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Required sequence elements for chloroplast DNA replication activity in vitro and in electroporated chloroplasts
Abstract
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.
... The purified oriA fragment was 3'end-labelled with biotin using the Biotin 3'end DNA Labeling Kit (Pierce) according to the manufacturer's instructions, and stored at -20°C. Some ctDNA ori fragments (Lugo et al. 2004, see legend to Fig. 1) were radioactively labeled (Sambrook and Russell 2001) for use as probes of a southwestern blot of total chloroplast proteins. ...
... After transfer, the proteins on the blot are renatured by incubating the blot in binding buffer (50 mM Tris pH 8.0, 1 mM dithiothreitol, 0.3% Tween 20, 150 mM NaCl). Radioactively labeled probes from different clones of either minimal ctDNA ori region (Lugo et al. 2004; see Figure 1 legend for details) or from pUC19 lacking an insert as a control were mixed with an excess of nonspecific poly (dIdC) competitor (10 lg/mL) in binding buffer and incubated with strips of individual lanes of the gel overnight at room temperature. The membrane strips were then washed three times (10 min each) with binding buffer, wrapped in clear plastic wrap and exposed to x-ray film. ...
... Detection of ori-binding proteins from total chloroplast extracts via southwestern blot. Lane 1 was probed with pUC19 DNA; lane 2 was probed with a 168 bp fragment of oriA; lane 3 was probed with a 1.3 kb fragment containing oriA; lane 4 was probed with a 248 bp fragment or oriB; lane 5 was probed with a 507 bp fragment containing oriB (SeeLugo et al. 2004 for details of ori fragments) ...
Replication of chloroplast DNA (ctDNA) in several plants and in Chlamydomonas reinhardii has been shown to occur by a double displacement loop (D-loop) mechanism and potentially also by a rolling circle mechanism. D-loop replication origins have been mapped in several species. Minimal replication origin sequences used as probes identified two potential binding proteins by southwestern blot analysis. A 28 kDa (apparent molecular weight by SDS-PAGE analysis) soybean protein has been isolated by origin sequence-specific DNA affinity chromatography from total chloroplast proteins. Mass spectrometry analysis identified this protein as the product of the soybean C6SY33 gene (accession number ACU14156), which is annotated as encoding a putative uncharacterized protein with a molecular weight of 25,897 Da, very near the observed molecular weight of the purified protein based on gel electrophoresis. Western blot analysis using an antibody against a homologous Arabidopsis protein indicates that this soybean protein is localized specifically in chloroplasts. The soybean protein shares some homology within a single-stranded DNA binding (SSB) domain of E. coli SSB and an Arabidopsis thaliana mitochondrial-localized SSB of about 21 kDa (mtSSB). However, the soybean protein induces a specific electrophoretic mobility shift only when incubated with a double-stranded fragment containing the previously mapped ctDNA replication oriA region. This protein has no electrophoretic mobility shift activity when incubated with single-stranded DNA. In contrast, the Arabidopsis mtSSB causes a mobility shift only with single-stranded DNA but not with the oriA fragment or with control dsDNA of unrelated sequence. These results suggest that the 26 kDa soybean protein is a specific origin binding protein that may be involved in initiation of ctDNA replication.
... In line with this model, two replication origins -oriA and oriBwere found in N. tabacum, on both of which, independently, r replication could be initiated in vitro and in isolated chloroplasts. h replication could be started in vitro and in isolated chloroplasts involving both oriA and oriB (Kunnimalaiyaan and Nielsen, 1997b;Kunnimalaiyaan et al., 1997;Lu et al., 1996;Lugo et al., 2004). Both oris are located in the inverted repeats, therefore there are two copies of both on one monomeric plastid DNA molecule. ...
... According to the Kolodner and Tewari (1975) model, plastid DNA replication of N. tabacum initiates on a pair of replication origins: oriA and oriB (Kunnimalaiyaan and Nielsen, 1997b;Kunnimalaiyaan et al., 1997;Lu et al., 1996;Lugo et al., 2004). Pairs of oris were also detected in other species: (Kunnimalaiyaan and Nielsen, 1997b), the suspension culture ori (Takeda et al., 1992;Wang et al., 2002), the putative homologue of oriA of Oenothera elata (Chiu and Sears, 1992), the putative homologue of an ori near psbA or rpl16, respectively, of Zea mays (Carrillo and Bogorad, 1988) and near rpl16 of Chlamydomonas reinhardtii (Lou et al., 1987) and Glycine max (Hedrick et al., 1993) are given. ...
... oriA is not essential in N. tabacum (Mü hlbauer et al., 2002). On the other hand, replication is more often initiated at oriA than at oriB (Kunnimalaiyaan and Nielsen, 1997b;Lugo et al., 2004). This could explain why, after co-transformation using deletion vectors A4 and B2 (Mü hlbauer et al., 2002), deletions of oriB2 were found more frequently than those of oriA. ...
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.
... The proposed tobacco OriA replication origin comprises 82 bases in the plastid tRNA Ile intron and includes an eight-base direct repeat and imperfect inverted repeat which forms a stem-loop structure. Although tobacco OriA exhibits in vitro evidence of replication activity, tobacco plastids with deleted OriA sequences become homoplastomic, suggesting that OriA is dispensable for replication (Kunnimalaiyaan and Nielsen 1997;Lugo et al. 2004;Muhlbauer et al. 2002). All 34 species exhibit strong conservation of the first ∼ 50 bases of the OriA sequence, which contain the direct repeat AGATCCAA and the 'up-stream stem' (Fig. 5). ...
... Both repeats are associated with protein binding and DNA melting in bacterial plasmid replication (del Solar et al. 1998). Although OriA is not considered essential for replication, initiation of replication has been demonstrated at OriA sites in vitro and in tobacco chloroplasts (Lugo et al. 2004;Muhlbauer et al. 2002). Moreover, variability in the usage of different plastid replication origins has been reported among cultured and primary cell types in rice (Wang et al. 2003). ...
Biomanufacturing by chloroplast transgene expression has the potential to produce significant amounts of biopharmaceuticals, endow plants with novel commercial or humanitarian capabilities, enhance phytoremediation methods and harden plants against adverse environments. Plastid bioengineering exploits the phenomenon of homologous recombination to specifically integrate heterologous sequences into the plastid genome. Previous research suggests the plastid genome 16S-23S internal transcribed spacer provides an advantageous integration site for transgene expression. To characterize the suitability of the 16S-23S region for interspecific recombination, we developed primers against conserved plastid sequences and amplified approximately 2.6 kb from 25 plant species. We analyzed the amplicons with nine species from Genbank for homeology, phylogenetic relationships, potential to form chimeric rDNA elements disruptive to translational/replication systems, and the potential number of recombination events for various minimal essential processing segments (MEPS) lengths. Multiple sequence alignment of the 34 species revealed considerable conservation, with identities exceeding 95% among the angiosperms. Substitutions were statistically clustered, generally in noncoding sites, although proposed functional elements such as the OriA region and 3' terminus of the 16S rRNA exhibited unexpected variation. The nonrandom distribution of substitutions undermines the established, statistical method of estimating the number of recombination initiation sites. This finding is further substantiated by comparing statistical estimates of the number of MEPS sites to a direct count at three different MEPS lengths. We frame this in silico analysis in terms of the potential of the 16S-23S region as a target for interspecific transformation, and describe a 'primer-to-plastid' system to rapidly generate species-specific flanking regions for transformation vectors.
... Cosa et al. (2001) showed that high protein expression levels can be obtained by inserting foreign genes into the inverted repeat region. Under the same conditions, the transcription level of the foreign gene inserted into trnI/trnA was 25 times higher than that inserted into the rbcL/accD locus (Krichevsky et al. 2010), which may be because the oriA located in the flanking region of trnI promotes the replication of the foreign gene in the chloroplast, and any gene inserted into one of the repeat sequences rapidly replicates into the second repeat sequence via gene transformation (Lugo et al. 2004). The intergenic region in the inverted repeat between the psbA and rrn operon is also usually used as the target site. ...
Tisochrysis lutea is a haptophyte microalga commonly used as a commercial feed for juvenile fish and shellfish larvae. Genetic modification is of considerable importance for developing the potential economic value of T. lutea. However, the genetic transformation system of T. lutea has not yet been established, which limits functional genomic studies and strain improvement of this important microalgal species. In the current study, a chloroplast transformation vector harboring the phosphinothricin resistance gene (bar) as a selectable marker was established, and two short peptide-encoding genes (ant1 and ant2) driven by the endogenous psbA and rbcL promoters were cloned in this vector. The recombinant plasmid was transformed using a biolistic method into the trnI/trnA locus of the chloroplast genome via homologous recombination. After continuous selection on phosphinothricin, the integration of foreign genes and the expression of specific products in the transformants were detected using polymerase chain reaction (PCR), Southern blotting, and western blot analysis. This is the first report of establishment of a stable transformation system in T. lutea, which is a prerequisite for functional genomics and applied research on this species.
... trnA/trnI is the most frequently used insertion site in plastid transformation experiments which directs the foreign gene sequences into inverted repeat region of plastome. trnI regions holds oriA within it that enables targeted foreign DNA replications in the chloroplasts and by the gene conversion the inserted foreign gene will be rapidly copied into another inverted repeat region (Lugo et al. 2004). ...
The high expression level of industrial and metabolically important proteins in plants can be achieved by plastid transformation. The CaIA vector, a Capsicum-specific vector harboring aadA (spectinomycin resistance), is a selectable marker controlled by the PsbA promoter, and the terminator is flanked by the trnA and trnI regions of the inverted repeat (IR) region of the plastid. The CaIA vector can introduce foreign genes into the IR region of the plastid genome. The biolistic method was used for chloroplast transformation in Scoparia dulcis with leaf explants followed by antibiotic selection on regeneration medium. Transplastomes were successfully screened, and the transformation efficiency of 3 transgenic lines from 25 bombarded leaf explants was determined. Transplastomic lines were evaluated by PCR and Southern blotting for the confirmation of aadA insertion and its integration into the chloroplast genome. Seeds collected from transplastomes were analyzed on spectinomycin medium with wild types to determine genetic stability. The increased chloroplast transformation efficiency (3 transplastomic lines from 25 bombarded explants) would be useful for expressing therapeutically and industrially important genes in Scoparia dulcis L.
Keywords : Scoparia dulcis L., Heterologous vectors, CaIA plastid vector, Inverted repeat region, Transplastomic lines
... The foreign gene inserted at the inverted repeat region of the chloroplast genome showed highest expression levels (Cosa et al. 2001), probably because oriA located within the trnI flanking region that facilitates replication of foreign genes within the chloroplasts and any gene inserted into one of the repeats is rapidly copied into the second repeat copy by gene conversion (Lugo et al. 2004). Hence, the trnI/trnA intergenic space regions were mostly selected for construction of chloroplast vector. ...
... The foreign gene inserted at the inverted repeat region of the chloroplast genome showed highest expression levels (Cosa et al. 2001), probably because oriA located within the trnI flanking region that facilitates replication of foreign genes within the chloroplasts and any gene inserted into one of the repeats is rapidly copied into the second repeat copy by gene conversion (Lugo et al. 2004). Hence, the trnI/trnA intergenic space regions were mostly selected for construction of chloroplast vector. ...
The plastid transformation is used for high level expression of certain metabolically and industrially important recombinant proteins in plants. The vector, pFaadAII, a tobacco based vector system, harbouring a chimeric gene consisting of aadA coding region from Escherichia coli with 5′ 16S rDNA promoter and 3′ untranslated transcript region (UTR) of chlamydomonas rbcL gene, located in between the intergenic regions of rp132 and trnL genes. This vector used for transformation of plastids targets the foreign sequences to the small single-copy region of the plastome. Biolistic mode of approach for chloroplast transformation in Scoparia dulcis L., was achieved by bombarding the leaf explants and spectinomycin based selection system was used for regeneration of transformed plants. Transplastomic lines have been successfully established with overall efficiency of two transgenic lines for twenty-five bombarded explants. Integration of aadA in selection based regenerants was characterized by PCR and protein accumulation analysis along with seedlings experiment obtained from selfing. The chloroplast transformation developed in this plant system will provide scope for research in plastid based metabolic engineering pathways.
... The foreign gene inserted at the inverted repeat region of the chloroplast genome showed highest expression levels (Cosa et al. 2001), probably because oriA located within the trnI flanking region that facilitates replication of foreign genes within the chloroplasts and any gene inserted into one of the repeats is rapidly copied into the second repeat copy by gene conversion (Lugo et al. 2004). Hence, the trnI/trnA intergenic space regions were mostly selected for construction of chloroplast vector. ...
Chloroplast transformation vectors require an expression cassette flanked by homologous plastid sequences to drive plastome recombination. The rrn16-rrn23 plastome region was selected and using this region, a new species-specific plastid transformation vector CuIA was developed with pKS+II as a backbone by inserting the rrn16-trnI and trnA-rrn23 sequences from Cucumis sativus L. An independent expression cassette with aadA gene encoding aminoglycoside 3′-adenylyltransferase with psbA controlling elements is added into the trnI-trnA intergenic region that confers resistance to spectinomycin. An efficient plastid transformation in bitter melon (Momordica charantia L.) was achieved by bombardment of petiole segments. The frequency of transplastomic plants yielded using standardized biolistic parameters with CuIA vector was two per 15 bombarded plates, each containing 20 petiole explants. Integration of aadA gene was verified by PCR analysis in transplastomes. Transplastomic technology developed may be a novel approach for high level expression of pharmaceutical traits.
... It appears that this preferred site is unique and allows highly efficient transgene integration and expression. One of the flanking regions used at this site of integration contains the chloroplast origin of replication (Kunnimalaiyaan, Shi, and Nielsen, 1997;Lugo et al., 2004) and this might facilitate replication of foreign vectors within chloroplasts (Daniell et al., 1990), enhance the probability of transgene integration and achieve homoplasmy even in the first round of selection (Guda, Lee, and Daniell, 2000). This is further illustrated by the first successful Rubisco engineering obtained by integrating the RbcS gene at this site (Dhingra et al., 2004). ...
Chloroplast genetic engineering offers a number of unique advantages, including a high-level of transgene expression, multi-gene engineering in a single transformation event, transgene containment via maternal inheritance, lack of gene silencing, position and pleiotropic effects, and undesirable foreign DNA. Thus far, over forty transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer important agronomic traits, as well as express industrially valuable biomaterials and therapeutic proteins. The hyperexpression of recombinant proteins within plastid engineered systems offers a cost effective solution for using plants as bioreactors. Additionally, the presence of chaperones and enzymes within the chloroplast help to assemble complex multi-subunit proteins and correctly fold proteins containing disulfide bonds, thereby drastically reducing the costs of in vitro processing. Oral delivery of vaccine antigens against cholera, tetanus, anthrax, plague, and canine parvovirus are made possible because of the high expression levels and antibiotic-free selection systems available in plastid transformation systems. Plastid genetic engineering also has become a powerful tool for basic research in plastid biogenesis and function. This approach has helped to unveil a wealth of information about plastid DNA replication origins, intron maturases, translation elements and proteolysis, import of proteins and several other processes. Although many successful examples of plastid engineering have set a foundation for various future applications, this technology has not been extended to many of the major crops. Highly efficient plastid transformation has been recently accomplished via somatic embryogenesis using species-specific chloroplast vectors in soybean, carrot, and cotton. Transgenic carrots were able to withstand salt concentrations that only halophytes could tolerate; more than twice the effectiveness of other engineering attempts. Recent advances in plastid engineering provide an efficient platform for the production of therapeutic proteins, vaccines, and biomaterials using an environmentally friendly approach. This review takes an in-depth look into the state of the art in plastid engineering and offers directions for further research and development.
... The expression of the foreign genes was verified by protein isolation and detection of the expected enzyme activity (). The electroporation method was further used to determine the DNA sequences necessary for replication in tobacco chloroplasts (Lugo et al., 2004) but in their study the degradation of introduced DNA started already 45 min. after transformation. ...
Sea slugs of the genus Elysia (e.g. E. chlorotica) are known for their ability to incorporate chloroplasts from the yellow-green alga Vaucheria litorea. These “kleptoplasts” stay active in the digestive tract of the sea slug for several months. Chloroplasts from Vaucheria litorea are also reported to be significantly more stable after in vitro isolation than chloroplasts of other algae or of higher plants. In organello assays with isolated chloroplasts are used in studies on photosynthetical and biochemical processes in these organelles, chloroplast-nucleus communication, plant development and plant responses to environmental changes. The major limiting factor of the chloroplast in organello systems is the rapid decline in function and intactness of the plastids. This study is investigating the stability and longevity of chloroplasts isolated from V. litorea and its relative V. bursata in comparison to an angiosperm Pisum sativum. The structural intactness of the chloroplasts was investigated by phase contrast microscopy and the overall transcriptional activities were analyzed by run-on transcription assays. The recently completed sequence of the plastid genome from V.litorea has made it possible to investigate also the specific changes in isolated chloroplasts on the transcriptional level. The expression patterns of chloroplast-encoded genes trnE, rrn23, rrn16, rbcL, psbD, psbA and psaA directly after isolation and 4 hours post-isolation time were analyzed using dot blot hybridization. The ability of isolated chloroplasts to incorporate 35S-methionine into de novo synthesized proteins at 0, 4 and 24 hours after isolation was tested by translational assays. The chloroplasts of Vaucheria litorea were both transcriptionally and translationally stable over an extended period of time. However, the culturing limitations and low plastid yields diminish the potential of this alga as a chloroplast donor for in organello assays. Chloroplasts of Pisum sativum, previously suspected to be more unstable, did not show a decline in transcription rate until 4 hours after isolation. The decrease in incorporation of 35S-methionine between time 0 and 4 hours after isolation was only minor, between 4 and 24 hours more significant but the radioactive signal was still readily detectable at 24 hours. Pisum sativum has simple growth requirements and offers high yields of isolated chloroplasts. Chloroplasts from this plant should therefore be in the centre of further investigations into their suitability to express foreign genes and their potential for in organello assays.
... In vitro replication analysis has revealed that the minimal sequences required for activity of single-ori ctDNA templates in tobacco is 82 bp for oriA and 243 bp for oriB (Kunnimalayaan and Nielsen, 1997b). There are conflicting reports on the effect of deletions of chloroplast oris (Muhlbauer et al., 2002; Kunnimalayaan and Nielsen, 1997b; Lu et al., 1996; Lugo et al., 2004). It is quite possible that disruption of an ori in vivo is not lethal if another mechanism can take over. ...
Chloroplast genetic engineering overcomes concerns of gene containment, low levels of transgene expression, gene silencing, positional and pleiotropic effects or presence of vector sequences in transformed genomes. Several therapeutic proteins and agronomic traits have been highly expressed via the tobacco chloroplast genome but extending this concept to important crops has been a major challenge; lack of 100 homologous species-specific chloroplast transformation vectors containing suitable selectable markers, ability to regulate transgene expression in developing plastids and inadequate tissue culture systems via somatic embryogenesis are major challenges. We employed a 'Double Gene/Single Selection (DGSS)' plastid transformation vector that harbors two selectable marker genes (aph A-6 and npt II) to detoxify the same antibiotic by two enzymes, irrespective of the type of tissues or plastids; by combining this with an efficient regeneration system via somatic embryogenesis, cotton plastid transformation was achieved for the first time. The DGSS transformation vector is at least 8-fold (1 event/2.4 bombarded plates) more efficient than 'Single Gene/Single Selection (SGSS)' vector (aph A-6; 1 event per 20 bombarded plates). Chloroplast transgenic lines were fertile, flowered and set seeds similar to untransformed plants. Transgenes stably integrated into the cotton chloroplast genome were maternally inherited and were not transmitted via pollen when out-crossed with untransformed female plants. Cotton is one of the most important genetically modified crops (120 billion US dollars US annual economy). Successful transformation of the chloroplast genome should address concerns about transgene escape, insects developing resistance, inadequate insect control and promote public acceptance of genetically modified cotton.
... Chloroplast vectors may also carry an origin of replication that facilitates replication of the plasmid inside the chloroplast, thereby increasing the template copy number for homologous recombination and consequently enhancing the probability of transgene integration. oriA is present within the trnI flanking region (Kunnimalaiyaan and Nielsen, 1997;Lugo et al., 2004), and this might facilitate replication of foreign vectors within chloroplasts , enhance the probability of transgene integration, and achieve homoplasmy even in the first round of selection (Guda et al., 2000). This is further confirmed by the first successful Rubisco engineering obtained by integrating the rbcS gene at this site (Dhingra et al., 2004). ...
Chloroplasts are ideal hosts for expression of trans- genes. Transgene integration into the chloroplast genome occurs via homologous recombination of flanking sequences used in chloroplast vectors. Iden- tification of spacer regions to integrate transgenes and endogenous regulatory sequences that support opti- mal expression is the first step in construction of chloroplast vectors. Thirty-five sequenced crop chlo- roplast genomes provide this essential information. Various steps involved in the design and construction of chloroplast vectors, DNA delivery, and multiple rounds of selection are described. Several crop species have stably integrated transgenes conferring agro- nomic traits, including herbicide, insect, and disease resistance, drought and salt tolerance, and phytore- mediation. Several crop chloroplast genomes have been transformed via organogenesis (cauliflower (Brassica oleracea), cabbage (Brassica capitata), lettuce (Lactuca sativa), oilseed rape (Brassica napus), petunia (Petunia hybrida), poplar (Populus spp.), potato (Sola- num tuberosum), tobacco (Nicotiana tabacum), and to- mato (Solanum lycopersicum)) or embryogenesis (carrot (Daucus carota), cotton (Gossypium hirsutum), rice (Oryza sativa), and soybean (Glycine max)), and maternal inher- itance of transgenes has been observed. Chloroplast- derived biopharmaceutical proteins, including insulin, interferons (IFNs), and somatotropin (ST), have been evaluated by in vitro studies. Human INFa2b trans- plastomic plants have been evaluated in field studies. Chloroplast-derived vaccine antigens against bacterial (cholera, tetanus, anthrax, plague, and Lyme disease), viral (canine parvovirus (CPV) and rotavirus), and protozoan (amoeba) pathogens have been evaluated by immune responses, neutralizing antibodies, and pathogen or toxin challenge in animals. Chloroplasts have been used as bioreactors for production of bio- polymers, amino acids, and industrial enzymes. Oral delivery of plant cells expressing proinsulin (Pins) in chloroplasts offered protection against development of insulitis in diabetic mice; such delivery eliminates expensive fermentation, purification, low temperature storage, and transportation. Chloroplast vector sys- tems used in these biotechnology applications are described. ADVANTAGES OF PLASTID TRANSFORMATION
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.
Chloroplast genetic engineering is becoming an attractive field in plant biotechnology. It offers several unique advantages,
including high-level transgene expression, multi-gene engineering in a single transformation event and transgene containment
by maternal inheritance, as well as lack of gene silencing, position and pleiotropic effects and undesirable foreign DNA.
The hyper-expression of recombinant proteins within plastids offers a cost effective solution for using plants as bioreactors.
Many transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer important agronomic
traits including herbicide, insect, and disease resistance, drought and salt tolerance, and phytoremediation. Moreover, many
vaccine antigens and biopharmaceutical proteins have been expressed at high levels via the chloroplast genome and their functionality
has been evaluated using in vitro cell cultures as well as challenge with bacterial or viral pathogens using animal models.
This technology has been extended to other crop species for the introduction of agronomic traits and production of low cost
vaccine antigens and therapeutic proteins. Production of therapeutic proteins in chloroplasts eliminates the expensive fermentation
technology. In addition, oral delivery of chloroplast-derived therapeutic proteins should eliminate expensive purification
steps, cold storage, cold transportation and delivery via sterile needles, thereby further decreasing their cost. Apart from
the successful productions of vaccines and therapeutic proteins, chloroplast technology is also a good platform for the production
of industrial products. Recently this technology has been extended for the production of enzymes used in ethanol production.
In this chapter, we describe the overall concept of the chloroplast technology and the current status of chloroplast-derived
vaccine antigens and biopharmaceutical proteins.
Recent advances in transcriptomics and bioinformatics, specifically strand-specific RNA sequencing, have allowed high-throughput, comprehensive detection of low-abundance transcripts typical of the non-coding RNAs studied in bacteria and eukaryotes. Before this, few plastid non-coding RNAs (pncRNAs) had been identified, and even fewer had been investigated for any functional role in gene regulation. Relaxed plastid transcription initiation and termination result in full transcription of both chloroplast DNA strands. Following this, post-transcriptional processing produces a pool of metastable RNA species, including distinct pncRNAs. Here we review pncRNA biogenesis and possible functionality, and speculate that this RNA class may have an underappreciated role in plastid gene regulation.
Introns and spacers are a rich and well-appreciated information source for evolutionary studies in plants. Compared to coding
sequences, the mutational dynamics of introns and spacers is very different, involving frequent microstructural changes in
addition to substitutions of individual nucleotides. An understanding of the biology of sequence change is required for correct
application of molecular characters in phylogenetic analyses, including homology assessment, alignment coding, and tree inference.
The widely used term “indel” is very general, and different kinds of microstructural mutations, such as simple sequence repeats,
short tandem repeats, homonucleotide repeats, inversions, inverted repeats, and deletions, need to be distinguished. Noncoding
DNA has been indispensable for analyses at the species level because coding sequences usually do not offer sufficient variability.
A variety of introns and spacers has been successfully applied for phylogeny inference at deeper levels (major lineages of
angiosperms and land plants) in past years, and phylogenetic structure R in intron and spacer data sets usually outperforms that of coding-sequence data sets. In order to fully utilize their potential,
the molecular evolution and applicability of the most important noncoding markers (the trnT–trnF region comprising two spacers and a group I intron; the trnS–G region comprising one spacer and a group II intron in trnG; the group II introns in petD, rpl16, rps16, and trnK; and the atpB–rbcL and psbA–trnG spacers) are reviewed. The study argues for the use of noncoding DNA in a spectrum of applications from deep-level phylogenetics
to speciation studies and barcoding, and aims at outlining molecular evolutionary principles needed for effective analysis.
KeywordsSpacers-Introns-Phylogenetic structure R
-Molecular evolution-SSRs-Inversions-Mutational hotspots-DNA barcoding
Chloroplast is a new hotspot in the field of plant transformation system of plant genetic engineering. Plastid transformation has several advantages: high expression, multiple expressed genes in a single transformation event, absence of gene silencing, et al. A series of elements for construction of dicistronic site-specific integration expression vector of rice chloroplast have been cloned, including trnl-trnA (rice chloroplast homologous recombination fragments), Prrn (16S rRNA operon promotor), PpsbA (the 3' untranslated region of the chloroplastpsbA gene), hptll gene (encoding hygromycin phosphotransferase) and EGFP (encoding enhanced green fluorescence protein). All the elements were constructed into a rice chloroplast dicistronic expression vector pCTE04 (-trnl-Prrn-RBS-hptlI-RBS-EGFP-PpsbA- trnA-). Then pCTE04 was introduced into chloroplasts of Dunaliella salina through particle bombardment. Strong green fluorescence was observed in chloroplasts of some bombarded Dunaliella salina cells under a stereo fluorescence microscope, indicating that pCTE04 could be expressed in Dunaliella salina chloroplasts transiently. It provides a solid foundation for further genetic engineering in rice chloroplast transformation.
Higher plant plastid DNA (ptDNA) is generally described as a double-stranded circular molecule of the size of the monomer of the plastid genome. Also, the substrates and products of ptDNA replication are generally assumed to be circular molecules. Linear or partly linear ptDNA molecules were detected in our present study using pulsed-field gel electrophoresis and Southern blotting of ptDNA restricted with 'single cutter' restriction enzymes. These linear DNA molecules show discrete end points which were mapped using appropriate probes. One possible explanation of discrete ends would be that they represent origins of replication. Indeed, some of the mapped ends correlate well with the known origins of replication of tobacco plastids, i.e. both of the oriA sequences and--less pronouncedly--with the oriB elements. Other ends correspond to replication origins that were described for Oenothera hookeri, Zea mays, Glycine max and Chlamydomonas reinhardtii, respectively, while some of the mapped ends were not described previously and might therefore represent additional origins of replication.
Chloroplasts contain multiple copies of a DNA molecule (the plastome) that encodes many of the gene products required to perform photosynthesis. The plastome is replicated by nuclear-encoded proteins and its copy number seems to be highly regulated by the cell in a tissue-specific and developmental manner. Our understanding of the biochemical mechanism by which the plastome is replicated and the molecular basis for its regulation is limited. In this commentary we review our present understanding of chloroplast DNA replication and examine current efforts to elucidate its mechanism at a molecular level.
Chloroplasts contain circular DNA molecules which are found in low copy number in proplastids but are amplified to very high copy number in actively dividing leaf cells. A double displacement loop (D-loop) mechanism for chloroplast DNA (ctDNA) replication has been proposed, and pairs of replication origins which fit this model have been identified in some species. It appears that ctDNA replication is under the control of at least some nuclear gene products, as genes for DNA polymerase, topoisomerases, DNA primase and other accessory replication proteins have not been reported in the sequenced chloroplast genomes, and ctDNA replication remains active in the absence of active chloroplast transcription or translation. Only a few chloroplast replication proteins have been isolated, and to date most have not been characterized in detail. The mechanism by which ctDNA copy number is regulated during plant development is not known. In this review we summarize the current understanding of ctDNA replication.
The mechanisms of chloroplast recombination are largely unknown. Using the chloroplast-encoded homing endonuclease I-CreI from Chlamydomonas reinhardtii, an experimental system is described that allows the study of double strand break (DSB)-induced recombination in chloroplasts.
The I-CreI endonuclease is encoded by the chloroplast ribosomal group I intron of C.reinhardtiiand cleaves specifically intronless copies of the large ribosomal RNA (23S) gene. To study DSB-induced recombination in chloroplast
DNA, the genes encoding the I-CreI endonuclease were deleted and a target site for I-CreI, embedded in a cDNA of the 23S gene, was integrated at an ectopic location. Endonuclease function was transiently provided
by mating the strains containing the recombination substrate to a wild-type strain. The outcome of DSB repair was analyzed
in haploid progeny of these crosses. Interestingly, resolution of DSB repair strictly depended upon the relative orientation
of the ectopic ribosomal cDNA and the adjacent copy of the 23S gene. Gene conversion was observed when the 23S cDNA and the
neighbouring copy of the 23S gene were in opposite orientation, leading to mobilization of the intron to the 23S cDNA. In
contrast, arrangement of the 23S cDNA in direct repeat orientation relative to the proximal 23S gene resulted in a deletion
between the 23S cDNA and the 23S gene. These results demonstrate that C.reinhardtii chloroplasts have an efficient system for DSB repair and that homologous recombination is strongly stimulated by DSBs in
chloroplast DNA.
Studies of chloroplast DNA variations, and several direct experimental observations, indicate the existence of recombination ability in algal and higher plant plastids. However, no studies have been done of the biochemical pathways involved. Using a part of a cyanobacterial recA gene as a probe in Southern blots, we have found homologous sequences in total DNA from Pisum sativum and Arabidopsis thaliana and in a cDNA library from Arabidopsis. A cDNA was cloned and sequenced, and its predicted amino acid sequence is 60.7% identical to that of the cyanobacterial RecA protein. This finding is consistent with our other results showing both DNA strand transfer activity and the existence of a protein of the predicted molecular mass crossreactive with antibodies to Escherichia coli RecA in the stroma of pea chloroplasts.
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 analyzed the mechanism of recombination-dependent DNA replication in bacteriophage T4-infected Escherichia coli using plasmids that have sequence homology to the infecting phage chromosome. Consistent with prior studies, a pBR322 plasmid, initially resident in the infected host cell, does not replicate following infection by T4. However, the resident plasmid can be induced to replicate when an integrated copy of pBR322 vector is present in the phage chromosome. As expected for recombination-dependent DNA replication, the induced replication of pBR322 required the phage-encoded UvsY protein. Therefore, recombination-dependent plasmid replication requires homology between the plasmid and phage genomes but does not depend on the presence of any particular T4 DNA sequence on the test plasmid. We next asked whether T4 recombination-dependent DNA replication can be triggered by a double-strand break (dsb). For these experiments, we generated a novel phage strain that cleaves its own genome within the nonessential frd gene by means of the I-TevI endonuclease (encoded within the intron of the wild-type td gene). The dsb within the phage chromosome substantially increased the replication of plasmids that carry T4 inserts homologous to the region of the dsb (the plasmids are not themselves cleaved by the endonuclease). The dsb stimulated replication when the plasmid was homologous to either or both sides of the break but did not stimulate the replication of plasmids with homology to distant regions of the phage chromosome. As expected for recombination-dependent replication, plasmid replication triggered by dsbs was dependent on T4-encoded recombination proteins. These results confirm two important predictions of the model for T4-encoded recombination-dependent DNA replication proposed by Gisela Mosig (p. 120-130, in C. K. Mathews, E. M. Kutter, G. Mosig, and P. B. Berget (ed.), Bacteriophage T4, 1983). In addition, replication stimulated by dsbs provides a site-specific version of the process, which should be very useful for mechanistic studies.
An electroporation-mediated method for the study of foreign gene expression within chloroplasts has been developed. The chloroplast expression vector pHD203-GUS, which consists of coding regions for beta-glucuronidase (GUS) and chloramphenicol acetyltransferase (CAT) separated by a double psbA promoter fragment from pea (in opposite orientation) was electroporated into spinach chloroplasts and the transient gene expression was examined. Conditions for the expression of the reporter genes have been optimized. Both CAT and GUS activities were detected in chloroplasts electroporated with pHD203-GUS, but not with nuclear expression vector pBI221 or negative control pUC18. No GUS activity was detected when pHD203-GUS was electroporated into spinach protoplasts. Dot immunoblot analysis using anti-GUS antibody confirmed the existence of GUS protein in chloroplasts electroporated with chloroplast-specific vector but not the negative controls, excluding the possibilities of endogenous GUS or bacterial contamination. The expression of GUS protein in treated chloroplasts was further confirmed by Western blot analysis.
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 complete nucleotide sequence (155 844 bp) of tobacco (Nicotiana tabacum var. Bright Yellow 4) chloroplast DNA has been determined. It contains two copies of an identical 25 339 bp inverted repeat, which are separated by a 86 684 bp and a 18 482 bp single-copy region. The genes for 4 different rRNAs, 30 different tRNAs, 39 different proteins and 11 other predicted protein coding genes have been located. Among them, 15 genes contain introns. Blot hybridization revealed that all rRNA and tRNA genes and 27 protein genes so far analysed are transcribed in the chloroplast and that primary transcripts of the split genes hitherto examined are spliced. Five sequences coding for proteins homologous to components of the respiratory-chain NADH dehydrogenase from human mitochondria have been found. The 30 tRNAs predicted from their genes are sufficient to read all codons if the ;two out of three' and ;U:N wobble' mechanisms operate in the chloroplast. Two sequences which autonomously replicate in yeast have also been mapped. The sequence and expression analyses indicate both prokaryotic and eukaryotic features of the chloroplast genes.
Studies of chloroplast DNA variations, and several direct experimental observations, indicate the existence of recombination ability in algal and higher plant plastids. However, no studies have been done of the biochemical pathways involved. Using a part of a cyanobacterial recA gene as a probe in Southern blots, we have found homologous sequences in total DNA from Pisum sativum and Arabidopsis thaliana and in a cDNA library from Arabidopsis. A cDNA was cloned and sequenced, and its predicted amino acid sequence is 60.7% identical to that of the cyanobacterial RecA protein. This finding is consistent with our other results showing both DNA strand transfer activity and the existence of a protein of the predicted molecular mass crossreactive with antibodies to Escherichia coli RecA in the stroma of pea chloroplasts.
DNA-dependent RNA polymerase of pea chloroplasts is tightly bound to a complex that contains chloroplast DNA, DNA polymerase, RNA polymerase, and many other proteins. This complex transcribes all the sequences of chloroplast DNA in vitro. The RNA polymerase has been solubilized and purified from this complex by fractionation in glycerol gradients and in DEAE-cellulose and Sepharose 6B columns. The purified enzyme was completely dependent on the exogenous DNA. The native molecular size of the enzyme was found to be more than 500 000 by native gel electrophoresis and glycerol gradients. Chloroplast RNA polymerase was obtained in 1500-fold purification, starting from the Triton-disrupted chloroplasts. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis has shown that chloroplast RNA polymerase may contain polypeptides of 180, 140, 110, 95, 65, 47, and 27 kdaltons. The enzyme has been found to be extremely sensitive to salt concentrations and had a temperature optimum of 30°C. The purified enzyme is not inhibited by either α-amanitin or rifampicin.
The origins of chloroplast DNA (cpDNA) replication were mapped in two plastome types of Oenothera in order to determine whether variation in the origin of cpDNA replication could account for the different transmission abilities associated with these plastomes. Two pairs of displacement loop (D-loop) initiation sites were observed on closed circular cpDNA molecules by electron microscopy. Each pair of D-loops was mapped to the inverted repeats of the Oenothera cpDNA by the analysis of restriction fragments. The starting points of the two adjacent D-loops are approximately 4 kb apart, bracketing the 16S rRNA gene. Although there are small DNA length variations near one of the D-loop initiation sites, no apparent differences in the number and the location of replication origins were observed between plastomes with the highest (type I) and lowest (type IV) transmission efficiencies.
Covalently closed circular chloroplast DNA (ctDNA) molecules have been isolated from pea, bean, spinach, lettuce, corn and oat plants by ethidium bromide/cesium chloride density-gradient centrifugation. As much as 30–40% of the total ctDNA could be isolated as closed circular DNA molecules and up to 80% of the total ctDNA was found in the form of circular molecules.The size of pea, spinach, lettuce, corn and oat ctDNA relative to an internal standard (ΦX174 replicative form II monomer DNA) was determined by electron microscopy. The ctDNAs showed significant differences in their sizes, and their molecular weights ranged from 85.4 · 106 for corn ctDNA to 96.7 · 106 for lettuce ctDNA. Each of these ctDNAs contained 3–4% of the circular molecules as circular dimers and 1–2% of the circular molecules as catenated dimers.The molecular complexity of these ctDNAs was studied by renaturation kinetics using T4 DNA as a standard. The molecular weights of the unique sequence of the ctDNAs ranged from 83.7 · 106 for oat ctDNA to 93.1 · 106 for lettuce ctDNA, which are in excellent agreement with the sizes of the circular ctDNA molecules. No repeating sequences were detected in any of the ctDNAs.The ctDNAs from pea, lettuce, corn, and spinach were studied by thermal denaturation using T4 DNA as a standard. All of the ctDNAs melted more broadly than T4 DNA and they all had a distinctly different . For example, the of pea ctDNA was 1°C below the of T4 DNA and the of corn was 1.9°C higher than the of T4 DNA.
In young spinach leaves, 1–4 mm long, 7–10% of the total DNA of the leaf was chloroplast (pt) DNA. Growth in these leaves was mainly by cell division with plastid division keeping pace with cell division and maintaining about 10 plastids per cell. About 1% of the leaf cells were formed in 4.0 mm leaves. Both cell division and cell expansion contribute to the next stage of leaf growth, which was quantitatively the major period of new cell formation, nuclear DNA synthesis and ptDNA synthesis. Relative to the nuclear DNA level ptDNA levels rose to 21% of the total DNA and chloroplast.plastome copy numbers from 1500 to 5000 per cell while chloroplast numbers rose from 10 to 30 per cell. In the final period of leaf growth, cell expansion was the main determinant of growth and chloroplast number per cell rose to 180. In contrast to young leaves, newly emerged cotyledons contained 20% of their DNA as ptDNA and, during cell expansion, cell number per cotyledon doubled. On average, the cells became octoploid, and chloroplast numbers and plastome copy numbers rose to 500 and 22 000 per cell respectively. Similar levels of nuclear ploidy, chloroplast number and plastome copy number were induced in the first leaf pair of spinach following decapitation. When senescence was induced in mature leaves by shading, no loss of nuclear or ptDNA occurred. Following the onset of leaf yellowing and a form of senescence induced by nitrogen deficiency in leaves which had not fully expanded, there was preferential loss of ptDNA which fell from 8200 to 3700 plastome copies per cell over an 11 d period.
The chloroplast DNA (ctDNA) from pea and corn plants contains both Cairns type and rolling circle replicative intermediates. Denaturation mapping studies with pea ctDNA molecules have shown that the rolling circles initiate replication at or near the site where the Cairns replicative intermediates terminate replication. These results suggest that the rolling circles are initiated by a Cairns round of replication. A model for the replication of the chloroplast DNA is based on these results.
Covalently closed circular choloroplast DNA (ctDNA) molecules have been isolated from pea, bean, spinach, lettuce, corn and oat plants by ethidium bromide/cesium choloride density-gradient entrifugation. As much as 30-40% of the total ctDNA could be isolated as closed circular DNA molecules and up to 80% of the total ctDNA was found in the form of circular molecules. The size of pea, spinach, lettuce, corn and oat ctDNA relative to an internal standard (phiX174 replicative form II monomer DNA) was determined by electron microscopy. The ctDNAs showed significant differences in their sizes, and their molecular weights ranged from 85.4 - 10(6) for corn ctDNA to 96.7 - 10(6) for lettuce ctDNA. Each of these ctDNAs contained 3-4% of the circular molecules as circular dimers and 1-2% of the circular molecules as catenated dimes. The molecular complexity of these ctDNAs was studied by renaturation kinetics using T4 DNA as a standard. The molecular weights of the unique sequences of the ctDNAs ranged from 83.7 - 10(6) for oat ctDNA to 93.1 - 10(6) for lettuce ctDNA, which are in excellent agreement with the sizes of the circular ctDNA molecules...
We have used a combination of one- and two-dimensional agarose gel electrophoresis, and solution hybridization to strand-specific probes, to map the replication origin of sea urchin mitochondrial DNA and to investigate the structure of replication intermediates. These assays are consistent with replication initiating unidirectionally from the D-loop region by D-loop expansion, as in vertebrates. A prominent site of initiation of lagging-strand synthesis lies at, or near to, the boundary between the genes for ATPase 6 and COIII, which is also close to a pause site for leading-strand synthesis. These findings suggest a role for pause sites in the regulation of mitochondrial transcription and replication, possibly involving template-binding proteins.
A partially purified replicative system of pea chloroplast that replicates recombinant DNAs containing pea chloroplast origin sequences has been characterised. Polymerisation by this system is very fast and insensitive to chain terminators like dideoxynucleotides, arabinosylcytosine 5′-triphosphate, etc. Both strands of template DNA are synthesized and single-stranded DNA templates undergo more than one round of replication.
When sequences of either of the two chloroplast origins of replication (OriA or OriB) are used as templates, the replicative intermediates are found to have sigma structures. Electron microscopic analysis of the sigma structures restricted with various enzymes reveals that the initiation site of in vitro replication maps near the displacement-loop regions where replication initiates also in vivo. Although the observed replication initiation in the OriA recombinant template is chloroplast-DNA-specific, the mode of replication is different from that observed in vivo with intact ctDNA. However, when the template DNA contains both the OriA and OriB sequences, the in vitro replication proceeds in the theta mode, the mode of replication usually observed in vivo.
Chloroplast DNA replication was studied in the green, autotrophic suspension culture line SB-1 of Glycine max. Three regions (restriction fragments Sac I 14.5, Pvu II 4.1 and Pvu II 14.8) on the plastome were identified that displayed significantly higher template activity in in vitro DNA replication assays than all other cloned restriction fragments of the organelle genome, suggesting that these clones contain sequences that are able to direct initiation of DNA replication in vitro. In order to confirm that the potential in vitro origin sites are functional in vivo as well, replication intermediates were analyzed by two-dimensional gel electrophoresis using cloned restriction fragments as probes. The two Pvu II fragments that supported deoxynucleotide incorporation in vitro apparently do not contain a functional in vivo replication origin since replication intermediates from these areas of the plastome represent only fork structures. The Sac I 14.5 chloroplast DNA fragment, on the other hand, showed intermediates consistent with a replication bubble originating within its borders, which is indicative of an active in vivo origin. Closer examination of cloned Sac I 14.5 sub-fragments confirmed high template activity in vitro for two, S/B 5 and S/B 3, which also seem to contain origin sites utilized in vivo as determined by two-dimensional gel electrophoresis. The types of replication intermediate patterns obtained for these sub-fragments are consistent with the double D-loop model for chloroplast DNA replication with both origins being located in the large unique region of the plastome [17, 18]. This is the first report of a chloroplast DNA replication origin in higher plants that has been directly tested for in vivo function.
One of the two origins of replication in pea chloroplast DNA (oriA) maps in the rRNA spacer region downstream of the 16S rRNA gene, and further characterization of this origin is presented here. End-labeling of nascent DNA strands from in vivo replicating ctDNA was used to generate probes for Southern hybridization. Hybridization data identified the same region that was previously mapped to contain D-loops by electron microscopy. Subclones of the oriA region were tested for their ability to support in vitro DNA replication using a partially purified pea ctDNA replication system. Two-dimensional agarose gel electrophoresis identified replication intermediates for clones from the region just downstream of the 16S rRNA gene, with a 450-bp SacI-EcoRI clone showing the strongest activity. The experiments presented in this paper identify the 940 base pair region in the rRNA spacer between the 3' end of the 16S rRNA gene and the EcoRI site as containing oriA. Previous studies by electron microscopy localized the D-loop in the spacer region just to the right of the BamHI site, but the experiments presented here show that sequences to the left of the BamHI site are required for replication initiation from oriA. DNA sequence analysis of this region of pea ctDNA shows the presence of characteristic elements of DNA replication origins, including several direct and inverted repeat sequences, an A + T rich region, and dnaA-like binding sites, most of which are unique to the pea ctDNA oriA region when compared with published rRNA spacer sequences from other chloroplast genomes.
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 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.
Initiation of chromosome replication is a key event in the life cycle of any organism. Little is known, however, about the regulatory mechanisms of this vital process. Conventionally, the initiation mechanism of chromosome replication in microorganisms has been studied using plasmids in which an origin of chromosome replication has been cloned, rather than using the chromosome itself. The reason for this is that even bacterial chromosomes are so large that biochemical and genetic manipulations become difficult and cumbersome. Recently, the combination of flow cytometry and genetic methods, in which modifications of the replication origin are systematically introduced onto the chromosome, has made possible detailed studies of the molecular events involved in the control of replication initiation in Escherichia coli. The results indicate that requirements for initiation at the chromosomal origin, oriC, are drastically different from those for initiation at cloned oriC.
We have developed a simple three-step method for transferring oriC mutations from plasmids to the Escherichia coli chromosome. Ten oriC mutations were used to replace the wild-type chromosomal origin of a recBCsbcB host by recombination. The mutations were subsequently transferred to a wild-type host by transduction. oriC mutants with a mutated DnaA box R1 were not obtained, suggesting that R1 is essential for chromosomal origin function. The other mutant strains showed the same growth rates, DNA contents and cell mass as wild-type cells. Mutations in the left half of oriC, in DnaA boxes M, R2 or R3 or in the Fis or IHF binding sites caused moderate asynchrony of the initiation of chromosome replication, as measured by flow cytometry. In mutants with a scrambled DnaA box R4 or with a modified distance between DnaA boxes R3 and R4, initiations were severely asynchronous. Except for oriC14 and oriC21, mutated oriCs could not, or could only poorly, support minichromosome replication, whereas most of them supported chromosome replication, showing that the classical definition of a minimal oriC is not valid for chromosome replication. We present evidence that the functionality of certain mutated oriCs is far better on the chromosome than on a minichromosome.
The occurrence of DNA recombination in plastids of higher plants is well documented. However, little is known at the enzymic level. To begin dissecting the biochemical mechanism(s) involved we focused on a key step: strand transfer between homologous parental DNAs. We detected a RecA-like strand transfer activity in stromal extracts from pea (Pisum sativum L.) chloroplasts. Formation of joint molecules requires Mg2+, ATP, and homologous substrates. This activity is inhibited by excess single-stranded DNA (ssDNA), suggesting a necessary stoichiometric relation between enzyme and ssDNA. In a novel assay with Triton X-100-permeabilized chloroplasts, we also detected strand invasion of the endogenous chloroplast DNA by 32P-labeled ssDNA complementary to the 16S rRNA gene. Joint molecules, analyzed by electron microscopy, contained the expected displacement loops.
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.
Chloroplast DNA conformation was analyzed by pulse-field gel electrophoresis. We found that spinach leaf chloroplast DNA molecules exist in at least four distinct forms with the apparent molecular weights of monomer, dimer, trimer, and tetramer. Two-dimensional gel analysis of DNA after UV nicking and in the presence of ethidium bromide indicates that they are not isomers that differ in superhelical density. DNA gyrase decatenation analysis demonstrates that the majority of the DNA molecules are oligomers rather than catenanes. The relative amounts of monomer, dimer, trimer, and tetramer forms, quantitated by molecular hybridization, are 1, 1/3, 1/9, and 1/27, respectively, and do not change during leaf maturation. The possible mechanisms of chloroplast DNA oligomer formation are discussed.
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.
In vitro synthesis and uptake of cytoplasmically-synthesized chloroplast proteins
- S G Bartlett
- A R Grossman
- N H Chua
S.G. Bartlett, A.R. Grossman, N.H. Chua, In vitro synthesis and
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Edelman, R.B. Hallick, N.H. Chua (Eds.), Methods in Chloroplast
Molecular Biology, Elsevier, Amsterdam, 1982, pp 1081-1091.
Fine mapping of replication origins (oriA and oriB) in Nicotiana tabacum chloroplast DNA
- Kunnimalaiyaan
M. Kunnimalaiyaan, B.L. Nielsen, Fine mapping of replication origins (oriA and oriB) in Nicotiana tabacum chloroplast DNA, Nucl.
Acids Res. 25 (1997) 3681-3686.