samples in some features, including nucleotide content, codon usage, RNA editing sites, and simple sequence repeats (SSRs). Phylogeny was inferred by the chloroplast genome data using maximum-likelihood (ML) and Bayesian inference methods. The generic relationship of Onagraceae was well resolved by the complete chloroplast genome sequences, showing potential value in inferring phylogeny within the family. Phylogenetic relationship in Oenothera was better resolved than other densely sampled genera, such as Circaea and Epilobium. Chloroplast genomes of Oenothera subsect. Oenothera, which are biparental inheritated, share a syndrome of characteristics that deviate from primitive pattern of the family, including slightly expanded inverted repeat region, intron loss in clpP, and presence of the inversion.
... This article is protected by copyright. All rights reserved Further refinement of the episomal strategy using chloroplast-specific ori from different organisms, including higher plants and algae (Carrillo and Bogorad, 1988;Chiu and Sears, 1992;Daniell et al., 1990;Karas et al., 2015;Kunnimalaiyaan and Nielsen, 1997;Meeker et al., 1988;Nisbet et al., 2004;Waddell et al., 1984;Wang et al., 1984), and the use of single or multiple ori with different activities could enable precise tuning of the copy number of the episomal constructs, adding another layer for control of gene expression in complex pathways. In essence, this work demonstrates a novel technology with significant improvements on the current state of plastid engineering, which should enable synthetic biology in plants. ...
In the age of synthetic biology, plastid engineering requires a nimble platform to introduce novel synthetic circuits in plants. While effective for integrating relatively small constructs into the plastome, plastid engineering via homologous recombination of transgenes is over thirty‐years‐old. Here we show the design‐build‐test of a novel synthetic genome structure that does not disturb the native plastome: the “mini‐synplastome.” The mini‐synplastome was inspired by dinoflagellate plastome organization, which is comprised of numerous minicircles residing in the plastid instead of a single organellar genome molecule. The first mini‐synplastome in plants was developed in vitro to meet the following criteria: 1) episomal replication in plastids; 2) facile cloning; 3) predictable transgene expression in plastids; 4) non‐integration of vector sequences into the endogenous plastome and; 5) autonomous persistence in the plant over generations in the absence of exogenous selection pressure. Mini‐synplastomes are anticipated to revolutionize chloroplast biotechnology, enable facile marker‐free plastid engineering, and provide an unparalleled platform for one‐step metabolic engineering in plants.
... The mechanism by which chloroplast DNA is replicated is also still a mystery. It appears that chloroplast DNA can be replicated by more than one mechanism [74], including recombination-dependent replication [47,48,75], a double D-loop mechanism [76,77], and rolling circle replication [78]. Studying DNA replication in polyploid cyanobacteria will provide useful information for understanding the mechanism of chloroplast DNA replication. ...
While the model bacteria Escherichia coli and Bacillus subtilis harbor single chromosomes, which is known as monoploidy, some freshwater cyanobacteria contain multiple chromosome copies per cell throughout their cell cycle, which is known as polyploidy. In the model cyanobacteria Synechococcus elongatus PCC 7942 and Synechocystis sp. PCC 6803, chromosome copy number (ploidy) is regulated in response to growth phase and environmental factors. In S. elongatus 7942, chromosome replication is asynchronous both among cells and chromosomes. Comparative analysis of S. elongatus 7942 and S. sp. 6803 revealed a variety of DNA replication mechanisms. In this review, the current knowledge of ploidy and DNA replication mechanisms in cyanobacteria is summarized together with information on the features common with plant chloroplasts. It is worth noting that the occurrence of polyploidy and its regulation are correlated with certain cyanobacterial lifestyles and are shared between some cyanobacteria and chloroplasts.
Abbreviations: NGS: next-generation sequencing; Repli-seq: replication sequencing; BrdU: 5-bromo-2′-deoxyuridine; TK: thymidine kinase; GCSI: GC skew index; PET: photosynthetic electron transport; RET: respiration electron transport; Cyt b6f complex: cytochrome b6f complex; PQ: plastoquinone; PC: plastocyanin.
... Some exceptions to the double D-loop replication model have been proposed. For example, Chlamydomonas and Oenothera possess two displacement loops, but discontinuous DNA replication begins shortly after initiation rather than after the fusion of the two D-loops [64,65]. Euglena possesses only one origin of replication site and appears to replicate bidirectionally from this site rather than forming two displacement loops [66]. ...
Mitochondria and chloroplasts perform essential functions in respiration, ATP production, and photosynthesis, and both organelles contain genomes that encode only some of the proteins that are required for these functions. The proteins and mechanisms for organelle DNA replication are very similar to bacterial or phage systems. The minimal replisome may consist of DNA polymerase, a primase/helicase, and a single-stranded DNA binding protein (SSB), similar to that found in bacteriophage T7. In Arabidopsis, there are two genes for organellar DNA polymerases and multiple potential genes for SSB, but there is only one known primase/helicase protein to date. Genome copy number varies widely between type and age of plant tissues. Replication mechanisms are only poorly understood at present, and may involve multiple processes, including recombination-dependent replication (RDR) in plant mitochondria and perhaps also in chloroplasts. There are still important questions remaining as to how the genomes are maintained in new organelles, and how genome copy number is determined. This review summarizes our current understanding of these processes.
... For details on the definition of the 1281 gene classes, see text. For macro repeat identification, see , for 1282 location of the origins of replication, see Chiu and Sears (1992). ...
The genus Anthriscus Pers. of the family Apiaceae is a small sized genus with 16 species, distributed in the world. In Turkey it is represented by 8 taxa, distributed in 4 sections. The aim of this study was to determine the genetic proximity and distances of taxa to each other and to identify interrelationships, systematic and phylogenetic relationships using the sequence analysis information of the non-coding trn region in the chloroplast genome of the Anthriscus species in Turkey. The phylogenetic tree showed that the taxa A. caucalis var. caucalis and A. tenerrima var. tenerrima (belonging to sect. Anthriscus) with A. cerefolium var. trichocarpa (belonging to sect. Cerefolium) had completed their speciations and isolation with other species in terms of speciation was provided. It can be said that A. kotschyi, the only representative of the sect. Caroides in Turkey, is isolated having completed its speciation also. The presence of the continuing gene exchange between the taxa can be mentioned, while the taxonomy of the two taxa of A. sylvestris and A. lamprocarpa, two members of the sect. Cacosciadium, cannot be determined more clearly yet. For this reason, it can be said that the A. sylvestris subsp. sylvestris and A. sylvestris subsp. nemorosa taxa, previously identified as two subspecies belonging to A. sylvestris, should be raised again to A. sylvestris and A. nemorosa taxa. In addition, an infrageneric arrangement and subsequent taxonomic regulation need to be made for the subspecies belonging to the A. lamprocarpa taxa.
... For details on the definition of the gene classes, see text. For macro repeat identification, see Greiner et al. (2008a), and for location of the origins of replication, see Chiu and Sears (1992). exclusively representing duplications; Figure 3), 11 with photosystem II, and six within the cytochrome b 6 f complex. ...
Spontaneous plastome mutants have been used as a research tool since the beginnings of genetics. However, technical restrictions have severely limited their contributions to research in physiology and molecular biology. Here, we used full plastome sequencing to systematically characterize a collection of 51 spontaneous chloroplast mutants in Oenothera (evening primrose). Most mutants carry a single mutation. Unexpectedly, the vast majority of mutations do not represent single nucleotide polymorphisms, but are insertions/deletions originating from DNA replication slippage events. Only very few mutations appear to be caused by imprecise double-strand break repair, nucleotide misincorporation during replication or incorrect nucleotide excision repair following oxidative damage. U-turn inversions were not detected. Replication slippage is induced at repetitive sequences that can be very small and tend to have high A/T content. Interestingly, the mutations are not distributed randomly in the genome. The underrepresentation of mutations caused by faulty double-strand break repair might explain the high structural conservation of seed plant plastomes throughout evolution. In addition to providing a fully characterized mutant collection for future research on plastid genetics, gene expression and photosynthesis, our work identified the spectrum of spontaneous mutations in plastids and reveals that this spectrum is very different from that in the nucleus.
... The basis for this is unclear given the nuclear control of chloroplast division (Rose etal. 1990); however, differential replication of Oenothera plastids has been suggested to be controlled by individual chloroplast genomes (Chiu and Sears 1992). ...
Medicago sativa L. cv Regen S is heteroplasmic for chloroplast DNA (cpDNA). Previous analyses of regenerated plants have shown a predominance of one of the cpDNAs which we have designated type A (the other we have designated type B). Studies of the replication of the two cpDNAs in tissue culture were carried out using leaflet expiants with defined cpDNA types and a distinguishing probe. The explants obtained showed a bias toward type A cpDNA during tissue culture. The data suggest that chloroplasts with different DNAs in a common nuclear background can multiply at different rates.
... The controls that regulate ctDNA replication initiation, replication, and copy number are not understood. From earlier publications on a number of plant species it appears that ctDNA may replicate by more than one mechanism, including a recombination-dependent replication mechanism (Rowan et al., 2010, this issue;Oldenburg and Bendich, 2004b;Marechal and Brisson, 2010), a double D-loop mechanism (Chiu and Sears, 1992;Kunnimalaiyaan and Nielsen, 1997a, b), and rolling circle replication (Kolodner and Tewari, 1975). ...
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.
... Gauly and Kössel (1989) suggest that methylation might play a role in regulation of replication, as it does in prokaryotes. Their hypothesis is supported by the fact that they revealed tissuespecific cytosine methylation in the plastid genome of maize, the position of which corresponds to the localisation of the replication origin in pea (Meeker et al. 1988), Oenothera (Chiu and Sears 1992) and tobacco chloroplasts (Lu et al. 1996). ...
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.
... CtDNA replication has been studied in several higher plant and algal species (reviewed in [4]). Methods used to study ctDNA replication origins in higher plants include EM and two-dimensional (2D) agarose gel electrophoresis, primer extension mapping of nascent 5 ends from total plastid DNA [3,[5][6][7][8][9], and in vitro replication analysis of ctDNA ori subclones [4,10]. These studies have identified two origins (oriA and oriB) that flank the 23S rRNA gene in tobacco ctDNA [4]. ...
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 the case of Euglena, it was shown that the replication origin inferred by the GC skew analysis (Morton 1999) matched the single locus previously identified by electron microscopic analysis of replication intermediates (Koller and Delius 1982;Ravel-Chapuis et al. 1982). In contrast, all other chloroplast genomes whose replication origin was studied by diverse biochemical methods were found to contain multiple origins either in the large IR, close to the ribosomal RNA operon, as in Pisum sativum ( Meeker et al. 1988), Nicotiana tabacum ( Nielsen 1997a, 1997b), and Oenothera hookeri ( Chiu and Sears 1992;Sears et al. 1996) or in the single-copy regions as in Chlamydomonas where oriA and oriB lie in the vicinity of rpl16 and chlL, respectively ( Waddell et al. 1984;Chang and Wu 2000). ...
In the Chlorophyceae, the chloroplast genome is extraordinarily fluid in architecture and displays unique features relative to other groups of green algae. For the Chaetophorales, 1 of the 5 major lineages of the Chlorophyceae, it has been shown that the distinctive architecture of the 223,902-bp genome of Stigeoclonium helveticum is consistent with bidirectional DNA replication from a single origin. Here, we report the 182,759-bp chloroplast genome sequence of Schizomeris leibleinii, a member of the earliest diverging lineage of the Chaetophorales. Like its Stigeoclonium homolog, the Schizomeris genome lacks a large inverted repeat encoding the rRNA operon and displays a striking bias in coding regions that is associated with a bias in base composition along each strand. Our results support the notion that these two chaetophoralean genomes replicate bidirectionally from a putative origin located in the vicinity of the small subunit ribosomal RNA gene. Their shared structural characteristics were most probably inherited from the common ancestor of all chaetophoralean algae. Short dispersed repeats account for most of the 41-kb size variation between the Schizomeris and Stigeoclonium genomes, and there is no indication that homologous recombination between these repeated elements led to the observed gene rearrangements. A comparison of the extent of variation sustained by the Stigeoclonium and Schizomeris chloroplast DNAs (cpDNAs) with that observed for the cpDNAs of the chlamydomonadalean Chlamydomonas and Volvox suggests that gene rearrangements as well as changes in the abundance of intergenic and intron sequences occurred at a slower pace in the Chaetophorales than in the Chlamydomonadales.
... As a result of this mechanism, half of each displaced parental strand (from either origin until the center of two origins) is left single-stranded on both sides of the pair of inverted repeats. This discovery of Cairn's replication mechanism [4] in pea and corn chloroplast genomes was followed by a series of studies independently confirming this model for various plant species (Euglena gra- cilis [5]; single D-loop]; Nicotiana tabacum [6]; Chlamydomonas reinhardtii [7]; Oenothera [8]; Zea mays [9]). The rolling circle mechanism could be initiated after one round of Cairns type of replication, so as to generate multiple copies of the chloroplast genome even though replication is initiated only once (pea and corn, [10]). ...
Electron microscopy analyses of replicating chloroplast molecules earlier predicted bidirectional Cairns replication as the prevalent mechanism, perhaps followed by rounds of a rolling circle mechanism. This standard model is being challenged by the recent proposition of homologous recombination-mediated replication in chloroplasts.
We address this issue in our current study by analyzing nucleotide composition in genome regions between known replication origins, with an aim to reveal any adenine to guanine deamination gradients. These gradual linear gradients typically result from the accumulation of deaminations over the time spent single-stranded by one of the strands of the circular molecule during replication and can, therefore, be used to model the course of replication. Our linear regression analyses on the nucleotide compositions of the non-coding regions and the synonymous third codon position of coding regions, between pairs of replication origins, reveal the existence of significant adenine to guanine deamination gradients in portions overlapping the Small Single Copy (SSC) and the Large Single Copy (LSC) regions between inverted repeats. These gradients increase bi-directionally from the center of each region towards the respective ends, suggesting that both the strands were left single-stranded during replication.
Single-stranded regions of the genome and gradients in time that these regions are left single-stranded, as revealed by our nucleotide composition analyses, appear to converge with the original bi-directional dual displacement loop model and restore evidence for its existence as the primary mechanism. Other proposed faster modes such as homologous recombination and rolling circle initiation could exist in addition to this primary mechanism to facilitate homoplasmy among the intra-cellular chloroplast population.
... It is unknown which, if any, of these mechanisms is involved in the autonomous replication of the extrachromosomal elements. Interestingly, one of the D-loops implicated in ptDNA replication was mapped to the region containing NICE1 sequences in other plant species (19,20), although not in tobacco (21). Alternatively, the engineered elements may replicate as part of the plastid Fig. 2A Lower). ...
The plastid genome of higher plants is a circular double-stranded DNA molecule which is present in multiple identical copies. We report here an 868-bp plastid DNA minicircle, NICE1, that formed in tobacco (Nicotiana tabacum) plastids during transformation, as an unexpected product of homologous recombination. Such extrachromosomal elements are normally absent in plastids of higher plants. We have constructed shuttle plasmids containing NICE1 sequences which are maintained extrachromosomally when reintroduced into plastids by particle bombardment. Furthermore, recombination between homologous sequences in the shuttle plasmids and the main plastid genome occurs. Recombination products were characterized after recovery of the shuttle plasmids in Escherichia coli and of recombinant plastid genomes in the progeny of transformed plants. Our findings indicate that shuttle plasmids can be used to engineer plastid genes without concomitant integration of foreign DNA and to recover plastid mutations in E. coli.
... The replicative intermediates of the covalently closed circular chloroplast (ct)' DNA molecules of 120-160 kilobase pairs from higher plants are well characterized (1)(2)(3). However, only few proteins, involved in generating the replicative intermediates of ctDNA, have been identified and purified (4). ...
A 69-kDa protein with topoisomerase I activity has been homogeneously purified from the chloroplasts of pea leaves. The topoisomerase properties are detected in crude lysate of pea chloroplasts using the technique of transferring 32P radioactivity from the 32P-labeled DNA to the protein. The purified enzyme relaxes both positive and negative supercoils in topological steps of unity without requiring magnesium ions. The enzyme is sensitive to topoisomerase I-specific inhibitors like camptothecin and berenil, and unaffected by reagents like novobiocin and doxorubicin at the topoisomerase II-inhibitory dosage. In the presence of the enzyme, supercoiled DNA is nicked, and the 3'-phosphoryl end of the nick becomes covalently linked with the enzyme. A tyrosine residue of the enzyme is responsible for the covalent linkage. Rabbit antiserum raised against the 16-mer peptide spanning the active residues of human topoisomerase I recognizes the 69-kDa protein within the crude lysate of pea chloroplasts as does the antiserum to the purified 69-kDa protein. From the enzymatic characteristics, the protein has been classified as a eukaryotic type I topoisomerase.
... 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). However, in a few species ctDNA replication origins have been mapped just outside the IR elements (Waddell et al., 1984;De Haas et al., 1987;Carrillo & Bogorad, 1988). ...
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 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). The location of oriA may be conserved, as the sequence of this region shows a high degree of homology among species (2,13), whereas oriB shows homology only between tobacco and petunia among the sequenced genomes. ...
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.
... The origin of replication has been mapped in the tobacco chloroplast genome and oriA is located within the trnI gene that forms the left flank in the chloroplast transformation vector used for cotton transformation. Several methods have been used to study ctDNA replication origins in higher plants including electron microscopy, two-dimensional (2D) agarose gel electrophoresis, primer extension mapping of nascent 5′ ends from total plastid DNA (Kolodner and Tewari, 1975; Meeker et al., 1988; Chiu and Sears, 1992; Hedrick et al., 1993; Nielsen et al., 1993; Kunnimalayaan et al., 1997), and in vitro replication analysis of ctDNA ori subclones (Kunnimalayaan and Nielsen, 1997 a, b). All these studies have identified two replication origins (oriA and oriB) that flank the 23S rRNA gene in tobacco ctDNA (Kunnimalayaan and Nielsen, 1997a,b). ...
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.
In most eukaryotes, organellar genomes are transmitted preferentially by the mother, but molecular mechanisms and evolutionary forces underlying this fundamental biological principle are far from understood. It is believed that biparental inheritance promotes competition between the cytoplasmic organelles and allows the spread of so-called selfish cytoplasmic elements. Those can be, for example, fast-replicating or aggressive chloroplasts (plastids) that are incompatible with the hybrid nuclear genome and therefore maladaptive. Here we show that the ability of plastids to compete against each other is a metabolic phenotype determined by extremely rapidly evolving genes in the plastid genome of the evening primrose Oenothera . Repeats in the regulatory region of accD (the plastid-encoded subunit of the acetyl-CoA carboxylase, which catalyzes the first and rate-limiting step of lipid biosynthesis), as well as in ycf2 (a giant reading frame of still unknown function), are responsible for the differences in competitive behavior of plastid genotypes. Polymorphisms in these genes influence lipid synthesis and most likely profiles of the plastid envelope membrane. These in turn determine plastid division and/or turnover rates and hence competitiveness. This work uncovers cytoplasmic drive loci controlling the outcome of biparental chloroplast transmission. Here, they define the mode of chloroplast inheritance, as plastid competitiveness can result in uniparental inheritance (through elimination of the “weak” plastid) or biparental inheritance (when two similarly “strong” plastids are transmitted).
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.
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.
Genetic engineering of proteins is a powerful tool used in both basic and applied research. In vitro alteration of the primary
structure of a gene and subsequent introduction of the mutated DNA into the genome of a living cell allows the directed manipulation
of protein structure. Such an approach, often termed ‘reverse genetics,’ has been widely used to investigate the complex relationship
between the structure of a protein and its function, and to explore the intricacies of biochemical and developmental pathways.
An obvious prerequisite for genetic engineering is the ability to introduce DNA into a living cell in such a way that it is
stably maintained and properly expressed in the appropriate genome of the host cell. The genome of a prokaryote is in the
cell cytoplasm and generally consists of one or a few copies of a large DNA molecule. In contrast, photosynthetic eukaryotes
contain three distinct genomes, each located within a subcellular organelle enveloped by one or more membranes and hence separated
from the cytoplasm. The three plant cell genomes are those of the nucleus, the mitochondrion, and the plastid. The genome
of the chloroplast, the plastid type found in photosynthetic cells, presents a complex genetic target because there are often
hundreds of copies of the circular chloroplast DNA molecule per chloroplast, and often hundreds of chloroplasts per cell.
Obtaining a plant cell in which every resident copy of a given chloroplast gene has been replaced by an engineered, mutant
gene copy is an essential step in experiments involving DNA-mediated chloroplast transformation. An ideal model organism for
such studies is provided by the unicellular green alga Chlamydomonas reinhardtii, which contains a single large chloroplast. This chapterpresents current results and future prospects for chloroplast transformation,
both in Chlamydomonas and in plants of agronomic interest.
Typescript (photocopy). Thesis (Ph. D.)--Oregon State University, 1994. Includes bibliographical references.
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.
We recently reported an 868-bp plastid DNA minicircle, NICE1, that formed during transformation in a transplastomic Nicotiana tabacum line. Shuttle plasmids containing NICE1 sequences were maintained extrachromosomally in plastids and shown to undergo recombination with NICE1 sequences on the plastid genome. To prove the general utility of the shuttle plasmids, we tested whether plastid genes outside the NICE1 region could be rescued in Escherichia coli. The NICE1-based rescue plasmid, pNICER1, carries NICE1 sequences for maintenance in plastids, the ColE1 ori for maintenance in E. coli and a spectinomycin resistance gene (aadA) for selection in both systems. In addition, pNICER1 carries a defective kanamycin resistance gene, kan*, to target the rescue of a functional kanamycin resistance gene, kan, from the recipient plastid genome. pNICER1 was introduced into plastids where recombination could occur between the homologous kan/kan* sequences, and subsequently rescued in E. coli to recover the products of recombination. Based on the expression of kanamycin resistance in E. coli and the analysis of three restriction fragment polymorphisms, recombinant kan genes were recovered at a high frequency. Efficient rescue of kan from the plastid genome in E. coli indicates that NICE1-based plasmids are suitable for rescuing mutations from any part of the plastid genome, expanding the repertoire of genetic tools available for plastid biology.
Approximately 4,200 nucleotides of the 16S/23S rDNA spacer and the 5' region flanking the rrn operon of the plastid chromosomes representing the five basic, phylogenetically related Euoenothera plastomes were sequenced and compared. The sequences that harbor the putative replication origins are almost identical except for a 785-bp intercistronic segment between the genes for the 16S rRNA and trnI. Differences are mainly caused by insertions/deletions and duplications; the predicted potential for formation of quite extensive secondary structure differs among the plastomes. Unexpected intraplastome variation has also been noted. Furthermore, the sequence-based and published genetically deduced plastome pedigrees differ significantly.
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.
We have determined the nucleotide sequences around the junction points of oligomeric-deleted ptDNAs possessing a head-to-head or tail-to-tail configuration from long-term cultured cell lines and albino plants. It was shown that DNA rearrangement occurred by direct fusion of deleted ptDNAs in an inverted orientation, which was linked by an asymmetrical sequence of 254-698 bp derived from either of the ptDNAs joined. It is notable that inverted repeats of 7-14 bp flank the asymmetrical sequences at each of the junction points. These features of the DNA sequence around the junction points are commonly observed in oligomeric ptDNA with a large-scale deletion regardless of the cell lines employed. It is suggested that the short inverted repeats are involved in the intermolecular recombination of ptDNA.
Careful coordination of cell multiplication with plastid multiplication and partition at cytokinesis is required to maintain the universal presence of plastids in the major photosynthetic lines of evolution. However, no cell cycle control points are known that might underlie this coordination. We review common properties, and their variants, of plastids and plastid DNA in germline, multiplying, and mature cells of phyla capable of photosynthesis. These suggest a basic level of control dictated perhaps by the same mechanisms that coordinate cell size with the nuclear ploidy level. No protein synthesis within the plastid appears to be necessary for this system to operate successfully at the level that maintains the presence of plastids in cells. A second, and superimposed, level of controls dictates expansion of the plastid in both size and number in response to signals associated with differentiation and with the environment. We also compare the germane properties of plastids with those of mitochondria. With the advent of genomics and new cell and molecular techniques, the players in these control mechanisms should now be identifiable.
The Phylum Apicomplexa comprises thousands of obligate intracellular parasites, some of which cause serious disease in man and other animals. Though not photosynthetic, some of them, including the malaria parasites (Plasmodium spp.) and the causative organism of Toxoplasmosis, Toxoplasma gondii, possess a remnant plastid partially determined by a highly derived residual genome encoded in 35 kb DNA. The genetic maps of the plastid genomes of these two organisms are extremely similar in nucleotide sequence, gene function and gene order. However, a study using pulsed field gel electrophoresis and electron microscopy has shown that in contrast to the malarial version, only a minority of the plastid DNA of Toxoplasma occurs as circular 35 kb molecules. The majority consists of a precise oligomeric series of linear tandem arrays of the genome, each oligomer terminating at the same site in the genetic map, i.e. in the centre of a large inverted repeat (IR) which encodes duplicated tRNA and rRNA genes. This overall topology strongly suggests that replication occurs by a rolling circle mechanism initiating at the centre of the IR, which is also the site at which the linear tails of the rolling circles are processed to yield the oligomers. A model is proposed which accounts for the quantitative structure of the molecular population. It is relevant that a somewhat similar structure has been reported for at least three land plant chloroplast genomes.
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.
Photosynthetic eukaryotes have evolved plastid division mechanisms since acquisition of plastids through endosymbiosis. The emerging evolutionary origin of the plastid division mechanism is remarkably complex. The constituents of the division apparatus of plastids may have complex origins. The one constituent is the plastid FtsZ ring taken over from the cyanobacteria-like ancestral endosymbionts. The second is the doublet of concentric plastid dividing rings (or triplet in red algae), possibly acquired by ancestral host eukaryotes following the primary endosymbiotic event. Placement of the division apparatus at the correct division site may involve a system analogous to the bacterial Min system. Plastid nucleoid partitioning may be mediated by binding to envelope or thylakoid membranes. Multiple copies of plastid DNA and symmetrical distribution of the nucleoids in the plastids may permit faithful transmission to daughter plastids via equal binary plastid divisions. Cyanelles retain peptidoglycan wall and cyanelle division occurs through septum formation such as bacterial cell division. Cyanelle division involves the cyanelle ring analogous to the inner stromal plastid-dividing (PD) ring. According to the prevailing hypothesis that primary endosymbiosis occurred only once, cyanelle division may represent an intermediate stage between cyanobacterial division and the well-known plastid division among extant plants. With the secondary plastids, which are surrounded by three or four membranes, the PD ring also participates in division of the inner two "true" plastid envelope membranes, and the third and the outermost membranes divide by unknown mechanisms.
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.
We used pulsed-field gel electrophoresis, restriction fragment mapping, and fluorescence microscopy of individual DNA molecules to analyze the structure of chloroplast DNA (cpDNA) from shoots of ten to 14 day old maize seedlings. We find that most of the cpDNA is in linear and complex branched forms, with only 3-4% as circles. We find the ends of linear genomic monomers and head-to-tail (h-t) concatemers within inverted repeat sequences (IRs) near probable origins of replication, not at random sites as expected from broken circles. Our results predict two major and three minor populations of linear molecules, each with different ends and putative origins of replication. Our mapping data predict equimolar populations of h-t linear concatemeric molecules differing only in the relative orientation (inversion) of the single copy regions. We show how recombination during replication can produce h-t linear concatemers containing an inversion of single copy sequences that has for 20 years been attributed to recombinational flipping between IRs in a circular chromosome. We propose that replication is initiated predominantly on linear, not circular, DNA, producing multi-genomic branched chromosomes and that most replication involves strand invasion of internal regions by the ends of linear molecules, rather than the generally accepted D-loop-to-theta mechanism. We speculate that if the minor amount of cpDNA in circular form is useful to the plant, its contribution to chloroplast function does not depend on the circularity of these cpDNA molecules.
We examined the DNA from chloroplasts obtained from different tissues of juvenile maize seedlings (from eight to 16 days old) and adult plants (50-58 days old). During plastid development, we found a striking progression from complex multigenomic DNA molecules to simple subgenomic molecules. The decrease in molecular size and complexity of the DNA paralleled a progressive decrease in DNA content per plastid. Most surprising, we were unable to detect DNA of any size in most chloroplasts from mature leaves, long before the onset of leaf senescence. Thus, the DNA content per plastid is not constant but varies during development from hundreds of genome copies in the proplastid to undetectable levels in the mature chloroplast. This loss of DNA from isolated, mature chloroplasts was monitored by three independent methods: staining intact chloroplasts with 4',6-diamidino-2-phenylindole (DAPI); staining at the single-molecule level with ethidium bromide after exhaustive deproteinization of lysed chloroplasts; and blot-hybridization after standard DNA isolation procedures. We propose a mechanism for the production of multigenomic chloroplast chromosomes that begins at paired DNA replication origins on linear molecules to generate a head-to-tail linear concatemer, followed by recombination-dependent replication.
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.
Chloroplast DNAs (ctDNA) from pea and corn plants were examined in the electron microscope for the presence of replicative intermediates. Pea and corn ctDNAs were each found to contain two displacement loops (D-loops). The D-loops were 820 (+/- 90) base pairs long in pea ctDNA and 860 (+/- 125) base pairs long in corn ctDNA. In each ctDNA, the two D-loops were located at positions that were 7100 +/- 240) base pairs apart. The displacing strands of the two D-loops were located on opposite strands of the parental DNA molecule and they were seen to expand toward each other. The D-loops in the ctDNA from pea and corn exhibited branch migration and thus were easily distinguished from the denatured regions that were also present in these closed circular ctDNAs. In addition, the positions of the D-loops were found to be distinct from the positions of the denaturation loops (Den-loops). The Den-loops were also shown to be located at AT-rich regions in these ctDNA molecules. D-loops and Den-loops were also found in the circular and catenated ctDNA oligomers from pea and corn plants. Mapping the positions of the D-loops relative to the positions of the Den-loops showed that the structure of the D-loop-containing region in the pea and corn ctDNAs has been conserved to a greater extent than the structure of the rest of the two ctDNA molecules.
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.
We have mapped all the cleavage sites for the restriction endonucleases BstEII, Kpn I, Pst I, Pvu II, Sac I, Sal I, Sma I, and Xho I on the circular chloroplast chromosomes from mung bean and pea. The mung bean chloroplast genome measures 150 kilobase pairs (kb) in length; it includes two identical sequences of 23 kb that contain the ribosomal genes and are arranged as an inverted repeat separated by single-copy regions of 21 and 83 kb. The pea chloroplast genome is only 120 kb in size, has only one set of ribosomal genes, and does not possess any detectable repeated sequences. The mung bean inverted repeat structure is common to all other nonleguminous higher plant chloroplast genomes studied, whereas the pea structure has been found only in the closely related legume Vicia faba. We conclude from these data that loss of one copy of the inverted repeat sequence has occurred only rarely during the evolution of the Angiosperms, and in the case of the legumes after the divergence of the mung bean line from the pea-Vicia line. We present hybridization data indicating that rearrangements that change the linear order of homologous sequences within the chloroplast genome have been quite frequent during the course of legume evolution.
Chloroplast DNA replication in Chlamydomonas reinhardtii is initiated by the formation of a displacement loop (D-loop) at a specific site. One D-loop site with its flanking sequence was cloned in recombinant plasmids SC3-1 and R-13. The sequence of the chloroplast DNA insert in SC3-1, which includes the 0.42-kilobase (kb) D-loop region, as well as 0.2 kb to the 5' end and 0.43 kb to the 3' end of the D-loop region, was determined. The sequence is A+T-rich and contains four large stem-loop stuctures. An open reading frame potentially coding for a polypeptide of 136 amino acids was detected in the D-loop region. One stem-loop structure and two back-to-back prokaryotic-type promoters were mapped within the open reading frame. The 5.5-kb EcoRI fragment cloned in R-13 contains the 1.05-kb SC3-1 insert and its flanking regions. A yeast autonomously replicating (ARS) sequence and an ARC sequence, which promotes autonomous replication in Chlamydomonas, have been mapped within the flanking regions [Vallet, J.-M. & Rochaix, J.-D. (1985) Curr. Genet. 9, 321-324]. Both R-13 and SC3-1 were active as templates in a crude algal preparation that supports DNA synthesis. In this in vitro system, chloroplast DNA synthesis initiated near the D-loop site.
Biosynthesis of the chloroplast depends upon the cooperative expression of both nuclear and plastid genes. Characterization of the integrative mechanisms involved in this interaction will necessarily first require more basic information about the component genomes. Despite its moderate complexity, our knowledge about the informational content of the plastid chromosome is still quite limited: only the genes for two polypeptides and the genes for structural RNAs of the organelle’s translation machinery have been identified and physically mapped, collectively covering approximately 10% of the chromosome’s coding capacity (reviewed in Herrmann and Possingham 1980).
Excerpt
INTRODUCTION
Autoradiography of Escherichia coli, labeled with tritiated thymidine and lysed with duponol, has shown that the bacterial chromosome comprises a single piece of DNA which is probably duplicated at a single growing point (Cairns, 1963). Further, it seemed likely from the variety of structures seen that this DNA is in the form of a circle while it is being replicated, even though no intact replicating circles had, at that time, been found.
There is no immediate prospect of proving that the bacterial chromosome is simply a continuous DNA double helix; the existence, for example, of protein linkers scattered along the chromosome (Freese, 1958) could be disproved only by a degree of purification of the intact chromosome that, at the moment, is technically impossible. So, rather than attempt any further purification, an effort was made to extract the chromosome as an intact but replicating circle by lysing labeled bacteria with...
An EcoRI 2.7 kbp fragment from Chlorella ellipsoidea chloroplast DNA (cpDNA) cloned in YIp5 was shown to promote autonomous replication in Saccharomyces cerevisiae. The fragment was localized in the small single copy region close to the inverted repeat. The ARS activity (autonomously replicating sequences in yeast) was found to be confined within a subclone of a ca. 300 bp HindIII fragment. Sequence analysis of this fragment revealed its high AT content and the presence of several direct and inverted repeats and a few elements that were related to the yeast ARS consensus sequence. Electron microscopic studies revealed that this sequence did not coincide with the primary replication origin of chloroplast DNA. The functioning of this sequence as a possible origin of plasmid replication in vivo is discussed. This is the first report on Chlorella cpDNA sequence. re]19850821 rv]19851211 ac]19851216.
1) DNA has been isolated from the five genetically distinguishable plastid types of Oenothera, subsection Euoenothera. DNA of plastomes I to IV was obtained from plants with identical nuclear backgrounds containing the genotype AA of Oenothera hookeri whereas the DNA of plastome V came from Oenothera argillicola (genotype CC). 2) The DNAs of the five basic Euoenothera wild-type plastomes can be distinguished by restriction endonuclease analysis with Sal I, Pst I, Kpn I, Eco RI and Bam HI. The fragment patterns exhibit distinct common features as well as some degree of variability. 3) Physical maps for the circular DNAs of plastome I, II, III and V could be constructed using the previously detailed map of plastome IV DNA (Gordon et al. 1981). This has been achieved by comparing the cleavage products generated by restriction endonucleases Sal I, Pst I and Kpn I which collectively result in 36 sites in each of the five plastome DNAs, and by hybridization of radioactively labelled chloroplast rRNA or chloroplast cRNA probes of spinach to Southern blots of appropriate restriction digests. The data show that the overall fragment order is the same for all five plastome DNAs. Each DNA molecule is segmentally organized into four regions represented by a large duplicated sequence in inverted orientation whose copies are separated by two single-copy segments. 4) The alterations in position of restriction sites among the Euoenothera plastome DNAs result primarily from insertions/deletions. Eleven size differences of individual fragments in the Sal I, Pst I and Kpn I patterns measuring 0.1-0.8 Md (150-1,200 bp) relative to plastome IV DNA have been located. Most changes were found in the larger of the two single-copy regions of the five plastomes. Changes in the duplication are always found in both copies. This suggests the existence of an editing mechanism that, in natural populations, equalizes or transposes any change in one copy of the repeat to the equivalent site of the other copy. 5) Detailed mapping of the two rDNA regions of the five plastomes, using the restriction endonucleases Eco RI and Bam HI which each recognize more than 60 cleavage sites per DNA molecule, disclosed a 0.3 Md deletion in plastome III DNA and a 0.1 Md insertion in plastome V DNA relative to DNA of plastome IV, I and II. These changes are most probably located in the spacer between the genes for 16S and 23S rRNA and are found in both rDNA units.
The levels of plastid DNA (pt DNA) in both the green and albino leaves of the barley mutant «albostrians» (Hordeum vulgare, Mutant 4205) are the same. The albino leaves of this plant are similar in size to green leaves and contain numerous undifferentiated plastid structures. They have previously been shown to contain no plastid ribosomes, (Borner et al., 1976). These plastids contain a single pt DNA nucleoid while fully differentiated chloroplasts in green leaves each contain a number of small, peripherally located nucleoids. We conclude that both the replication of pt DNA and the formation of simple undifferentiated plastids does not require plastid protein synthesis, is controlled by nuclear DNA, and is mediated by the cytoplasmic protein synthesizing system. We suggest that during differentiation pt DNA is redistributed through the plastid matrix. The implications of these observations in relation to the standing of the concept that plastids are semi-autonomous, self-replicating organelles is discussed.
The relationship between nuclear and plastid DNA synthesis in cultured tobacco cells was measured by following3H-thymidine incorporation into total cellular DNA in the absence or presence of specific inhibitors. Plastid DNA synthesis
was determined by hybridization of total radiolabeled cellular DNA to cloned chloroplast DNA.
Cycloheximide, an inhibitor of nuclear encoded cytoplasmic protein synthesis, caused a rapid and severe inhibition of nuclear
DNA synthesis and a delayed inhibition of plastid DNA synthesis. By contrast, chloramphenicol which only inhibits plastid
and mitochondrial protein production, shows little inhibition of either nuclear or plastid DNA synthesis even after 24 h of
exposure to the cells.
The inhibition of nuclear DNA synthesis by aphidicolin, which specifically blocks the nuclear DNA polymeraseα, has no significant effect on plastid DNA formation. Conversely, the restraint of plastid DNA synthesis exerted by low levels
of ethidium bromide has no effect on nuclear DNA synthesis.
These results show that the synthesis of plastid and nuclear DNA are not coupled to one another. However, both genomes require
the formation of cytoplasmic proteins for their replication, though our data suggest that different proteins regulate the
biosynthesis of nuclear and plastid DNA.
Complete sequences are now available for the chloroplast genomes of two green plants. The information that can be gleaned from these sequences should help us to understand not only how chloroplasts function within present-day plants, but may also yield insights into the evolutionary relationships of photosynthetic organisms and into the gene movement that has occurred among the varius genetic compartments of eukaryotic cells.
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.
The transmission abilities of four out of the five major plastome types of Oenothera (I–V) were analyzed in a constant nuclear background by assessing both the frequency of biparental inheritance and the extent of variegation in the progeny. Reciprocal crosses were performed between plants carrying one of four wild-type plastomes and plants carrying one of seven white plastid mutants. The frequency of biparental plastid transmission ranged from 0 to 56% depending on the plastid types involved in the crosses. The transmission abilities of the four representative wild-type plastids appear to be in the order of I > III > II > IV in the nuclear background of O. hookeri str. Johansen. In general, variegated seedlings from crosses that produced a higher frequency of biparental plastid transmission also had an increased abundance of tissue containing plastids of paternal origin. Although the transmission abilities of most Oenothera plastid mutants are comparable to the wild-type plastids, three mutant plastids derived from species having different type I plastids show three distinguishable transmission patterns. This study confirms the significant role of the plastome in the process of plastid transmission and possibly in plastid multiplication. However, the hypothesis of differential plastid multiplication rates suggested by earlier studies can explain the results only partially. The initiation of plastid multiplication within the newly formed zygote also seems to be plastome-dependent.
Four different base-specific chemical reactions generate a means of directly sequencing RNA terminally labeled with 32P. After a partial, specific modification of each kind of RNA base, an amine-catalyzed strand scission generates labeled fragments whose lengths determine the position of each nucleotide in the sequence. Dimethyl sulfate modifies guanosine. Diethyl pyrocarbonate attacks primarily adenosine. Hydrazine attacks uridine and cytidine, but salt suppresses the reaction with uridine. In all cases, aniline induces a subsequent strand scission. The electrophoretic fractionation of the labeled fragments on a polyacrylamide gel, followed by autoradiography, determines the RNA sequence. RNA labeled at the 3' end yields clean cleavage patterns for each purine and pyrimidine and allows a determination of the entire RNA sequence out to 100-200 bases from the labeled terminus.
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.
This chapter explores how animal mitochondrial DNA (mtDNA) is expressed and replicated. The mtDNA contain only one significant stretch of nucleotide sequence that does not code for RNA or a protein molecule. This region is the displacement-loop (Dloop) portion, which is a signature feature of vertebrate mtDNA. The essential major cis-acting elements necessary for transcriptional initiation are located within the confines of the D-loop region, which is defined explicitly as that sequence bounded by the genes for transfer RNA (tRNA)-phenylalanine and tRNA-proline. The D-loop region also contains all of the required template information necessary for the initiation of nascent heavy (H)-strand DNA synthesis, which marks the beginning of a round of DNA replication in this system. Human mtDNA promoters include a short region encompassing the transcriptional start sites that has important sequence requirements, as evidenced by the fact that mutations in these start sites have serious consequences for promoter function. Efficient transcription requires the presence and action of the only identified trans-acting factor in vertebrate mitochondrial transcription. This factor, termed “mtTFl,” functions by binding immediately upstream of the transcriptional start sites from positions –10 to –40 at each promoter.
In a previous publication it was shown that the output of yeast mitochondrial loci lacking nearby intergenic sequences (encompassing ori/rep elements) was reduced in crosses to strains with wild-type mtDNAs. In the present work, mitochondrial genomes carrying the intergenic deletions were marked at unlinked loci by introducing specific antibiotic resistance mutations against erythromycin, oligomycin and paromomycin. These marked genomes were used to follow the output of unlinked regions of the genome from crosses between the intergenic deletion mutants and wild-type strains. Transmission of genetically unlinked markers in coding regions was substantially reduced when an intergenic deletion was present on the same genome. In general the transmission of the antibiotic markers was the same as or slightly higher than the corresponding intergenic marker. These results indicate that the presence of an intergenic deletion in the regions studied impairs the transmission to progeny of a mitochondrial genome as a whole. More specifically, the results suggest that ori/rep sequences, present in the regions that have been deleted, confer a competitive advantage over genomes lacking a full complement of such sequences. These results support the hypothesis that intergenic sequences, and specifically ori/rep elements, have a biological role in the mitochondrial genome. However, because of the exclusive presence of ori/rep sequences in the genus Saccharomyces, it may be that these sequences evolved in (or invaded) the mitochondrial genome relatively late in the evolution of the yeasts.(ABSTRACT TRUNCATED AT 250 WORDS)
Maize chloroplast DNA sequences representing 94% of the chromosome have been surveyed for their activity as autonomously replicating sequences in yeast and as templates for DNA synthesis in vitro by a partially purified chloroplast DNA polymerase. A maize chloroplast DNA region extending over about 9 kilobase pairs is especially active as a template for the DNA synthesis reaction. Fragments from within this region are much more active than DNA from elsewhere in the chromosome and 50- to 100-fold more active than DNA of the cloning vector pBR322. The smallest of the strongly active subfragments that we have studied, the 1368-base-pair EcoRI fragment x, has been sequenced and found to contain the coding region of chloroplast ribosomal protein L16. EcoRI fragment x shows sequence homology with a portion of the Chlamydomonas reinhardtii chloroplast chromosome that forms a displacement loop [Wang, X.-M., Chang, C.H., Waddell, J. & Wu, M. (1984) Nucleic Acids Res. 12, 3857-3872]. Maize chloroplast DNA fragments that permit autonomous replication of DNA in yeast are not active as templates for DNA synthesis in the in vitro assay. The template active region we have identified may represent one of the origins of replication of maize chloroplast DNA.
Mitochondrial DNAs (mtDNA) from four stable revertant strains generated from high frequency petite forming strains of Saccharomyces cerevisiae have been shown to contain deletions which have eliminated intergenic sequences encompassing ori1, ori2 and ori7. The deleted sequences are dispensable for expression of the respiratory phenotype and mutant strains exhibit the same relative amount of mtDNA per cell as the wild-type (wt) parental strain. These deletion mutants were also used to study the influence of particular intergenic sequences on the transmission of closely linked mitochondrial loci. When the mutant strains were crossed with the parental wt strains, there was a strong bias towards the transmission into the progeny of mitochondrial genomes lacking the intergenic deletions. The deficiency in the transmission of the mutant regions was not a simple function of deletion length and varied between different loci. In crosses between mutant strains which had non-overlapping deletions, wt mtDNA molecules were formed by recombination. The wt recombinants were present at high frequencies among the progeny of such crosses, but recombinants containing both deletions were not detected at all. The results indicate that mitochondrial genomes can be selectively transmitted to progeny and that two particular intergenic regions positively influence transmission. Within these regions other sequences in addition to ori/rep affect transmission.
A rapid and simple method for constructing restriction maps of large DNAs (100–200 kb) is presented. The utility of this method
is illustrated by mapping the Sal I, Sac I, and Hpa I sites of the 152 kb Atriplex triangularis chloroplast genome, and the Sal I and Pvu II sites of the 155 kb Cucumis sativa chloroplast genome. These two chloroplast DNAs are very similar in organization; both feature the near-universal chloroplast
DNA inverted repeat sequence of 22–25 kb.
The positions of four different genes have been localized on these chloroplast DNAs. In both genomes the 16S and 23S ribosomal
RNAs are encoded by duplicate genes situated at one end of the inverted repeat, while genes for the large subunit of ribulose-1,5-bisphosphate
carboxylaae and a 32 kilo-dalton photosystem II polypeptide are separated by 55 kb of DNA within the large single copy region.
The physical and genetic organization of these DNAs is compared to that of spinach chloroplast DNA.
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.
Chloroplast DNA (cpDNA), containing 10% replicative molecules, was isolated 2 h after onset of the dark period from cultures of Euglena gracilis strain Z. The DNA was digested with the restriction enzymes PvuII, SalI, BamHI, or EcoRI. Fragments that contained intact replicative loops were measured to determine the position of replicated sequences in relation to the restriction enzyme sites. It was found that replication starts at a unique position near one of the palindromic sequences I(2) (Koller and Delius, 1982a) which is located upstream (with respect to the direction of rRNA transcription) of the AT-rich region of variable size (Jenni et al., 1981; Schlunegger et al., in preparation). In the majority of cases DNA synthesis proceeds unidirectionally away from this region for 5000 nucleotides before it starts in the other direction (in the same sense as the rRNA transcription) through the Z-region and the second palindromic sequence.
The recessive nuclear gene iojap of Zea mays conditions a permanent, heritable deficiency in the ability of the plastid to differentiate. iojap-affected plastids contain a normal genome as evidenced by comparison of the restriction endonuclease digestion patterns of affected and normal plastids. iojap-affected plastids contain neither detectable ribosomes nor high molecular weight RNA; the affected plastids do not incorporate exogenous amino acids into protein. The lesion in plastid ribosome content occurs early in organ ontogeny because iojap-mediated albino stripes can occupy entire clones within affected leaves.
Determinations were made of the percentage of chloroplast DNA (ct DNA) in total cell DNA isolated from shoots of pea at different stages of development. Labeled pea ct DNA was reassociated with a high concentration of total DNA; the percentage of ct DNA was estimated by comparing the rate of reassociation of this reaction with that of a model reaction containing a known concentration of unlabeled ct DNA. The maximum change in ct DNA content was from 1.3% of total DNA in young shoots to 7.3% in fully greened shoots. Analyses were also performed on DNA from embryos, etiolated tissue, roots, and leaves. The first leaf set to develop in pea was excised over a growth period of 8 days during which leaf length increased from 4 to 12 millimeters. Young leaves contained about 8% ct DNA; in fully greened leaves the level of ct DNA approached 12%, equivalent to as many as 9,575 copies of ct DNA per cell. Root tissue contained only 0.4% ct DNA.
Absolute DNA amounts of individual chloroplasts from mesophyll and epidermal cells of developing spinach leaves were measured by microspectrofluorometry using the DNA-specific stain, 4,6-diamidino-2-phenyl indole, and the bacterium, Pediococcus damnosus, as an internal standard. Values obtained by this method showed that DNA amounts of individual chloroplasts from mesophyll cells fell within a normal distribution curve, although mean DNA amounts changed during leaf development and also differed from the levels in epidermal chloroplasts. There was no evidence in the data of plastids containing either the high or low levels of DNA which would be indicative of discontinuous polyploidy of plastids, or of division occurring in only a small subpopulation of chloroplasts. By contrast, the distribution of nuclear DNA amounts in the same leaf tissues in which cell division was known to be occurring showed a clear bimodal distribution. We consider that the distribution of chloroplast DNA in the plastid population shows that there is no S-phase of chloroplast DNA synthesis, all chloroplasts in the population in young leaf cells synthesize DNA, and all chloroplasts divide.
The chloroplast division cycle and its relationship to the cell division cycle The cell division cycle in plants
- Sa Boffey
Cairns replicative intermediates in Acetabularia chloroplast DNA
- A Santulli
- A Casale
- A Mazza
The chloroplast division cycle and its relationship to the cell division cycle
- S A Boffey
- SA Boffey
Rearrangements in the chloroplast genome of mung bean and pea
- J D Palmer
- W E Thompson
- JD Palmer