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Analysis of the tobacco chloroplast DNA replication origin (oriB) downstream of the 23 S rRNA gene
Abstract
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.
... Considering the highly conserved structure and gene content of ctDNA, the observed differences in number and location of plastome D-loops are somewhat surprising. Two-dimensional gel electrophoresis of in vivo and in vitro ctDNA replication intermediates and other experimental approaches to further define plastome ori regions (Hedrick et al., 1993; Kunnimalaiyaan and Nielsen, 1997b; Kunnimalaiyaan et al., 1997; Lu et al., 1996; Nielsen et al., 1993; Wang, Y. et al., 2002) for the most part support the presence of two D-loops but also failed to establish a consensus ori location on the plastome of higher plants. In keeping with these results is the general lack of primary structure homology between those ori regions that have been sequenced (Kunnimalaiyaan and Nielsen, 1997b; Lu et al., 1996; Nielsen et al., 1993; Takeda et al., 1992; Wu, M. et al., 1986); rev in (Heinhorst and Cannon, 1993; Kunnimalaiyaan and Nielsen, 1997a). ...
... Such secondary structure elements may be important for initiation site selection, like the in vitro D-loop extension site that was mapped to the base of a (G+C)-rich potential stem/loop in the cloned oriA region of the tobacco plastome (Lu et al., 1996). Hairpin structures might also mediate specific interactions with the organellar DNA replication machinery, since gel mobility shift analysis revealed that the cloned oriB from tobacco (Kunnimalaiyaan et al., 1997) and oriA from Chlamydomonas (Nie, Z.Q. et al., 1987) specifically interact with a small subset of chloroplast proteins. ...
... Such interaction between ori and a redox-active protein might explain the observed sensitivity of Chlamydomonas ctDNA copy numbers to the redox state of the cell (Lau et al., 2000). The different DNA replication initiation sites used in proplastids versus leaf chloroplasts of rice and tobacco (Kunnimalaiyaan and Nielsen, 1997b; Kunnimalaiyaan et al., 1997; Lu et al., 1996; Takeda et al., 1992;) raise the interesting possibility that plastome ori regions and maybe replication mode might change during organelle differentiation. One can envision that the rapid plastome amplification in very young, developing chloroplasts proceeds by a different mechanism and initiates from a different origin than the slower maintenance replication in proplastids and in more mature chloroplasts. ...
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.
... 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]. ...
... Z00044). Direct repeats in each origin are underlined and italicized, restriction sites are in bold, and the location and direction of DNA synthesis initiation for each origin is indicated by the open arrows [9,13]. For oriB, two separate repeats are indicated as R1 and R2 (R2 is an imperfect repeat). ...
... AUM 13, which is a derivative of AUM 12 with a 6.1 kbp internal deletion but with both origins still intact (the elements from oriA and oriB are each functional in the respective single-ori clones; see Fig. 1), exhibits simple Y patterns indicative of rolling circle replication (Fig. 6B). This is similar to patterns observed for single-ori clones [9]. AUM 1.0, which lacks parts of both ori, showed only the monomer spot and no patterns characteristic of replication activity, and shows a weaker signal due to the absence of replication (Fig. 6B). ...
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.
... Using this approach partially purified chloroplast extracts from several plant sources have been shown to prefer specific cloned regions of ctDNA as in vitro DNA synthesis templates, yet to date it has been difficult to distinguish between true DNA replication and random repair synthesis (2,9). Using 2D gel electrophoresis (5) and other techniques we have recently reported the identification and localization of ctDNA replication origins (oriA and oriB) in each inverted repeat (IR) of tobacco (10,11). Four oris have been reported for Oenothera due to their location as identical pairs in each IR (12). ...
... The bound proteins were eluted with buffer A containing 600 mM KCl. Ten milliliter fractions were collected and tested for ctDNA polymerase activity (11). Active fractions were pooled and dialyzed against buffer B (20 mM potassium phosphate buffer, pH 8.0, 25% glycerol, 10 mM B-mercaptoethanol and protease inhibitors: 10 mM benzamide, 1 mM PMSF, 10 mM sodium metabisulfite), with several changes of buffer. ...
... These clones were confirmed by standard sequence analysis to ensure the correct sequence. After initial comparison by [ 3 H]TTP incorporation in vitro replication products labeled with [ 32 P]dCTP were prepared and analyzed by 2D gel electrophoresis as described (11). ...
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.
... Furthermore, the weak thread-like signals joining the ∼2 kb, ∼4 kb and ∼4.5 kb spots indicated the presence of minicircular DNA in a continuum of sizes, which were likely replication intermediates of the minicircles. However, a replicative arc resembling the bubble-forming mechanism was not observed in the 2D-gel (41,43). Although it is possible that these intermediate forms were too rare to be detected, the 2D-gel patterns were similar even after overnight exposure. ...
... The mechanism of DNA replication can be deduced from these arcs. In general, replicative arc generated by bubble-forming type replication intermediates extend from one-unit mass (1n) to double-unit mass (2n) (43,44). Theoretical rolling circle replication intermediates contain branched tail with various lengths which sometimes can be larger than 1-U mass. ...
Plastid genomes of peridinin-containing dinoflagellates are unique in that its genes are found on multiple circular DNA molecules
known as ‘minicircles’ of ∼2–3 kb in size, carrying from one to three genes. The non-coding regions (NCRs) of these minicircles
share a conserved core region (250–500 bp) that are AT-rich and have several inverted or direct repeats. Southern blot analysis
using an NCR probe, after resolving a dinoflagellate whole DNA extract in pulsed-field gel electrophoresis (PFGE), revealed
additional positive bands (APBs) of 6–8 kb in size. APBs preferentially diminished from cells treated with the DNA-replication
inhibitor aphidicolin, when compared with 2–3 kb minicircles, implicating they are not large minicircles. The APBs are also
exonuclease III-sensitive, implicating the presence of linear DNA. These properties and the migration pattern of the APBs
in a 2D-gel electrophoresis were in agreement with a rolling circle type of replication, rather than the bubble-forming type.
Atomic force microscopy of 6–8 kb DNA separated by PFGE revealed DNA intermediates with rolling circle shapes. Accumulating
data thus supports the involvement of rolling circle intermediates in the replication of the minicircles.
... Chloroplast replication origins generally map to noncoding regions in the neighborhood of the rRNA genes (Sears, Stoike, and Chiu 1996; Kunnimalaiyaan, Shi, and Nielsen 1997), although the core region of the Chlamydomonas origin partly overlaps a ribosomal protein gene (Chang and Wu 2000 ). Replication origins frequently contain predicted stem-loop structures, inverted repeats, and multiple direct repeats, not only in plastids (Sears, Stoike, and Chiu 1996; Kunnimalaiyaan, Shi, and Nielsen 1997) but also in a number of systems ranging from bacteria to animals (reviewed in Pearson et al. 1996). ...
... Chloroplast replication origins generally map to noncoding regions in the neighborhood of the rRNA genes (Sears, Stoike, and Chiu 1996; Kunnimalaiyaan, Shi, and Nielsen 1997), although the core region of the Chlamydomonas origin partly overlaps a ribosomal protein gene (Chang and Wu 2000 ). Replication origins frequently contain predicted stem-loop structures, inverted repeats, and multiple direct repeats, not only in plastids (Sears, Stoike, and Chiu 1996; Kunnimalaiyaan, Shi, and Nielsen 1997) but also in a number of systems ranging from bacteria to animals (reviewed in Pearson et al. 1996). We previously suggested that the 9A region of H. triquetra could contain the replication origin because it is present both in normal minicircles and in aberrant chimeric ones containing multiple fragments of different genes (Zhang, Cavalier-Smith, and Green 2001). ...
Dinoflagellate chloroplast genes are unique in that each gene is on a separate minicircular chromosome. To understand the origin and evolution of this exceptional genomic organization we completely sequenced chloroplast psbA and 23S rRNA gene minicircles from four dinoflagellates: three closely related Heterocapsa species (H. pygmaea, H. rotundata, and H. niei) and the very distantly related Amphidinium carterae. We also completely sequenced a Protoceratium reticulatum minicircle with a 23S rRNA gene of novel structure. Comparison of these minicircles with those previously sequenced from H. triquetra and A. operculatum shows that in addition to the single gene all have noncoding regions of approximately a kilobase, which are likely to include a replication origin, promoter, and perhaps segregation sequences. The noncoding regions always have a high potential for folding into hairpins and loops. In all six dinoflagellate strains for which multiple minicircles are fully sequenced, parts of the noncoding regions, designated cores, are almost identical between the psbA and 23S rRNA minicircles, but the remainder is very different. There are two, three, or four cores per circle, sometimes highly related in sequence, but no sequence identity is detectable between cores of different species, even within one genus. This contrast between very high core conservation within a species, but none among species, indicates that cores are diverging relatively rapidly in a concerted manner. This is the first well-established case of concerted evolution of noncoding regions on numerous separate chromosomes. It differs from concerted evolution among tandemly repeated spacers between rRNA genes, and that of inverted repeats in plant chloroplast genomes, in involving only the noncoding DNA cores. We present two models for the origin of chloroplast gene minicircles in dinoflagellates from a typical ancestral multigenic chloroplast genome. Both involve substantial genomic reduction and gene transfer to the nucleus. One assumes differential gene deletion within a multicopy population of the resulting oligogenic circles. The other postulates active transposition of putative replicon origins and formation of minicircles by homologous recombination between them.
... oriB in tobacco has been described as a 243-bp element, also capable of forming a stem-loop and containing repeated elements (Kunnimalaiyaan and Nielsen, 1997b). It is located downstream of the trnN gene (111778-112020 and 130606-130848), close to the border of the IR regions at the small single copy region (Kunnimalaiyaan et al., 1997). Therefore, the two copies of oriB in the plastome have a different surrounding: In IR A , oriB is located within the coding sequence of the ycf1 gene, which starts within the Inverted Repeat. ...
... Fine mapping of the sequences of both tobacco replication origins has been made in in vitro replication experiments (Kunnimalaiyaan and Nielsen, 1997b), which do not necessarily reproduce the situation in the plastid. In vivo analysis of replication origins has been restricted to the analysis of displacement loops up to now, which does not allow precise localisation (Kunnimalaiyaan et al., 1997;Lu et al., 1996). In an earlier study with proplastids of cultured tobacco cells, displacement loops were mapped to the ends of the Inverted Repeat near to, but not identical to the described oriB locus, and to the large single copy region of the plastome, but not to the region of oriA (Takeda et al., 1992). ...
Sequences described as chloroplast DNA replication origins were analysed in vivo by creating deletion and insertion mutants via plastid transformation in tobacco. Deletion of the described oriA sequence, which is located within the intron of the trnI gene, resulted in heteroplastomic transformants, when the selection marker was inserted within the intron. Removal of the complete intron sequence together with the oriA sequence, however, yielded homoplastomic transformants of normal phenotype, in which wild-type signals were no longer detectable through Southern analysis, thus bringing the role of the described oriA sequence for plastome replication into question. Similarly, deletion of sequence elements upstream of trnI, which have a possible ori function in Oenothera, did not show any effect in tobacco. The two copies of oriB, which are located at the very end of the plastome Inverted Repeats, were targeted with two different transformation vectors in a cotransformation approach. While in initial transformants integration of the selection marker could be detected at both sites, the transgene was found exclusively at one site or the other after additional rounds of regeneration. Whereas the copy of oriB in Inverted Repeat B could be completely deleted, targeting of the copy in Inverted Repeat A resulted in heteroplastomic lines, as the essential ycf1 gene was also affected. Due to the strong selection against cotransformants we conclude that at least one copy of the oriB sequence is essential for plastome replication, whereas replication appears possible without oriA elements.
... In line with this model, two replication origins -oriA and oriBwere found in N. tabacum, on both of which, independently, r replication could be initiated in vitro and in isolated chloroplasts. h replication could be started in vitro and in isolated chloroplasts involving both oriA and oriB (Kunnimalaiyaan and Nielsen, 1997b;Kunnimalaiyaan et al., 1997;Lu et al., 1996;Lugo et al., 2004). Both oris are located in the inverted repeats, therefore there are two copies of both on one monomeric plastid DNA molecule. ...
... According to the Kolodner and Tewari (1975) model, plastid DNA replication of N. tabacum initiates on a pair of replication origins: oriA and oriB (Kunnimalaiyaan and Nielsen, 1997b;Kunnimalaiyaan et al., 1997;Lu et al., 1996;Lugo et al., 2004). Pairs of oris were also detected in other species: (Kunnimalaiyaan and Nielsen, 1997b), the suspension culture ori (Takeda et al., 1992;Wang et al., 2002), the putative homologue of oriA of Oenothera elata (Chiu and Sears, 1992), the putative homologue of an ori near psbA or rpl16, respectively, of Zea mays (Carrillo and Bogorad, 1988) and near rpl16 of Chlamydomonas reinhardtii (Lou et al., 1987) and Glycine max (Hedrick et al., 1993) are given. ...
According to the Kolodner and Tewari model [Kolodner, R.D. and Tewari, K.K. (1975) Nature, 256, 708.], plastid DNA replication involves displacement-loop and rolling-circle modes of replication, which are initiated on a pair of origins of replication (ori). In accordance with the model, such a pair of oris -oriA and oriB- was described in Nicotiana tabacum [Kunnimalaiyaan, M. and Nielsen B.L. (1997b) Nucl. Acids Res. 25, 3681.]. However, as reported previously, both copies of oriA can be deleted without abolishing replication. Deletion of both oriBs was not found [Mühlbauer, S.K. et al. (2002) Plant J. 32, 175.]. Here we describe new ori inactivation lines, in which one oriB is deleted and the other copy is strongly mutated. In addition, lines oriA and oriB were deleted from the same inverted repeat. In contrast to the expectations of the model, neither oriA nor oriB is essential. Some of the deletions led to reduced growth of plants and reduced plastid DNA copy number in later stages of leaf development. The gross structure of plastid DNA was unchanged; however, the location of the ends of branched plastid DNA complexes was different in the inactivation mutants. Taken together, the results indicate that there are additional mechanisms of plastid DNA replication and/or additional origins of replication. These mechanisms seem to be different from those found in eubacteria, which, according to the endosymbiont theory, are the progenitors of plastids.
... Another known element in cpDNA is the origins of replication (ori), which has been reported to be bound by proteins 30 . We obtained the sequences of ori in the previous study 31,32 . ...
Although the chloroplast genome (cpDNA) of higher plants is known to exist as a large protein-DNA complex called ‘plastid nucleoid’, researches on its DNA state and regulatory elements are limited. In this study, we performed the assay for transposase-accessible chromatin sequencing (ATAC-seq) on five common tissues across five grasses, and found that the accessibility of different regions in cpDNA varied widely, with the transcribed regions being highly accessible and accessibility patterns around gene start and end sites varying depending on the level of gene expression. Further analysis identified a total of 3970 putative protein binding footprints on cpDNAs of five grasses. These footprints were enriched in intergenic regions and co-localized with known functional elements. Footprints and their flanking accessibility varied dynamically among tissues. Cross-species analysis showed that footprints in coding regions tended to overlap non-degenerate sites and contain a high proportion of highly conserved sites, indicating that they are subject to evolutionary constraints. Taken together, our results suggest that the accessibility of cpDNA has biological implications and provide new insights into the transcriptional regulation of chloroplasts.
... 2D-AGE has been extensively used to investigate genome maintenance in prokaryotes [1,12], yeast [3,4], and plant organelles [9,11]. While the method has proven itself extremely valuable in understanding vertebrate [5,13,14], fruit fly [8], and nematode [10] mtDNA replication, it is not yet widely adopted in the field. ...
Two-dimensional neutral/neutral agarose gel electrophoresis (2D-AGE) has been employed for nearly two decades in the analysis of replication and maintenance processes of animal mitochondrial DNA, but the method's potential has not been fully exploited. Here, we describe the various steps involved in this technique, from DNA isolation, to two-dimensional neutral/neutral agarose gel electrophoresis (2D-AGE), Southern hybridization and interpretation. We also provide examples of the applicability of 2D-AGE to investigate the different features of mtDNA maintenance and regulation.
... Although we still know little of cpDNA replication in plants, two replication origins (oriA and oriB) of tobacco cpDNA were described to initiate DNA replication, with oriA located within the intron of trnI gene and oriB located downstream of trnN close to the border of the IR and SSC regions [53][54][55]. ...
Transcription activator-like effector nuclease (TALEN) technology has been widely used to edit nuclear genomes in plants but rarely for editing organellar genomes. In addition, ciprofloxacin, commonly used to cause the double-strand break of organellar DNA for studying the repair mechanism in plants, confers no organellar selectivity and site-specificity. To demonstrate the feasibility of TALEN-mediated chloroplast DNA editing and to use it for studying the repair mechanism in plastids, we developed a TALEN-mediated editing technology fused with chloroplast transit peptide (cpTALEN) to site-specifically edit the rpoB gene via Agrobacteria-mediated transformation of tobacco leaf. Transgenic plants showed various degrees of chlorotic phenotype. Repairing damaged plastid DNA resulted in point mutation, large deletion and small inversion surrounding the rpoB gene by homologous recombination and/or microhomology-mediated recombination. In an albino line, microhomology-mediated recombination via a pair of 12-bp direct repeats between rpoC2 and ycf2 genes generated the chimeric ycf2-rpoC2 subgenome, with the level about 3- to 5-fold higher for subgenomic DNA than ycf2. Additionally, the expression of chimeric ycf2-rpoC2 transcripts versus ycf2 mRNA agreed well with the level of corresponding DNA. The ycf2-rpoC2 subgenomic DNA might independently and preferentially replicate in plastids.
... Proposed models include displacement-loop and rolling circle (θ and σ; [186]) replication of circular templates, and recombination dependent mechanisms acting on circular or linear templates [94, 187,188]. There is strong experimental evidence for multiple replication origins mapping to both the IR and single copy regions [189][190][191][192][193][194][195][196]. Several different groups using distinct approaches have demonstrated that ptDNA is present in vivo in many forms. ...
The plastid genome (plastome) has proved a valuable source of data for evaluating evolutionary relationships among angiosperms. Through basic and applied approaches, plastid transformation technology offers the potential to understand and improve plant productivity, providing food, fiber, energy, and medicines to meet the needs of a burgeoning global population. The growing genomic resources available to both phylogenetic and biotechnological investigations is allowing novel insights and expanding the scope of plastome research to encompass new species. In this chapter, we present an overview of some of the seminal and contemporary research that has contributed to our current understanding of plastome evolution and attempt to highlight the relationship between evolutionary mechanisms and the tools of plastid genetic engineering.
... 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.
... N. tabacum OriA mapped to the intron of the trn I(GAU) gene located between the 16S and 23S rRNA genes (Lu et al. 1996;Kunnimalaiyaan and Nielsen 1997a). OriB mapped to the large inverted repeat close to the border of the small single copy region in orf350 or ycf 1 (Kunnimalaiyaan and Nielsen 1997a;Kunnimalaiyaan et al. 1997b). (Meeker et al. 1988) lacks a large inverted repeat, b) Dicots containing large inverted repeats. ...
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.
... Redigieren der Tabaksequenz die Positionen besonders stark verschoben haben, konnte erst nach Veröffentlichung der Sequenz eindeutig zugeordnet werden, an welcher Stelle er tatsächlich liegt, und ob er für die " knock-out " -Mutanten von Bedeutung ist (Kunnimalaiyaan & Nielsen 1997) zichtet werden kann: in verschiedenen Ansätzen wurden oriA und oriB durch Plastidentransformation deletiert, wobei lediglich der Ansatz, in dem gleichzeitig ycf1 deletiert wurde, keine homoplastomischen Linien hervorbrachte (Mühlbauer et al. 2002 ). Im Falle des ...
... It has been proposed that differences in origins of replication between different species affect the specificity and efficiency of replication (Hornung et al. 1996 ). In tobacco, two replication origins have been mapped using electron microscopy of replication intermediates and an in vitro replication approach ( Kunnimalaiyaan, Shi, and Nielsen 1997 ). One of them, designated OriA, is situated within the trnI-GAU intron. ...
The nuclear and plastid genomes of the plant cell form a coevolving unit which in interspecific combinations can lead to genetic incompatibility of compartments even between closely related taxa. This phenomenon has been observed for instance in Atropa-Nicotiana cybrids. We have sequenced the plastid chromosome of Atropa belladonna (deadly nightshade), a circular DNA molecule of 156,688 bp, and compared it with the corresponding published sequence of its relative Nicotiana tabacum (tobacco) to understand how divergence at the level of this genome can contribute to nuclear-plastid incompatibilities and to speciation. It appears that (1) regulatory elements, i.e., promoters as well as translational and replicational signal elements, are well conserved between the two species; (2) genes--including introns--are even more highly conserved, with differences residing predominantly in regions of low functional importance; and (3) RNA editotypes differ between the two species, which makes this process an intriguing candidate for causing rapid reproductive isolation of populations.
... However, incomplete methylation occurred at the three sites within the BglII fragment from the inverted repeats. This incomplete methylation in the IR may simply be a result of stocheometry (there are two copies of the IR sequence for every one copy of the LSC sequence), or it may be an indication that sequences within the IR are less accessible to the methylase–perhaps due to their relative proximity to the IR sequences involved in initiating DNA replication (Kunnimalaiyaan et al. 1997). For the transgenic plants expressing a nuclear encoded, plastid targeted Dcm methylase, a total of 23 Dcm target sites were tested by ScrF1 digestion. ...
We report the integration of a type II restriction-methylase, mFokI, into the tobacco chloroplast genome and we demonstrate that the introduced enzyme effectively directs the methylation of its target sequence in vivo and does not affect maternal inheritance. We further report the transformation of tobacco with an E. coli dcm methylase targeted to plastids and we demonstrate efficient cytosine methylation of the plastid genome. Both adenosine methylation of FokI sites and cytosine methylation of dcm sites appeared phenotypically neutral. The ability to tolerate such plastid genome methylation is a pre-requisite for a proposed plant transgene containment system. In such a system, a chloroplast located, maternally inherited restriction methylase would provide protection from a nuclear-encoded, plastid targeted restriction endonuclease. As plastids are not paternally inherited in most crop species, pollen from such plants would carry the endonuclease transgene but not the corresponding methylase; the consequence of this should be containment of all nuclear transgenes, as pollination will only be viable in crosses to the appropriate transplastomic maternal background.
Plastids, like mitochondria, result from an ancient endosymbiosis event and contain a distinct genome. Though many plastid genes have since been transferred to the nuclear genome, the small plastid genome still encodes between 90 and 100 genes, which are notably involved in translation, transcription, and energy metabolism in the plastid. The many roles of this organelle, the most familiar being photosynthesis in chloroplasts, make it essential for the development of higher plants. As such, the ability of the plastid to maintain the stability of its genome represents a crucial element of plant life. The physical organization of the genome itself can have an influence on DNA metabolism, with its large inverted repeats acting as templates for recombination. Furthermore, the localization of chloroplast DNA near elements of the electron transport chain increases the importance of DNA repair mechanisms, as reactive oxygen species (ROS) appear as by-products of photosynthesis. These ROS, along with UV radiation and DNA double-strand breaks, create a genotoxic stress through their respective ability to oxidize nucleotides, link DNA bases, or rearrange the structure of the genome. To minimize the deleterious effects of these events, different mechanisms present in the nucleus such as homologous recombination exist in plastids. Some less conservative mechanisms based on sequences of microhomology are also found, and sometimes lead to copy-number variation in certain areas of the plastid genome. While some of these changes can remain silent, others can be linked to phenotypes such as variegation.
DNA replication, transcription of the circular plastid genome and plastid division represent fundamental processes that take place in all types of the multifunctional, multiple-form plastids. The components of the plastid genetic system are highly expressed during early phases of chloroplast development corresponding to the establishment of 'housekeeping' functions before the onset of photosynthesis. A survey of fundamental and recent work is presented concerning plastid division, the organization of the plastid genome and the characterization of enzymes implicated in replication and transcription.
To maintain and to differentiate into various plastid lineages, replication of the plastid DNA (ptDNA) and division of the plastid must take place. However, replication initiation of the ptDNA has been less understood. The present study describes identification of the initiation region (origin) of ptDNA replication in the rice cultured cells. RNA-primed newly replicated DNA strands pulse-labeled with bromodeoxyuridine were isolated and size-fractionated. Locations of these nascent strands on the ptDNA determined the two major origin regions around the 3′ region of each 23S rDNA in the inverted repeats (IRA and IRB). Two-dimensional agarose gel electrophoresis of the replication intermediates suggested that replication from each origin proceeds bidirectionally. This contrasted to replication by the double D - loop mechanism.
The plastid genome (plastome) has proved a valuable source of data for evaluating evolutionary relationships among angiosperms. Through basic and applied approaches, plastid transformation technology offers the potential to understand and improve plant productivity, providing food, fiber, energy and medicines to meet the needs of a burgeoning global population. The growing genomic resources available to both phylogenetic and biotechnological investigations are allowing novel insights and expanding the scope of plastome research to encompass new species. In this chapter we present an overview of some of the seminal and contemporary research that has contributed to our current understanding of plastome evolution and attempt to highlight the relationship between evolutionary mechanisms and tools of plastid genetic engineering.
Stagnation in absolute yield increases of major food and fiber crops around the world is raising awareness that breeding for
quantitative yield characteristics will most likely be insufficient to meet the needs of the burgeoning global population.
Additionally the attractive agronomic characteristics of high absolute yield and disease resistance have been challenging
to stack into new cultivars by classical breeding. Losses incurred along the production-consumption continuum are not considered
in evaluations of yield stability. Reports from the Food and Agriculture Organization indicate that up to 25% of all food
productivity is lost due to post-harvest variables including pre-harvest infestations which result in accumulation of toxic
metabolites in storage. Annual losses caused by mycotoxigenic fungi, such as members of the genus Aspergillus, can potentially reach up to a billion dollars due to market rejection and animal health impacts. Aflatoxin B1, the most
prevalent of the toxins produced by a number of Aspergillus and Emericella species, is classified as a class 1 carcinogen. Around the globe effective management programs for the detection and elimination
of aflatoxins presents a major challenge. Much attention has been focused on implementing strategies to prevent pre-harvest
infestation by Aspergillus flavus, the major source of aflatoxin contamination in food and feed crops. Genetic improvement of susceptible crop species may
enhance resistance to microbial pathogens and facilitate reduced pesticide load, yet the possibility for transmission of novel
genes to wild relatives has hampered acceptance of GM crops in some markets. Chloroplast transformation presents an attractive
alternative to nuclear transformation and offers the potential to ameliorate this and other environmental concerns. Most agronomically
important species exhibit maternal inheritance of organellar genomes eliminating the threat of transgene escape through pollen.
Additionally, gene silencing is absent due to site directed, single copy insertion by homologous recombination. Foreign proteins
can accumulate to high levels and are retained within the chloroplast envelope protecting them from degradation by host cytoplasmic
proteases. In this study, a bacterial chloroperoxidase gene (cpo-p) was transformed into the tobacco chloroplast genome to test its efficacy against several plant pathogens and Aspergillus flavus.
Mitokondrid on kõrgemate organismide nagu loomade, taimede ja seente rakkudes olevad väikesed organellid. Mitokondrid on esmatähtsad ainevahetusel ja üks nende peamisi ülesandeid on energia tootmine. Selleks on mitokondritel vaja mitmeid valke, mis on koondunud membraaniga seotud kompleksidesse. Enamus nendest valkudest on kodeeritud tuuma kromosoomide poolt, kuid mõned hädavajalikud subühikud mitokondri DNA poolt. Mitokondrid peavad seega oma väikest genoomi hästi säilitama, et vigadeta DNA-d saaks transkribeerida ja funktsionaalseid valke toota, näiteks energia saamiseks. Kaua aega usuti, et kõikide eukarüootsete rakkude mitokondrite DNA näeb samasugune välja ning seda paljundatakse ja säilitatakse sarnaselt nii loomades, taimedes kui seentes. Pärmseeni, mille uurimiseks on olemas head geneetilise vahendid, kasutatakse seepärast eukarüootide mudelina. Selles väitekirjas kirjeldatud analüüsid tõestavad, et kuigi loomade ja pärmide mitokondriaalse DNA (mtDNA) säilitamises on sarnasusi, on seal ka olulisi erinevusi, mida ei ole siiani selgelt välja toodud. Näiteks on loomade mtDNA ringikujuline, pärmide mtDNA aga lineaarne ja harunenud. Loomade DNA süntees algab spetsiaalse RNA-praimeri sünteesiga kindlas DNA punktis. Pärmides paarduvad ja rekombineeruvad homoloogsed DNA järjestused omavahel, mille käigus üks homoloogne DNA invaseerub teise struktuuri. See invasioon tekitab DNA sünteesi alguspunkti, mis funktsionaalselt vastab loomade RNA-praimerile. Need erinevad DNA säilitamise süsteemid vajavad spetsiifilisi valke. Viimastel aastatel on näidatud, et pärmide ja loomade mitokondrites on palju sarnaseid, aga ka mitmeid erinevaid valke. Need leiud ja käesoleva väitekirja tulemused on olulised ka väljaspool baasteadust. Käesolevas uurimuses kasutati mudelorganismina pärmi Candida albicans. Erinevalt enam uuritud pagaripärmist Saccharomyces cerevisiae põhjustab C. albicans naha ja limaskestade nakkusi. Nõrgestunud immuunsüsteemiga patsientidele (nt. vähi ja AIDSi haigetele) võivad need nakkused olla eluohtlikud. Käesoleva töö tulemused võivad aidata leida ravimeid, mis tõrjuvad seenhaigusi, samas inimesele vähe või üldse mitte kahju tegemata. Mitochondria are small separate organelles within cells of higher organisms such as animals, plants or fungi. They are of major importance for the metabolism, and one of their main functions is the production of energy. In order to produce energy, mitochondria need a number of proteins which are grouped to membrane associated complexes to carry out the energy production. Many of these proteins are encoded by genes of the main chromosomes in the nucleus, but a few essential subunits are encoded on DNA within the mitochondria. Mitochondria therefore need to maintain their own small genome with great care to guarantee error-free DNA, that is transcribed and translated in order to deliver functional proteins products e.g. for energy production. It has been a longstanding belief, that mitochondria in different eukaryotic cells carry an equally looking DNA which is reproduced and maintained in the same ways in animals, plants and fungi. Therefore, yeast-fungi are widely used as eukaryotic model organisms because of the availability of genetics tools. The investigations conducted in the course of this thesis show, that although there are indeed similarities between animal and yeast mitochondrial DNA (mtDNA) maintenance, there are also important differences that have not been clearly shown until now. For example, while the mtDNA in animals appears in form of a circular molecule, yeast mitochondria contain linear and branched ones. In animals the DNA reproduction is started by making a specific starter molecule, termed RNA-primer, at a certain point on the DNA. In yeast, homologous DNA sequences are paired and recombined, in the course of which one homologous DNA invades the other. This invasion provides a starting point for DNA synthesis analogous to the RNA-primer that is used in mammals. These differing maintenance systems require specific protein machineries. In recent years it was shown that mitochondria in yeast and animals contain many similar but also a number of differing proteins. Such findings and also the results of this thesis have implications beyond the general use of basic research. The yeast model organism used in experiments of the current work was Candida albicans. Other than the commonly known baker´s yeast Saccharomyces cerevisiae, the potential pathogen C. albicans causes skin and mucosal infections. These can be life threatening for patients whose immune system is compromised (e.g. through cancer or AIDS). Findings like the ones in this thesis open ways to find unique targets for drugs that may specifically attack the pathogen without or little side effects for a patient. Väitekirja elektrooniline versioon ei sisalda publikatsioone.
Molecular recombination and transcription are proposed mechanisms to initiate mitochondrial DNA (mtDNA) replication in yeast. We conducted a comprehensive analysis of mtDNA from the yeast Candida albicans. Two-dimensional agarose gel electrophoresis of mtDNA intermediates reveals no bubble structures diagnostic of specific replication origins, but rather supports recombination-driven replication initiation of mtDNA in yeast. Specific species of Y structures together with DNA copy number analyses of a C. albicans mutant strain provide evidence that a region in a mainly noncoding inverted repeat is predominantly involved in replication initiation via homologous recombination. Our further findings show that the C. albicans mtDNA forms a complex branched network that does not contain detectable amounts of circular molecules. We provide topological evidence for recombination-driven mtDNA replication initiation and introduce C. albicans as a suitable model organism to study wild-type mtDNA maintenance in yeast.
Die Pflanzenzelle enthält ein integriertes, kompartimentiertes genetisches System, mit den Subgenomen im Zellkern, in den Mitochondrien und den Plastiden, das aus Endocytobioseereignissen mit prokaryotischen Zellen hervorgegangen ist. Im Laufe der Evolution der eukaryotischen Zelle wurden die genetischen Potentiale der symbiontischen Partnerzellen vermischt. Dabei ging ein Teil genetischer Information verloren, ein anderer wurde aus den Organellen in den Kern transferiert, und außerdem wurde neue Information hinzugewonnen. Dies ging einher mit der Einbettung von Mitochondrien und Plastiden in die Signaltransduktionsketten und Regelkreise der Wirtszelle. Heute interagieren die Subgenome auf vielen Ebenen; ihre Expression wird in der Pflanzenzelle koordiniert in Raum, Zeit und Quantität reguliert. Die Interdependenz der Subgenome hatte ihre Koevolution zur Folge, so daß die genetischen Kompartimente der Zelle nicht mehr ohne weiteres zwischen Arten ausgetauscht werden können. Kombinationen von artfremden Organellen können zu Entwicklungsstörungen führen, wie sie sowohl von "kompartimentellen" (Genom/Plastom-) Hybriden als auch von Cybriden beschrieben worden sind (Bastardbleichheit, Bastardscheckung). In dieser Arbeit wurden reziproke Cybriden der Arten Atropa belladonna und Nicotiana tabacum auf molekulare Determinanten von Genom/Plastom-Inkompatibilität untersucht. Die Cybriden sind je nach Kombination elterlicher Organellen entweder albinotisch [Kern von Atropa, Plastide vom Tabak; Ab(Nt)-Cybride] oder gleichen dem Wildtyp [Kern von Tabak; Plastide von Atropa, Nt(Ab)-Cybride]. 1. Als Voraussetzung für einen Sequenzvergleich der plastidären Chromosomen beider Solanaceen-Arten wurde das Plastidenchromosom von Atropa komplett sequenziert. Der Vergleich der (Atropa)-Sequenz mit der bekannten des Chromosoms aus dem Tabak und anschließende molekularbiologische Untersuchungen führten zur Identifizierung von zwei potenziellen Ursachen für die Defekte im albinotischen Material. 2. Die Ab(Nt)-Cybride zeigt eine gestörte Akkumulation von Transkripten für eine Reihe von Operonen. Das resultierende aberrante Transkriptmuster ähnelte verblüffend dem von Tabakpflanzen mit Defizienz der plastidenkodierten RNA-Polymerase (PEP). Möglicherweise ist in der Cybride die Interaktion des PEP-Apoenzyms mit einem oder mehreren der kernkodierten Sigmafaktoren gestört. Tatsächlich unterscheiden sich die für eine Untereinheit der PEP kodierenden (plastidären) rpoC2-Gene von Tabak und Atropa durch eine Insertion/Deletion an einer Stelle im Molekül, die mit Sigmafaktoren interagieren kann. Transformation der Plastiden der Ab(Nt)-Cybride mit dem rpoC2-Gen aus Tabak führte in der Tat zu einer partiellen Reversion zum WT und macht Transkriptionsdefekte als eines von offenbar mehreren Determinanten für die Genom/Plastom-Inkompatibilität in Artbastarden wahrscheinlich. 3. Neben der Transkription ist im albinotischen Material auch die RNA-Edierung gestört. Die plastidären Editotypen beider Solanaceen ähneln einander, doch gibt es für beide Arten spezifische Edierungsstellen. Von den fünf tabakspezifischen Stellen in der Ab(Nt)-Cybride werden vier nicht ediert. Offensichtlich besitzt der Atropa-Kern nicht die notwendigen Kernfaktoren zur Prozessierung dieser Stellen. Da Edierung generell hochkonservierte und funktionell wichtige Aminosäurepositionen betrifft, trägt der Ausfall der Edierung sehr wahrscheinlich ebenfalls zum beobachteten Defekt in der Plastidenentwicklung bei. 4. Auf der anderen Seite werden die Stellen der grünen Nt(Ab)-Cybride, bemerkenswerterweise auch Atropa-spezifische, heterolog vom Tabakkern ediert. Der erstmalige Befund von heterologem Edieren stellte sich als Folge der Allotetraploidie von Tabak heraus. Untersuchungen dieser Stellen in den diploiden Eltern des allotetraploiden Tabaks, N. tomentosiformis als Nachkomme des Vaters und N. sylvestris als Nachkomme der Mutter, zeigten, daß der Tabak die Fähigkeit zur heterologen Edierung von Atropa-spezifischen Stellen wohl vom Vater ererbt hat. Dies wurde auch durch einen transplastomischen Ansatz bestätigt. In diesen Experimenten wurde die intronnahe ndhA-Edierungsstelle aus Spinat, die es auch in N. tomentosiformis gibt, nicht aber in N. sylvestris, in Tabak über ballistische Transformation eingebracht. 5. Über Konstruktionen, die entweder der gespleißen oder ungespleißten ndhA-mRNA inklusive der Edierungsstelle entsprachen, konnte gezeigt werden, daß die Edierung an dieser Stelle immer erst nach dem Spleißen erfolgt. Dies ist der erste Nachweis einer strikten kinetischen Verknüpfung von RNA-Edierung mit einem anderen mRNA-Reifungsschritt in Plastiden. Er zeigt an, daß das ndhA-Intron phylogenetisch älter als die ndhA-Edierungsstelle ist. Mechanistische Implikationen dieses Befundes werden diskutiert.
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.
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.
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.
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.
Protein-nucleic acid interactions are crucial in the regulation of many fundamental cellular processes. The nature of these interactions is susceptible to analysis by a variety of methods, but the combination of high analytical power and technical simplicity offered by the gel retardation (band shift) technique has made this perhaps the most widely used such method over the last decade. This procedure is based on the observation that the formation of protein-nucleic complexes generally reduces the electrophoretic mobility of the nucleic acid component in the gel matrix. This review attempts to give a simplified account of the physical basis of the behavior of protein-nucleic acid complexes in gels and an overview of many of the applications in which the technique has proved especially useful. The factors which contribute most to the resolution of the complex from the naked nucleic acid are the gel pore size, the relative mass of protein compared with nucleic acid, and changes in nucleic acid conformation (bending) induced by binding. The consequences of induced bending on the mobility of double-strand DNA fragments are similar to those arising from sequence-directed bends, and the latter can be used to help characterize the angle and direction of protein-induced bends. Whether a complex formed in solution is actually detected as a retarded band on a gel depends not only on resolution but also on complex stability within the gel. This is strongly influenced by the composition and, particularly, the ionic strength of the gel buffer. We discuss the applications of the technique to analyzing complex formation and stability, including characterizing cooperative binding, defining binding sites on nucleic acids, analyzing DNA conformation in complexes, assessing binding to supercoiled DNA, defining protein complexes by using cell extracts, and analyzing biological processes such as transcription and splicing.
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.
We have compared the sequence organization of four previously uncharacterized legume chloroplast DNAs - from alfalfa, lupine, wisteria and subclover — to that of legume chloroplast DNAs that either retain a large, ribosomal RNA-encoding inverted repeat (mung bean) or have deleted one half of this repeat (broad bean). The circular, 126 kilobase pair (kb) alfalfa chloroplast genome, like those of broad bean and pea, lacks any detectable repeated sequences and contains only a single set of ribosomal RNA genes. However, in contrast to broad bean and pea, alfalfa chloroplast DNA is unrearranged (except for the deletion of one segment of the inverted repeat) relative to chloroplast DNA from mung bean. Together with other findings reported here, these results allow us to determine which of the four possible inverted repeat configurations was deleted in the alfalfa-pea-broad bean lineage, and to show how the present-day broad bean genome may have been derived from an alfalfa-like ancestral genome by two major sequence inversions. The 147 kb lupine chloroplast genome contains a 22 kb inverted repeat and has essentially complete colinearity with the mung bean genome. In contrast, the 130 kb wisteria genome has deleted one half of the inverted repeat and appears colinear with the alfalfa genome. The 140 kb subclover genome has been extensively rearranged and contains a family of at least five dispersed repetitive sequence elements, each several hundred by in size; this is the first report of dispersed repeats of this size in a land plant chloroplast genome. We conclude that the inverted repeat has been lost only once among legumes and that this loss occurred prior to all the other rearrangements observed in subclover, broad bean and pea. Of those lineages that lack the inverted repeat, some are stable and unrearranged, other have undergone a moderate amount of rearrangement, while still others have sustained a complex series of rearrangement either with or without major sequence duplications and transpositions. Peer Reviewed http://deepblue.lib.umich.edu/bitstream/2027.42/46960/1/294_2004_Article_BF00355401.pdf
We have investigated the frequency of replication origin usage in bacterial plasmids containing more than one potential origin. Escherichia coli recA- cells were selectively transformed with pBR322 monomers, dimers, or trimers. Plasmid DNA was isolated and digested with a restriction enzyme that cut the monomer only once, and the replicative intermediates (RIs) were analyzed by neutral/neutral two-dimensional agarose gel electrophoresis. Evidence for initiation outside the linearized plasmid was found only for oligomers. Moreover, in dimers, the intensity of the signal indicative for external initiation was equivalent to that reflecting internal initiation, whereas it was approximately twice as strong in trimers. To determine whether initiation could occur simultaneously at two origins in a single plasmid, we studied the replication of a neodimer in which both units could be unambiguously distinguished. The results showed that although both origins were equally competent to initiate replication, only one was active per plasmid. These observations strongly suggest that in ColE1 plasmids, replication initiates at a single site even when there are several identical potential origins per plasmid. In addition to the conventional two-dimensional gel patterns, novel specific patterns were observed with intensities that varied from one DNA sample to another. These unique patterns were the result of breakage of the RIs at a replication fork. This type of breakage changes both the mass and shape of RIs. When the entire population of RIs is affected, a new population of molecules is formed that may generate a novel pattern in two-dimensional gels.
Protein-nucleic acid interactions are crucial in the regulation of many fundamental cellular processes. The nature of these interactions is susceptible to analysis by a variety of methods, but the combination of high analytical power and technical simplicity offered by the gel retardation (band shift) technique has made this perhaps the most widely used such method over the last decade. This procedure is based on the observation that the formation of protein-nucleic complexes generally reduces the electrophoretic mobility of the nucleic acid component in the gel matrix. This review attempts to give a simplified account of the physical basis of the behavior of protein-nucleic acid complexes in gels and an overview of many of the applications in which the technique has proved especially useful. The factors which contribute most to the resolution of the complex from the naked nucleic acid are the gel pore size, the relative mass of protein compared with nucleic acid, and changes in nucleic acid conformation (bending) induced by binding. The consequences of induced bending on the mobility of double-strand DNA fragments are similar to those arising from sequence-directed bends, and the latter can be used to help characterize the angle and direction of protein-induced bends. Whether a complex formed in solution is actually detected as a retarded band on a gel depends not only on resolution but also on complex stability within the gel. This is strongly influenced by the composition and, particularly, the ionic strength of the gel buffer. We discuss the applications of the technique to analyzing complex formation and stability, including characterizing cooperative binding, defining binding sites on nucleic acids, analyzing DNA conformation in complexes, assessing binding to supercoiled DNA, defining protein complexes by using cell extracts, and analyzing biological processes such as transcription and splicing.
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.
By means of mobility-shift assays and Exonuclease III mapping we have determined a 14 bp sequence (named CDF2 binding site)
located in front of the 16S rRNA initiation start site which is protected by a spinach chloroplast extract. This region does
not include neither one of the two ‘−35’ not of the two ‘−10’ E.coli-like promoter elements which are recognised by E.coli RNA polymerase in vitro. The CDF2 binding site is specifically recognized by two small polypeptides which migrate corresponding to 35 and 33 kDa
respectively as shown by UV cross-linking experiments. In vivo transcription initiation of the 16S rRNA gene occurs 13 nucleotides downstream of the 14 bp sequence and is different from
the transcription start site which is used by E.coli polymerase in vitro.
Recently, two 2-dimensional (2D) gel techniques, termed neutral/neutral and neutral/alkaline, have been developed and employed
to map replication origins in eukaryotic plasmids and chromosomal DNA (1–11). The neutral/neutral technique, which requires
less DNA for analysis, has been preferentially used in recent studies. We show here that the signal predicted for an origin
is not detected using the neutral/neutral technique if the origin is located near the end of the analyzed restriction fragment.
We also demonstrate that analysis of the same batch of DNA by the two different mapping techniques can generate apparently
contradictory results: in some situations where neutral/alkaline 2D analysis indicates that a certain origin is always used,
neutral/neutral 2D analysis suggests that the origin is not always used. Several possible explanations for this type of disagreement
between the two techniques are discussed, and we conclude that it is important to use both techniques in combination in order
to minimize possible misinterpretations.
The locations of the two replication origins in pea chloroplast DNA (ctDNA) have been mapped by electron microscopic analysis of restriction digests of supercoiled ctDNA cross-linked with trioxalen. Both origins of replication, identified as displacement loops (D-loops), were present in the 44-kilobase-pair (kbp) SalI A fragment. The first D-loop was located at 9.0 kbp from the closest SalI restriction site. The average size of this D-loop was about 0.7 kbp. The second D-loop started 14.2 kbp in from the same restriction site and ended at about 15.5 kbp, giving it a size of about 1.3 kbp. The orientation of these two D-loops on the restriction map of pea ctDNA was determined by analyzing SmaI, PstI, and SalI-SmaI restriction digests of pea ctDNA. One D-loop has been mapped in the spacer region between the 16S and 23S rRNA genes. The second D-loop was located downstream of the 23S rRNA gene. Denaturation mapping of recombinants pCP 12-7 and pCB 1-12, which contain both D-loops, confirmed the location of the D-loops in the restriction map of pea ctDNA. Denaturation-mapping studies also showed that the two D-loops had different base compositions; the one closest to a SalI restriction site denatured readily compared with the other D-loop. The recombinants pCP 12-7 and pCB 1-12 were found to be highly active in DNA synthesis when used as templates in a partially purified replication system from pea chloroplasts. Analysis of in vitro-synthesized DNA with either of these recombinants showed that full-length template DNA was synthesized. Recombinants from other regions of the pea chloroplast genome showed no significant DNA synthesis activity in vitro.
Equilibrium and kinetic rate constants were determined for the binding of the initiator protein DnaA of Escherichia coli to its binding site, the non-palindromic 9-bp DnaA box, using gel retardation techniques. The dissociation constant for specific binding was between 1 and 50 nM for individual DnaA boxes on 21-bp double-stranded oligonucleotides. Only DnaA boxes of the sequence TT(A/T)TNCACA resulted in specific fragment retention. Both the 9-bp consensus sequence and flanking sequences determined the binding efficiency. One DnaA monomer was found to bind to a DnaA box and to induce a bend of about 40 degrees.
Spinach chloroplasts contain two types of RNA polymerases. One is multimeric and Escherichia coli-like. The other one is not E. coli-like and might represent a monomeric enzyme of 110 kD. The quantitative relation of the two polymerases changes during plant development. This raises the question, how are plastid genes transcribed that contain E. coli-like and non-E. coli-like promoter elements during developmental phases when both enzymes are present? Transcription of the spinach plastid rrn operon promoter is initiated at three sites: P1, PC, and P2. P1 and P2 are preceded by E. coli-like promoter elements that are recognized by E. coli RNA polymerase in vitro. However, in vivo, transcription starts exclusively at PC. We analyzed different promoter constructions using in vitro transcription and gel mobility-shift studies to understand why P1 and P2 are not used in vivo. Our results suggest that the sequence-specific DNA-binding factor CDF2 functions as a repressor for transcription initiation of the E. coli-like enzyme at P1 and P2. We propose a mechanism of constitutive repression to keep the rrn operon in all developmental phases under the transcriptional control of the non-E. coli-like RNA polymerase.
Since the publication of the first edition five years ago, a wide range of new methodologies have been developed to facilitate studies on both isolated parts of the genome and the genome as a whole. This new edition has been updated and expanded so that it provides a comprehensive guide to the methods currently available to characterize the function and activity of an individual transcription factor. All the original chapters have been fully updated or rewritten and additional chapters cover the use of in vitro transcription assays, analysis of chromatin structure, use of the genomic binding site assay and analysis of transcription factor modifications. As with the previous edition, the book starts with a series of chapters concerned with characterizing the proteins binding to a specific DNA sequence and then a chapter on more detailed characterization of the protein itself. The next two chapters describe the isolation of cDNA clones encoding a transcription factor using oligonucleotides predicted from protein sequence and screening of a cDNA expression library. Chapter 6 deals with identification of transcription factors based on sequence homology analysis by both experimental screening and database searches. Chapter 7 is a new chapter that describes methods of identifying the target genes of a previously uncharacterized factor. The next chapters deal with analysis of transcription factor function. Chapter 8 deals with general techniques, and then the following chapters cover the specialized techniques of in vitro transcription assays using transcriptionally active nuclear extracts derived from rat brain, and analysis of the effect of transcription factors on chromatin structure. The final chapter describes methods for detecting the phosphorylation and glycosylation state of transcription factors.
The DNA mobility-shift assay relies on the principle that a DNA fragment to which a protein has bound will move more slowly in gel electrophoresis than the DNA fragment alone. Hence it represents the simplest means of studying a DNA-protein interaction. This article describes the methods of preparing the DNA probe and protein extract for this assay as well as for performing the binding assay itself. The uses of this assay in the initial characterization of a novel factor, in determining its relationship to previously characterized factors, and in studying the DNA-binding domain of cloned factors are discussed.
Plants are unique among eukaryotes in possessing two DNA-containing organelles—the plastid and the mitochondrion. Moreover, the green alga Chlamydomonas reinhardtii has recently been shown to contain a third extranuclear genome—that of the basal body (Hall et al., 1989). Nothing is known about the origin, phylogenetic distribution and evolution of basal body DNA, and therefore this genome will not be considered in this chapter. In contrast, we now possess a rather detailed picture of the tempo and mode of evolution of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) in land plants. Review of this topic will form the heart of this chapter, as presented in Sects. III–V. Data for both genomes will be presented in an integrated format in order to highlight the striking contrasts in their evolution in land plants. The much more limited evolutionary data base available for algal organelle genomes will be discussed in Sect. VI. All plastid and mitochondrial genomes are of endosymbiotic, bacterial origin. However, as discussed in the next section, considerable uncertainty remains as to the precise number and nature of endosymbioses that have taken place.
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.
Chloroplast DNA (ctDNA) generally occurs as circular molecules with molecular weights (MWs) in the range 70–130 × 106 depending on the species1,2. In Euglena gracilis, ctDNA (44 µm, 92 × 106 MW3) replicates through Cairns-type intermediates4 having structural aspects suggesting bidirectional replication5. Pea and corn ctDNA were shown to contain two displacement loops (D-loops) located 7,100 base pairs (bp) apart6. The displacing strands of the two D-loops are located on opposite strands of the parental DNA; they expand towards each other and form a Cairns replicative intermediate when the two strands elongate past each other. Rolling circle intermediates7, apparently resulting from the continuation of Cairns rounds of replication, have also been observed8. The origins of replication were not located on physical maps for any of these studies. We present here the results of an electron microscopic (EM) study indicating that replication of ctDNA in Euglena is initiated near the 5′ end of the supplementary 16S ribosomal RNA gene9.
Publisher Summary This chapter describes a filter hybridization approach for mapping chloroplast DNA (cpDNA) restriction sites along with several strategies for generating restriction fragments for use as hybridization probes. Approaches for visualizing and mapping cpDNAs that are difficult or impossible to isolate in very pure form are also discussed. The chloroplast genome is densely packed with genes and many of them have been isolated and sequenced. Chloroplast gene products identified thus far function either in photosynthesis or as components of the chloroplast protein synthesizing system. The basic strategy for mapping by overlap hybridization is to hybridize each one of a set of restriction fragments that together cover an entire chloroplast genome to replica membranes containing various single and double digests of total cpDNA. Three classes of individually purified cpDNA restriction fragments that can be used as hybridization probes are homologous, uncloned fragments; homologous, cloned fragments; and heterologous, cloned fragments. Procedures to obtain each class of probe fragments and the relative merits of each class are discussed.
An in vitro chloroplast DNA synthesizing lysate system prepared from purified chloroplasts of Petunia hybrida leaves has been developed. Both co-isolated endogeneous chloroplast (cp)DNA and externally added DNA can be used as DNA templates in the system. The system contains a -like DNA polymerase as determined by using DNA polymerase-specific inhibitors and synthetic templates. The molecular weight of this enzyme is about 85 kd. Part of the DNA synthesizing activity is repair synthesis. When a chimaeric plasmid containing a fragment with a potential cpDNA replication origin is used as a template (pPCY62), specific initiation of DNA synthesis is observed on this fragment which strongly suggests that the in vitro chloroplast lysate system is also capable of replication initiation.
The origins of chloroplast DNA (cpDNA) replication were mapped in two plastome types of Oenothera in order to determine whether variation in the origin of cpDNA replication could account for the different transmission abilities associated with these plastomes. Two pairs of displacement loop (D-loop) initiation sites were observed on closed circular cpDNA molecules by electron microscopy. Each pair of D-loops was mapped to the inverted repeats of the Oenothera cpDNA by the analysis of restriction fragments. The starting points of the two adjacent D-loops are approximately 4 kb apart, bracketing the 16S rRNA gene. Although there are small DNA length variations near one of the D-loop initiation sites, no apparent differences in the number and the location of replication origins were observed between plastomes with the highest (type I) and lowest (type IV) transmission efficiencies.
Covalently closed circular chloroplast DNA (ctDNA) molecules have been isolated from pea, bean, spinach, lettuce, corn and oat plants by ethidium bromide/cesium chloride density-gradient centrifugation. As much as 30–40% of the total ctDNA could be isolated as closed circular DNA molecules and up to 80% of the total ctDNA was found in the form of circular molecules.The size of pea, spinach, lettuce, corn and oat ctDNA relative to an internal standard (ΦX174 replicative form II monomer DNA) was determined by electron microscopy. The ctDNAs showed significant differences in their sizes, and their molecular weights ranged from 85.4 · 106 for corn ctDNA to 96.7 · 106 for lettuce ctDNA. Each of these ctDNAs contained 3–4% of the circular molecules as circular dimers and 1–2% of the circular molecules as catenated dimers.The molecular complexity of these ctDNAs was studied by renaturation kinetics using T4 DNA as a standard. The molecular weights of the unique sequence of the ctDNAs ranged from 83.7 · 106 for oat ctDNA to 93.1 · 106 for lettuce ctDNA, which are in excellent agreement with the sizes of the circular ctDNA molecules. No repeating sequences were detected in any of the ctDNAs.The ctDNAs from pea, lettuce, corn, and spinach were studied by thermal denaturation using T4 DNA as a standard. All of the ctDNAs melted more broadly than T4 DNA and they all had a distinctly different . For example, the of pea ctDNA was 1°C below the of T4 DNA and the of corn was 1.9°C higher than the of T4 DNA.
A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.
The chloroplast DNA (ctDNA) from pea and corn plants contains both Cairns type and rolling circle replicative intermediates. Denaturation mapping studies with pea ctDNA molecules have shown that the rolling circles initiate replication at or near the site where the Cairns replicative intermediates terminate replication. These results suggest that the rolling circles are initiated by a Cairns round of replication. A model for the replication of the chloroplast DNA is based on these results.
Covalently closed circular choloroplast DNA (ctDNA) molecules have been isolated from pea, bean, spinach, lettuce, corn and oat plants by ethidium bromide/cesium choloride density-gradient entrifugation. As much as 30-40% of the total ctDNA could be isolated as closed circular DNA molecules and up to 80% of the total ctDNA was found in the form of circular molecules. The size of pea, spinach, lettuce, corn and oat ctDNA relative to an internal standard (phiX174 replicative form II monomer DNA) was determined by electron microscopy. The ctDNAs showed significant differences in their sizes, and their molecular weights ranged from 85.4 - 10(6) for corn ctDNA to 96.7 - 10(6) for lettuce ctDNA. Each of these ctDNAs contained 3-4% of the circular molecules as circular dimers and 1-2% of the circular molecules as catenated dimes. The molecular complexity of these ctDNAs was studied by renaturation kinetics using T4 DNA as a standard. The molecular weights of the unique sequences of the ctDNAs ranged from 83.7 - 10(6) for oat ctDNA to 93.1 - 10(6) for lettuce ctDNA, which are in excellent agreement with the sizes of the circular ctDNA molecules...
We have used a combination of one- and two-dimensional agarose gel electrophoresis, and solution hybridization to strand-specific probes, to map the replication origin of sea urchin mitochondrial DNA and to investigate the structure of replication intermediates. These assays are consistent with replication initiating unidirectionally from the D-loop region by D-loop expansion, as in vertebrates. A prominent site of initiation of lagging-strand synthesis lies at, or near to, the boundary between the genes for ATPase 6 and COIII, which is also close to a pause site for leading-strand synthesis. These findings suggest a role for pause sites in the regulation of mitochondrial transcription and replication, possibly involving template-binding proteins.
When tobacco suspension culture line BY2 cells in stationary phase are transferred into fresh medium, replication of proplastid DNA proceeds for 24 h in the absence of nuclear DNA replication. Replicative intermediates of the proplastid DNA concentrated by benzoylated, naphthoylated DEAE cellulose chromatography, were radioactively labelled and hybridized to several sets of restriction endonuclease fragments of tobacco chloroplast DNA. The intermediates hybridized preferentially to restriction fragments in the two large inverted repeats. Mapping of D-loops and of restriction fragment lengths by electron microscopy permitted the localization of the replication origin, which was close to the 23S rRNA gene in the inverted repeats. The replication origins in both segments of the inverted repeat in tobacco proplastid DNA were active in vivo.
Mammalian mitochondrial DNA maintains a novel displacement-loop region containing the major sites of transcriptional initiation and the origin of heavy strand DNA replication. Because the exact map positions of the 5' termini of nascent mouse displacement-loop strands are known, it is possible to examine directly a potential relationship between replication priming and transcription. Analyses of in vivo nucleic acids complementary to the displacement-loop region reveal two species with identical 5' ends at map position 16 183. One is entirely RNA and the other is RNA covalently linked to DNA. In the latter the transition from RNA to DNA is sharp, occurring near or within a series of previously identified conserved sequences 74-163 nucleotides downstream from the transcriptional initiation site. These data suggest that the initial events in replication priming and transcription are the same and that the decision to synthesize DNA or RNA is a downstream event under the control of short, conserved displacement-loop template sequences.
From a high-salt extract of the purified thylakoid membrane, an 18-kD protein was detected. This protein was translated by the chloroplast ribosomes and could form a stable DNA-protein complex with a cloned chloroplast DNA replicative origin [Nie, Z.Q., Chang, D.Y., and Wu, M. (1987) Mol. Gen. Genet. 209, 265-269]. In this paper, the 18-kD protein is linked to frxB, a chloroplast-encoded, ferredoxin-type, iron-sulfur protein, by N-terminal microsequencing of the purified protein and computer analysis. The identification is further supported empirically by the fact that the electron paramagnetic resonance spectra of the protein indicate the presence of iron-sulfur clusters. A polyclonal antibody raised against a synthetic pentadecameric peptide with amino acid sequence corresponds to the highly conserved region of the frxB protein and reacts strongly and specifically with the 18-kD protein band in protein gel blot analyses. The 18-kD iron-sulfur protein is found to be related to a subunit of the respiratory chain NADH dehydrogenase by its cross-reaction with a polyclonal antibody raised against highly purified NADH-ubiquinone oxidoreductase, a key enzyme of the respiratory chain. These data are consistent with chlororespiration, and, thus, possible implication of chlororespiration in regulating the initiation of chloroplast DNA replication is discussed.
Replication intermediates from the yeast 2 microns plasmid and a recombinant plasmid containing the yeast autonomous replication sequence ARS1 have been analyzed by two-dimensional agarose gel electrophoresis. Plasmid replication proceeds through theta-shaped (Cairns) intermediates, terminating in multiply interlocked catenanes that are resolved during S phase to monomer plasmids. Restriction fragments derived from the Cairns forms contain replication forks and bubbles that behave differently from one another when subjected to high voltage and agarose concentrations. The two-dimensional gel patterns observed for different restriction fragments from these two plasmids indicate that in each plasmid there is a single, specific origin of replication that maps, within the limits of our resolution, to the ARS element. Our results strongly support the long-standing assumption that in Saccharomyces cerevisiae an ARS is an origin of replication.
An enzyme system prepared from maize chloroplasts catalyzes the synthesis of DNA from maize chloroplast DNA sequences cloned
in bacterial plasmids. Cloned maize chloroplast DNA fragments Bam HI 17′ (2470 bp) and Eco RI x (1368 bp) have been shown
to be preferred templates for in vitro DNA synthesis catalyzed by pea chloroplast DNA polymerase preparations [Gold et al. (1987) Proc. Natl. Acad. Sci. USA 84, 194–198]. Analysis of replicative intermediates indicates that although the template
activity of the recombinant plasmid pZmcBam 17′ is substantially greater than that of the pZmcEco ×, replication in both cases
originates from within a 455 bp region which overlaps the two plasmids. The remaining approximately 1500 basepair portion
of maize chloroplast BamHI fragment 17′ is not more active because it contains additional origins for replication. The overlapping
region shows sequence homology with a portion of the Chlamydomonas reinhardtii chloroplast chromosome that contains a replication origin. Replication is shown to proceed bidirectionally within the 455
bp origin region. Recombinant plasmid pZmc 427, which is also active in the in vitro DNA synthesis assay, promoted localized replication initiation within a 1 kbp Bg1II-Eco RI fragment of the chloroplast DNA
insert, a region that includes the 3′ terminal part of the psbA gene.
Individual promoters for transcription of each strand of human mtDNA are located near the origin of heavy-strand DNA replication in the displacement-loop region. Initiation of heavy-strand synthesis represents the first event in mtDNA replication. Analyses of the 5' and 3' map positions of displacement-loop nucleic acids from mitochondria of cultured human cells reveal a close correspondence between the 3' ends of RNA, whose 5' ends map at a unique site, and the 5' ends of DNA strands. The 5' ends of the RNA species all map at nucleotide position 407 in the genomic sequence, which corresponds exactly to the major 5' transcriptional start site, determined previously in vitro, that is contained within the light-strand promoter sequence. Displacement-loop heavy-strand DNAs map immediately adjacent to the 3' termini of these RNAs, and these transition points between RNA and DNA lie within short conserved sequence blocks in the template sequence. The simplest interpretation of these data is that replication is initiated at the major transcriptional promoter with subsequent precise cleavage of primary transcripts to provide the appropriate primer species.
Replication of the approximately 200 tandem copies of yeast ribosomal RNA genes (rDNA) is known to be initiated within a subset of the repeats, with transcription continuing during the replication process. To examine replication fork movement in this gene cluster, we used a two-dimensional (2D) agarose gel electrophoresis procedure that distinguishes molecules with different branched structures. Replication forks move through most of the rDNA in the same direction in which RNA polymerase I transcribes the 35S rRNA precursor: the 3' end of this transcription unit acts as a barrier to replication forks moving in the direction opposite to RNA polymerase I. The replication fork barrier (RFB) is observed as the accumulation of branched intermediates of specific size. We propose that the act of transcription may influence the movement of replication forks, creating barriers at the 3' ends of actively transcribed genes.
Chloroplast DNA, isolated from a synchronized culture of Chlamydomonas reinhardii , was digested with restriction endonucleases and examined in the electron microscope. Restriction fragments containing displacement
loops (D-loop) were photographed and measured to determine the position of replicated sequences in relation to the restriction
enzyme sites. D-loops were located at two positions on the physical map of chloroplast DNA. One replication origin was mapped
at about 10 kb upstream of the 5′ end of a 16s rRNA gene. The second origin was spaced 6.5kb apart from the first origin and
was about 16.5 kb upstream of the same 16s rRNA. Initiations at those two sites were not always synchronized. Replication
initiated with the formation of a D-loop resulting from the synthesis of one daughter strand. After a short initial lag phase,
corresponding to the synthesis of 350±130 bp of one daughter strand, DNA synthesis then proceeded in both directions. Both
D-loop regions were preferred binding sites of undetermined protein complexes.
The entire mitochondrial genome of Schizosaccharomyces pombe ura4-294h- was analyzed by the 2D pulsed field gel electrophoresis technique developed by Brewer and Fangman. The genome consists of multimers with an average size of 100 kb and analysis of the overlapping restriction fragments of the complete mitochondrial DNA (mtDNA) genome resulted in simple Y 2D gel patterns. Large single-stranded DNA molecules or double-stranded DNA molecules containing large or numerous single-stranded regions were found in the S. pombe mtDNA preparation. The replication of mtDNA monomers was found to occur in either direction. On the basis of these results, a replication mechanism for S. pombe mtDNA that is most consistent with a rolling circle model is suggested.
A partially purified replicative system of pea chloroplast that replicates recombinant DNAs containing pea chloroplast origin sequences has been characterised. Polymerisation by this system is very fast and insensitive to chain terminators like dideoxynucleotides, arabinosylcytosine 5′-triphosphate, etc. Both strands of template DNA are synthesized and single-stranded DNA templates undergo more than one round of replication.
When sequences of either of the two chloroplast origins of replication (OriA or OriB) are used as templates, the replicative intermediates are found to have sigma structures. Electron microscopic analysis of the sigma structures restricted with various enzymes reveals that the initiation site of in vitro replication maps near the displacement-loop regions where replication initiates also in vivo. Although the observed replication initiation in the OriA recombinant template is chloroplast-DNA-specific, the mode of replication is different from that observed in vivo with intact ctDNA. However, when the template DNA contains both the OriA and OriB sequences, the in vitro replication proceeds in the theta mode, the mode of replication usually observed in vivo.
Chloroplast DNA replication was studied in the green, autotrophic suspension culture line SB-1 of Glycine max. Three regions (restriction fragments Sac I 14.5, Pvu II 4.1 and Pvu II 14.8) on the plastome were identified that displayed significantly higher template activity in in vitro DNA replication assays than all other cloned restriction fragments of the organelle genome, suggesting that these clones contain sequences that are able to direct initiation of DNA replication in vitro. In order to confirm that the potential in vitro origin sites are functional in vivo as well, replication intermediates were analyzed by two-dimensional gel electrophoresis using cloned restriction fragments as probes. The two Pvu II fragments that supported deoxynucleotide incorporation in vitro apparently do not contain a functional in vivo replication origin since replication intermediates from these areas of the plastome represent only fork structures. The Sac I 14.5 chloroplast DNA fragment, on the other hand, showed intermediates consistent with a replication bubble originating within its borders, which is indicative of an active in vivo origin. Closer examination of cloned Sac I 14.5 sub-fragments confirmed high template activity in vitro for two, S/B 5 and S/B 3, which also seem to contain origin sites utilized in vivo as determined by two-dimensional gel electrophoresis. The types of replication intermediate patterns obtained for these sub-fragments are consistent with the double D-loop model for chloroplast DNA replication with both origins being located in the large unique region of the plastome [17, 18]. This is the first report of a chloroplast DNA replication origin in higher plants that has been directly tested for in vivo function.
One of the two origins of replication in pea chloroplast DNA (oriA) maps in the rRNA spacer region downstream of the 16S rRNA gene, and further characterization of this origin is presented here. End-labeling of nascent DNA strands from in vivo replicating ctDNA was used to generate probes for Southern hybridization. Hybridization data identified the same region that was previously mapped to contain D-loops by electron microscopy. Subclones of the oriA region were tested for their ability to support in vitro DNA replication using a partially purified pea ctDNA replication system. Two-dimensional agarose gel electrophoresis identified replication intermediates for clones from the region just downstream of the 16S rRNA gene, with a 450-bp SacI-EcoRI clone showing the strongest activity. The experiments presented in this paper identify the 940 base pair region in the rRNA spacer between the 3' end of the 16S rRNA gene and the EcoRI site as containing oriA. Previous studies by electron microscopy localized the D-loop in the spacer region just to the right of the BamHI site, but the experiments presented here show that sequences to the left of the BamHI site are required for replication initiation from oriA. DNA sequence analysis of this region of pea ctDNA shows the presence of characteristic elements of DNA replication origins, including several direct and inverted repeat sequences, an A + T rich region, and dnaA-like binding sites, most of which are unique to the pea ctDNA oriA region when compared with published rRNA spacer sequences from other chloroplast genomes.
We have Identified a second DNA-binding protein in sea urchin embryo mitochondria, which interacts with a binding site in
the major replication pause region, at the junction of the genes for ATP synthase subunit 6 and cytochrome c oxidase subunit III (COIII). We provisionally designate this protein mtPBP2, to distinguish it from the previously characterized
mito-chondrial pause-region binding protein mtPBP2, whose properties and binding site are quite distinct. The high-affinity
binding site for mtPBP2 lies at the 5′ end of the COIII gene, and exhibits partial dyad symmetry, although modification Interference
analysis Indicates that recognition is complex. Binding of mtPBP2 to this site induces a bend of approximately 45° in the
DNA. Southwestern blots show that mtPBP1 and 2 are both single polypeptides, of apparent molecular weights 25 kD and 18 kD
respectively. In vitro, mtPBP1 and mtPBP2 bind independently to their high-affinity sites, which are separated by about 50 bp.
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.
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.