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Analysis of soybean chloroplast DNA replication by two-dimensional gel electrophoresis
Abstract
Chloroplast DNA replication was studied in the green, autotrophic suspension culture line SB-1 of Glycine max. Three regions (restriction fragments Sac I 14.5, Pvu II 4.1 and Pvu II 14.8) on the plastome were identified that displayed significantly higher template activity in in vitro DNA replication assays than all other cloned restriction fragments of the organelle genome, suggesting that these clones contain sequences that are able to direct initiation of DNA replication in vitro. In order to confirm that the potential in vitro origin sites are functional in vivo as well, replication intermediates were analyzed by two-dimensional gel electrophoresis using cloned restriction fragments as probes. The two Pvu II fragments that supported deoxynucleotide incorporation in vitro apparently do not contain a functional in vivo replication origin since replication intermediates from these areas of the plastome represent only fork structures. The Sac I 14.5 chloroplast DNA fragment, on the other hand, showed intermediates consistent with a replication bubble originating within its borders, which is indicative of an active in vivo origin. Closer examination of cloned Sac I 14.5 sub-fragments confirmed high template activity in vitro for two, S/B 5 and S/B 3, which also seem to contain origin sites utilized in vivo as determined by two-dimensional gel electrophoresis. The types of replication intermediate patterns obtained for these sub-fragments are consistent with the double D-loop model for chloroplast DNA replication with both origins being located in the large unique region of the plastome [17, 18]. This is the first report of a chloroplast DNA replication origin in higher plants that has been directly tested for in vivo function.
... Replication of plastid and nuclear DNA do not appear to be tightly co-ordinated in N. tabacum (Heinhorst et al. 1985) or the green alga Chlamydomonas reinhardtii (Chiang and Sueoka 1967). In contrast to the stringent controls restricting nuclear DNA synthesis to one round of replication during the S phase of each cell cycle, plastid DNA replication appears to be less stringent and is not limited to the S phase (Heinhorst and Cannon 1993). Moreover, plastid genomes appear to be chosen randomly for replication (Birky 1994). ...
... (Meeker et al. 1988) lacks a large inverted repeat, b) Dicots containing large inverted repeats. N. tabacum Nt (Ori A) and Nt (Ori B) (Kunnimalaiyaan and Nielsen 1997;Kunnimalaiyaan et al. 1997), N. tabacum D-loops Nt (pro) in proplastids (Takeda et al. 1992), Oenothera hookeri Oe (Ori A) and Oe (Ori B) (Chiu and Sears 1992;Sears et al. 1996), Glycine max bubbles Gm (Hedrick et al. 1993). c) Oryza sativa (Os) replication origins in suspension culture cells (Os 1 ), leaf blades (Os 2 ), and coleoptiles (Os 3 ) mapped by Wang et al. 2003, Z. mays (Zm, Gold et al. 1987, linear DNA replicons (Ellis and Day 1986;Harada et al. 1992;Zubko and Day 2002), Hordeum vulgare (Hv), Oryza sativa (Os). ...
... Two replication bubbles were mapped to 1.8 kbp Sac I-Bam HI and 2.5 kbp Bam HI fragments in the large single copy region of Glycine max (soybean) chloroplast DNA by two-dimensional gel electrophoresis ( Fig. 6b; Hedrick et al. 1993). The recently published G. max plastid genome (Saski et al. 2005) locates these bubble-containing fragments to regions containing the rps 12-clp P1 and pet B-pet D genes in the large single copy region. ...
Plastid DNA is conserved, highly polyploid and uniform within aplant reflecting efficient
plastid DNA replication/recombination/repair (DNA-RRR) pathways. We will review the current understanding
of the DNA sequences, proteins, and mechanisms involved in plastid genome maintenance. This includes
analysis of the topological forms of plastid DNA, models of plastid DNA replication, homologous recombination,
replication slippage, DNA repair, and plastid DNA-RRR-proteins. We will focus on flowering plants
but include information from algae when relevant. Plastid DNA is comprised of amultimeric series
of circular, linear, and branched forms. Variant plastid DNA molecules include small linear palindromes
with hairpin ends. Plastid transformation has demonstrated an efficient homologous recombination
pathway, acting on short ∼200 bp sequences, that is active throughout shoot development.
These functional studies involving plastid transformation to manipulate DNA sequences, combined with
genomics and reverse genetics to isolate mutants in plastid DNA-RRR proteins, will be particularly
important for making progress in this field.
... In the last few years 2DE of DNA molecules has been developed into a powerful tool for the detection of replication intermediates and for determining the replication type (36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47). This technique takes advantage of the fact that DNA molecules are separated according to their molecular mass in the first dimension and that a non-linear DNA molecule does not migrate at the same rate as a linear molecule of equal mass in the second dimension, i.e. migration is additionally dependent on the structure (36,46). ...
... By using this method the origin of mp1 replication was mapped and sequenced (Fig. 4). The position of a site-and strand-specific nick could only be identified when primer extensions were carried out for multiple cycles (40), reflecting the scarcity of replication intermediates of mp1 in mtDNA. The effectivness and precision of the applied method was controlled by the identification of cleavage products of recombinant plasmid DNA, as shown for BamHI (primers 1 and 3, lanes c) and SmaI (primer 2, lane c). ...
We analyzed the structure and replication of the mitochondrial (mt) circular DNA plasmid mpl (1309 bp) from the higher plant
Chenopodium album (L.). Two dimensional gel electrophoresis (2DE) revealed the existence of oligomers of up to a decamer in addition to the
prevailing monomeric form. The migration behavior of cut replication intermediates during 2DE was consistent with a rolling
circle (RC) type of replication. We detected entirely single-stranded (ss) plasmid copies hybridizing only with one of the
two DNA strands. This result indicates the occurence of an asymmetric RC replication mechanism. mp1 has, with respect to its
replication, some unique features compared with bacterial RC plasmids. We identified and localized a strand-specific nicking
site (origin of RC replication) on the plasmid by primer extension studies. Nicks in the plasmid were found to occur at any
one of six nucleotides (TAAG/GG) around position 735 of the leading strand. This sequence shows no homology to origin motifs
from known bacterial RC replicons. mp1 is the first described RC plasmid in a higher plant.
... Two-dimensional (2D) agarose gel electrophoresis has been successfully employed to examine in vivo and in vitro replication intermediates in several systems (5)(6)(7)(8). 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). ...
... We report here the usefulness of this technique to identify minimal sequences required for replication in vitro of oriA and oriB from tobacco ctDNA. Although there are a few previous reports on in vitro replication analysis using partially purified chloroplast protein fractions (2,9,13), it is not always clear whether the activity observed reflected a true replicative type of DNA synthesis or repair synthesis. In addition, a reliable method for the purification of a faithful replication system is necessary to carry out biochemical studies. ...
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.
... According to the Kolodner and Tewari (1975) model, plastid DNA replication of N. tabacum initiates on a pair of replication origins: oriA and oriB (Kunnimalaiyaan and Nielsen, 1997b;Kunnimalaiyaan et al., 1997;Lu et al., 1996;Lugo et al., 2004). Pairs of oris were also detected in other species: (Kunnimalaiyaan and Nielsen, 1997b), the suspension culture ori (Takeda et al., 1992;Wang et al., 2002), the putative homologue of oriA of Oenothera elata (Chiu and Sears, 1992), the putative homologue of an ori near psbA or rpl16, respectively, of Zea mays (Carrillo and Bogorad, 1988) and near rpl16 of Chlamydomonas reinhardtii (Lou et al., 1987) and Glycine max (Hedrick et al., 1993) are given. (Waddell et al., 1984), G. max (Hedrick et al., 1993), O. elata (Chiu and Sears, 1992) and Pisum sativum (Meeker et al., 1988). ...
... Pairs of oris were also detected in other species: (Kunnimalaiyaan and Nielsen, 1997b), the suspension culture ori (Takeda et al., 1992;Wang et al., 2002), the putative homologue of oriA of Oenothera elata (Chiu and Sears, 1992), the putative homologue of an ori near psbA or rpl16, respectively, of Zea mays (Carrillo and Bogorad, 1988) and near rpl16 of Chlamydomonas reinhardtii (Lou et al., 1987) and Glycine max (Hedrick et al., 1993) are given. (Waddell et al., 1984), G. max (Hedrick et al., 1993), O. elata (Chiu and Sears, 1992) and Pisum sativum (Meeker et al., 1988). The pair is located in the inverted repeats in N. tabacum and O. elata (in P. sativum, lacking an inverted repeat, the pair of origins is also located in the same gene-order context), whereas in G. max and C. reinhardtii it is found in the large single copy region. ...
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.
... 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). ...
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]. ...
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.
... Two-dimensional agarose gel electrophoresis has been used to map and characterize DNA replication origins in animals, yeast and bacterial plasmids (reviewed by Kunnimalaiyaan & Nielsen, 1997) and has recently been applied to the analysis of in vivo and in vitro ctDNA replication intermediates (Hedrick et al., 1993;Nielsen et al., 1993;Lu et al., 1996). Analysis of the tobacco ctDNA 4.29 kb SspI fragment containing the 23 S rRNA gene and the region downstream to the end of the IR by this technique showed the presence of E-arc (extended arc) and simple Y patterns. ...
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.
... The origin of replication has been mapped in the tobacco chloroplast genome and oriA is located within the trnI gene that forms the left flank in the chloroplast transformation vector used for cotton transformation. Several methods have been used to study ctDNA replication origins in higher plants including electron microscopy, two-dimensional (2D) agarose gel electrophoresis, primer extension mapping of nascent 5′ ends from total plastid DNA (Kolodner and Tewari, 1975; Meeker et al., 1988; Chiu and Sears, 1992; Hedrick et al., 1993; Nielsen et al., 1993; Kunnimalayaan et al., 1997), and in vitro replication analysis of ctDNA ori subclones (Kunnimalayaan and Nielsen, 1997 a, b). All these studies have identified two replication origins (oriA and oriB) that flank the 23S rRNA gene in tobacco ctDNA (Kunnimalayaan and Nielsen, 1997a,b). ...
Chloroplast genetic engineering overcomes concerns of gene containment, low levels of transgene expression, gene silencing, positional and pleiotropic effects or presence of vector sequences in transformed genomes. Several therapeutic proteins and agronomic traits have been highly expressed via the tobacco chloroplast genome but extending this concept to important crops has been a major challenge; lack of 100 homologous species-specific chloroplast transformation vectors containing suitable selectable markers, ability to regulate transgene expression in developing plastids and inadequate tissue culture systems via somatic embryogenesis are major challenges. We employed a 'Double Gene/Single Selection (DGSS)' plastid transformation vector that harbors two selectable marker genes (aph A-6 and npt II) to detoxify the same antibiotic by two enzymes, irrespective of the type of tissues or plastids; by combining this with an efficient regeneration system via somatic embryogenesis, cotton plastid transformation was achieved for the first time. The DGSS transformation vector is at least 8-fold (1 event/2.4 bombarded plates) more efficient than 'Single Gene/Single Selection (SGSS)' vector (aph A-6; 1 event per 20 bombarded plates). Chloroplast transgenic lines were fertile, flowered and set seeds similar to untransformed plants. Transgenes stably integrated into the cotton chloroplast genome were maternally inherited and were not transmitted via pollen when out-crossed with untransformed female plants. Cotton is one of the most important genetically modified crops (120 billion US dollars US annual economy). Successful transformation of the chloroplast genome should address concerns about transgene escape, insects developing resistance, inadequate insect control and promote public acceptance of genetically modified cotton.
Plastids, like mitochondria, result from an ancient endosymbiosis event and contain a distinct genome. Though many plastid genes have since been transferred to the nuclear genome, the small plastid genome still encodes between 90 and 100 genes, which are notably involved in translation, transcription, and energy metabolism in the plastid. The many roles of this organelle, the most familiar being photosynthesis in chloroplasts, make it essential for the development of higher plants. As such, the ability of the plastid to maintain the stability of its genome represents a crucial element of plant life. The physical organization of the genome itself can have an influence on DNA metabolism, with its large inverted repeats acting as templates for recombination. Furthermore, the localization of chloroplast DNA near elements of the electron transport chain increases the importance of DNA repair mechanisms, as reactive oxygen species (ROS) appear as by-products of photosynthesis. These ROS, along with UV radiation and DNA double-strand breaks, create a genotoxic stress through their respective ability to oxidize nucleotides, link DNA bases, or rearrange the structure of the genome. To minimize the deleterious effects of these events, different mechanisms present in the nucleus such as homologous recombination exist in plastids. Some less conservative mechanisms based on sequences of microhomology are also found, and sometimes lead to copy-number variation in certain areas of the plastid genome. While some of these changes can remain silent, others can be linked to phenotypes such as variegation.
To maintain and to differentiate into various plastid lineages, replication of the plastid DNA (ptDNA) and division of the plastid must take place. However, replication initiation of the ptDNA has been less understood. The present study describes identification of the initiation region (origin) of ptDNA replication in the rice cultured cells. RNA-primed newly replicated DNA strands pulse-labeled with bromodeoxyuridine were isolated and size-fractionated. Locations of these nascent strands on the ptDNA determined the two major origin regions around the 3′ region of each 23S rDNA in the inverted repeats (IRA and IRB). Two-dimensional agarose gel electrophoresis of the replication intermediates suggested that replication from each origin proceeds bidirectionally. This contrasted to replication by the double D - loop mechanism.
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.
Photoautotrophic (PA) tissue cultures that rely entirely upon light for energy and CO2 for carbon have been initiated from a total of 29 species of higher plants. The species include C4 and Crassulacean acid metabolism species, legumes, and a number of important crop plants. However, no grasses have been grown successfully. PA culture initiation is time-consuming and appears to be associated with long-term adaptation physiologically and apparently genetically, since once initiated the cultures can be bleached and then rapidly become photosynthetically competent again. In addition, a series of unusual mutations have also been found in the psbA gene leading to triazine herbicide resistance in PA cultures in contrast to those found with triazine-resistant weeds. A large number of physiological, biochemical, and molecular biological studies have been carried out especially with the Chenopodium rubrum and Glycine max cell lines. The cultures can be grown in fermenters, hence, seem ideal for compound and macromolecule production, although the overall record of valuable secondary compound production has been disappointing. PA cultures are useful for many studies since they are axenic, grow relatively rapidly, are easy to view microscopically, can be heavily ¹⁴C labeled using ¹⁴CO2, are in easily changed medium for compound addition and removal and other conditions are readily altered, and the cells are easily extracted.
The spacer between the 16S and 23S rRNA genes of the chloroplast DNA has been implicated as an origin of replication in several
species of plants. In the evening primrose, Oenothera, this site was found to vary greatly in size, with plastid genomes (plastomes)
being readily distinguished. To determine whether plastome "strength" in transmission could be correlated with variation at
oriB, the 16S rRNA-trnI spacer was sequenced from five plastomes. The size variation was found to be due to differential amplification
(and deletion) of combinations of sequences belonging to seven families of direct repeats. From these comparisons, one short
series of direct repeats and one region capable of forming a hairpin structure were identified as candidates for the factor
that could be responsible for the differences between strong and weak plastome types. Ample sequence variation allowed phylogenetic
inferences to be made about the relationships among the plastomes. Phylogenetic trees also could be constructed for most of
the families of direct repeats. The amplifications and deletions of repeats that account for the size variation at oriB are
proposed to have occurred through extensive replication slippage at this site.
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.
According to the endosymbiotic theory, mitochondrial genomes evolved from the chromosome of an alpha-proteobacterium-like ancestor and developed during evolution an extraordinary variation in size, structure and replication. We studied in vitro DNA replication of the mitochondrial circular plasmid mp1 (1309 bp) from the higher plant Chenopodium album (L.) as a model system that replicates in a manner reminiscent of bacterial rolling circle plasmids. Several mp1 subclones were tested for their ability to support DNA replication using a newly developed in vitro system. Neutral/neutral two-dimensional gel electrophoresis of the in vitro products revealed typical simple Y patterns of intermediates consistent with a rolling circle type of replication. Replication activity was very high for a BamHI-restricted total plasmid DNA clone, a 464 bp BamHI/KpnI fragment and a 363 bp BamHI/SmaI fragment. Further subcloning of a 148 bp BamHI/EcoRI fragment resulted in the strongest in vitro DNA replication activity, while a 1161 bp-template outside of this region resulted in a substantial loss of activity. Electron microscopic studies of in vitro DNA replication products from the highly active clones also revealed sigma-shaped molecules. These results support our in vivo data for the presence of a predominant replication origin between positions 628 and 776 on the plasmid map. This sequence shares homology with double-stranded rolling circle origin (dso) or transfer origin (oriT) nicking motifs from bacterial plasmids. mp1 is the first described rolling circle plasmid in eukaryotes.
Nucleoids were purified from chloroplasts of dividing soybean cells and their polypeptide composition analyzed by SDS-polyacrylamide gel electrophoresis. Of the 15-20 nucleoid-associated polypeptides, several demonstrated DNA binding activity. Upon disruption of the nucleoids with high concentrations of NaCl, a subset of these proteins and the majority of chloroplast DNA were recovered in the supernatant after centrifugation. Removal of the salt by dialysis resulted in formation of nucleoprotein complexes resembling genuine nucleoids. Purification of these structures revealed three major proteins of 68, 35 and 18 kDa. After purification of the 68 kDa protein to homogeneity, this protein was able to compact purified chloroplast DNA into a nucleoid-like structure in a protein concentration-dependent fashion. Addition of the 68 kDa protein to an in vitro chloroplast DNA replication system resulted in complete inhibition of nucleotide incorporation at concentrations above 300 ng of 68 kDa protein per microg of template DNA. These results led to in situ immunofluorescence studies of chloroplasts replicating DNA which suggested that newly synthesized DNA is not co-localized with nucleoids. Presumably, either the plastid replication machinery has means of removing nucleoid proteins prior to replication or the concentration of nucleoid proteins is tightly regulated and the proteins turned over in order to allow replication to proceed.
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.
In a previous study, we mapped replication origin regions of the plastid DNA around the 3' end of the 23S rRNA gene in rice suspension-cultured cells. Here, we examined initiation of the plastid DNA replication in different rice cells by two-dimensional agarose gel electrophoresis. We show for the first time, to our knowledge, that the replication origin region of the plastid DNA differs among cultured cells, coleoptiles and mature leaves. In addition, digestion of the replication intermediates from the rice cultured cells with mung bean nuclease, a single-strand-specific nuclease, revealed that both two single strands of the double-stranded parental DNA were simultaneously replicated in the origin region. This was further confirmed by two-dimensional agarose gel analysis with single-stranded RNA probes. Thus, the mode of plastid DNA replication presented here differs from the unidirectional replication started by forming displacement loops (D-loops), in which the two D-loops on the opposite strands expand toward each other and only one parental strand serves as a template.
Higher plant plastid DNA (ptDNA) is generally described as a double-stranded circular molecule of the size of the monomer of the plastid genome. Also, the substrates and products of ptDNA replication are generally assumed to be circular molecules. Linear or partly linear ptDNA molecules were detected in our present study using pulsed-field gel electrophoresis and Southern blotting of ptDNA restricted with 'single cutter' restriction enzymes. These linear DNA molecules show discrete end points which were mapped using appropriate probes. One possible explanation of discrete ends would be that they represent origins of replication. Indeed, some of the mapped ends correlate well with the known origins of replication of tobacco plastids, i.e. both of the oriA sequences and--less pronouncedly--with the oriB elements. Other ends correspond to replication origins that were described for Oenothera hookeri, Zea mays, Glycine max and Chlamydomonas reinhardtii, respectively, while some of the mapped ends were not described previously and might therefore represent additional origins of replication.
Chloroplasts contain multiple copies of a DNA molecule (the plastome) that encodes many of the gene products required to perform photosynthesis. The plastome is replicated by nuclear-encoded proteins and its copy number seems to be highly regulated by the cell in a tissue-specific and developmental manner. Our understanding of the biochemical mechanism by which the plastome is replicated and the molecular basis for its regulation is limited. In this commentary we review our present understanding of chloroplast DNA replication and examine current efforts to elucidate its mechanism at a molecular level.
Chloroplast DNAs (ctDNA) from pea and corn plants were examined in the electron microscope for the presence of replicative intermediates. Pea and corn ctDNAs were each found to contain two displacement loops (D-loops). The D-loops were 820 (+/- 90) base pairs long in pea ctDNA and 860 (+/- 125) base pairs long in corn ctDNA. In each ctDNA, the two D-loops were located at positions that were 7100 +/- 240) base pairs apart. The displacing strands of the two D-loops were located on opposite strands of the parental DNA molecule and they were seen to expand toward each other. The D-loops in the ctDNA from pea and corn exhibited branch migration and thus were easily distinguished from the denatured regions that were also present in these closed circular ctDNAs. In addition, the positions of the D-loops were found to be distinct from the positions of the denaturation loops (Den-loops). The Den-loops were also shown to be located at AT-rich regions in these ctDNA molecules. D-loops and Den-loops were also found in the circular and catenated ctDNA oligomers from pea and corn plants. Mapping the positions of the D-loops relative to the positions of the Den-loops showed that the structure of the D-loop-containing region in the pea and corn ctDNAs has been conserved to a greater extent than the structure of the rest of the two ctDNA molecules.
We have used a two-dimensional gel electrophoresis mapping technique to determine where DNA replication initiates on a plasmid
which utilizes a fragment of human DNA to replicate autonomously in human cells. Replication was found to initiate at multiple
locations on the plasmid carrying the human sequence, in contrast to the pattern seen for an Epstein-Barr virus vector which
served as a control with a fixed origin. The family of repeats, a portion of the Epstein-Barr virus origin of replication
which is present our plasmid, was shown to function as a replication fork barrier. The nature of the stalled replicative intermediates
on the human DNA-based plasmid further indicated that replication did not initiate at a single fixed position each time the
plasmid replicated. The results suggest that the replication apparatus used to duplicate DNA in human cells may not have precise
sequence requirements which target initiation to specific locations.
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.
Chloroplast DNA replication in Chlamydomonas reinhardtii is initiated by the formation of a displacement loop (D-loop) at a specific site. One D-loop site with its flanking sequence was cloned in recombinant plasmids SC3-1 and R-13. The sequence of the chloroplast DNA insert in SC3-1, which includes the 0.42-kilobase (kb) D-loop region, as well as 0.2 kb to the 5' end and 0.43 kb to the 3' end of the D-loop region, was determined. The sequence is A+T-rich and contains four large stem-loop stuctures. An open reading frame potentially coding for a polypeptide of 136 amino acids was detected in the D-loop region. One stem-loop structure and two back-to-back prokaryotic-type promoters were mapped within the open reading frame. The 5.5-kb EcoRI fragment cloned in R-13 contains the 1.05-kb SC3-1 insert and its flanking regions. A yeast autonomously replicating (ARS) sequence and an ARC sequence, which promotes autonomous replication in Chlamydomonas, have been mapped within the flanking regions [Vallet, J.-M. & Rochaix, J.-D. (1985) Curr. Genet. 9, 321-324]. Both R-13 and SC3-1 were active as templates in a crude algal preparation that supports DNA synthesis. In this in vitro system, chloroplast DNA synthesis initiated near the D-loop site.
The levels of chloroplast DNA in a cultured photoautotrophic soybean (Glycine max [L.] Merr. v Corsoy) cell line were determined by molecular hybridization. The cells were also grown photomixotrophically and heterotrophically as suspension cultures and the level of plastid DNA was found to be constant at approximately 26% of the total cellular DNA in all three growth modes. By comparison, total cellular DNA extracted from plants of the same variety used as the explant source for the cultured cells contained 12.3 to 18.9% (leaves and seeds) and 6.1 to 8.9% (roots) plastid DNA.
DNA polymerases were purified from chloroplasts and mitochondria of cultured Glycine max cells. The chloroplast enzyme exists in two forms which are indistinguishable from each other biochemically. All three organellar enzymes have an estimated molecular weight of 85,000 to 90,000 and prefer poly(rA)dT(12-18) over activated DNA as a template in vitro. Maximum activity of the chloroplast and mitochondrial DNA polymerases requires KCl and a reducing agent, and the enzymes are completely resistant to inhibitors of DNA polymerase alpha. Taken together, these properties classify the soybean organellar enzymes as DNA polymerases gamma. A unique feature that distinguishes the plant enzymes from their animal counterparts is their resistance to dideoxyribonucleotides.
The nucleotide sequence of a spacer region between 16S and 23S rRNA genes from soybean chloroplasts has been determined. The spacer region is over 3000 bp long and contains two tRNA genes, coding for rRNA(Ile) and tRNA(Ala) which contain intervening sequences of 953 and 811 base pairs respectively. There is a strong homology between the two introns suggesting that they have a common origin. These spacer tRNAs are synthesized as part of a kb precursor molecule containing 16S and 23S rRNA sequences.
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.
Chloroplast DNAs from soybean (Glycine max), common bean (Phaseolus vulgaris) and mung bean (Vigna radiata) have been compared in overall structure and nucleotide sequence homologies. Detailed restriction maps demonstrate that the soybean and common bean genomes possess the classical large chloroplast DNA inverted repeat, encoding ribosomal RNA genes, as found previously in mung bean (Palmer and Thompson 1981 a). Heterologous filter hybridizations indicate essentially complete colinearity between mung bean and common bean chloroplast DNAs. Although the linear order of sequence elements is also conserved between soybean and mung bean DNAs, two regions of deletions/additions, each totaling almost 5 kilobase pairs in size, have been identified at the ends of the large single copy DNA region.
Alignment and comparison of restriction maps has allowed calculation of nucleotide sequence divergence values for the three DNAs. Mung bean and soybean chloroplast DNAs differ by an average of 10–13% in nucleotide sequence, while mung bean and common bean are significantly more closely related, differing by only 5–6% in base sequence. Base substitutions are distributed non-randomly in these chloroplast DNAs; chloroplast ribosomal DNA is relatively conserved and the two deletion/addition regions relatively diverged in base sequence.
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.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
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.
The time course of replication of the 9 kb ribosomal DNA repeats of synchronized root cells of pea was followed by two-dimensional gel electrophoresis. The temporal order of appearance of single-stranded replication intermediates shows that replication begins within the subrepeats located in the intergenic spacer region about 1.5 kb downstream from the 3' end of the 25S gene. Hybridization to specific probes indicated that this location is identical to that established earlier by a different method.
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.
Two-dimensional (2D) agarose gel electrophoresis is progressively replacing electron microscopy as the technique of choice to map the initiation and termination sites for DNA replication. Two different versions were originally developed to analyze the replication of the yeast 2 microns plasmid. Neutral/Neutral (N/N) 2D agarose gel electrophoresis has subsequently been used to study the replication of other eukaryotic plasmids, viruses and chromosomal DNAs. In some cases, however, the results do not conform to the expected 2D gel patterns. In order to better understand this technique, we employed it to study the replication of the colE1-like plasmid, pBR322. This was the first time replicative intermediates from a unidirectionally replicated plasmid have been analyzed by means of N/N 2D agarose gel electrophoresis. The patterns obtained were significantly different from those obtained in the case of bidirectional replication. We showed that identification of a complete are corresponding to molecules containing an internal bubble is not sufficient to distinguish a symmetrically located bidirectional origin from an asymmetrically located unidirectional origin. We also showed that unidirectionally replicated fragments containing a stalled fork can produce a pattern with an inflection point. Finally, replication appeared to initiate at only some of the potential origins in each multimer of pBR322 DNA.
A DNA helicase activity was detected in extracts of purified chloroplasts from the SB-1 cell line of Glycine max and partially purified by column chromatography on DEAE cellulose, phosphocellulose, and single-stranded DNA cellulose. The chloroplast helicase has a DNA-dependent ATPase activity, and its strand displacement activity is strictly dependent upon the presence of a nucleoside triphosphate and Mg2+ or Mn2+. Strand displacement activity does not require a free unannealed single-strand or replication fork-like structure.
Replication of ribosomal DNA replicons in cells of Pisum sativum (cv. Alaska) occurs bidirectionally by displacement loops. Replication is initiated on opposite parental strands and nascent chains are elongated moving 5'----3' along each parental template. Replicative intermediates were analyzed by 2-dimensional agarose gel electrophoresis under neutral--neutral and neutral--alkaline conditions. Southern blots of ribosomal DNA fragments separated in the second dimension under neutral conditions show slowly migrating replicative fragments that hybridize with specific probes in a manner consistent with bidirectional replication. The replicative fragments are present in root meristems with cells in S phase; they are absent or few in number in meristems with cells in G2 phase. The following observations indicate that the replicative fragments are single stranded. The apparent length of the replicative fragments is not the same when separated under neutral and alkaline conditions. They contain rDNA without breaks and they do not exhibit the smaller nascent chains expected from replication bubbles and forks. They are not cleaved by restriction enzymes that require duplex DNA as substrate and they are digestible by S1 nuclease.
We have used two complementary two-dimensional gel electrophoretic methods to localize replication inititation sites and to determine replication fork direction in the amplified 240 kb dihydrofolate reductase domain of the methotrexate-resistant CHO cell line CHOC 400. Surprisingly, our analysis indicates that replication begins at many sites in several restriction fragments distributed throughout a previously defined 28 kb initiation locus, including a fragment containing a matrix attachment region. Initiation sites were not detected in regions lying upstream or downstream of this locus. Our results suggest that initiation reactions in mammalian chromosomal origins may be more complex than in the origins of simple microorganisms.
To study initiation of DNA replication in mammalian chromosomes, we have established a methotrexate-resistant Chinese hamster ovary cell line (CHOC 400) that contains approximately 1,000 copies of the early replicating dihydrofolate reductase (DHFR) domain. We have previously shown that DNA replication in the prevalent 243-kilobase (kb) amplicon type in this cell line initiates somewhere within a 28-kb region located downstream from the DHFR gene. In an attempt to localize the origin of replication with more precision, we blocked the progress of replication forks emanating from origins at the beginning of the S phase by the introduction of trioxsalen cross-links at 1- to 5-kb intervals in the parental double-stranded DNA. The small DNA fragments synthesized under these conditions (which should be centered around replication origins) were then used as hybridization probes on digests of cosmids and plasmids from the DHFR domain. These studies suggested that in cells synchronized by this regimen, DNA replication initiates at two separate sites within the previously defined 28-kb replication initiation locus, in general agreement with results described in the accompanying paper (T.-H. Leu and J. L. Hamlin, Mol. Cell. Biol. 9:523-531, 1989). One of these sites contains a repeated DNA sequence element that is found at or near many other initiation sites in the genome, since it was also highly enriched in the early replicating DNA isolated from cross-linked CHO cells that contain only two copies of the DHFR domain.
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.
Maize chloroplast DNA sequences representing 94% of the chromosome have been surveyed for their activity as autonomously replicating sequences in yeast and as templates for DNA synthesis in vitro by a partially purified chloroplast DNA polymerase. A maize chloroplast DNA region extending over about 9 kilobase pairs is especially active as a template for the DNA synthesis reaction. Fragments from within this region are much more active than DNA from elsewhere in the chromosome and 50- to 100-fold more active than DNA of the cloning vector pBR322. The smallest of the strongly active subfragments that we have studied, the 1368-base-pair EcoRI fragment x, has been sequenced and found to contain the coding region of chloroplast ribosomal protein L16. EcoRI fragment x shows sequence homology with a portion of the Chlamydomonas reinhardtii chloroplast chromosome that forms a displacement loop [Wang, X.-M., Chang, C.H., Waddell, J. & Wu, M. (1984) Nucleic Acids Res. 12, 3857-3872]. Maize chloroplast DNA fragments that permit autonomous replication of DNA in yeast are not active as templates for DNA synthesis in the in vitro assay. The template active region we have identified may represent one of the origins of replication of maize chloroplast DNA.
In our previous study of chloroplast (Cp) DNA replication in Chlamydomonas reinhardtii, one D-loop site with its flanking regions was cloned and sequenced. The D-loop site mapped by electron microscopy (EM) overlaps with an open reading frame (ORF) potentially coding for a polypeptide of 136 amino acids. In this report, the corresponding D-loop isolated from another species of Chlamydomonas was sequenced. An ORF was also detected. Sequence comparison indicated that most conserved sequences between these two cloned origins are located within the ORF. Amino acid sequences of these two ORFs are highly conserved. The corresponding sequence for this ORF in the tobacco Cp genome was located by a Southern blotting analysis. Since the complete sequence data of Cp DNAs from a liverwort and from tobacco have been determined in 2 Japanese laboratories recently, it has been possible for us to show that this ORF encodes a protein homologous to the Cp ribosomal protein (r-protein) L16, by sequence comparison.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
All of the PstI restriction fragments of the chloroplast DNA of Nicotiana tabacum have been cloned in the plasmid vector pBR322. The cloned fragment sizes range from 0.8 to 26 kb, are stable, and can be amplified by chloramphenicol with varying efficiencies. Using these clones we have detailed a PstI physical map of the tobacco chloroplast genome. Selected clones of SalI, BamHI and PstI fragments were used to localize the map positions of the alpha, beta, and epsilon subunits of the chloroplast ATPase coupling factor, the large subunit of ribulosediphosphate carboxylase and the 32-kDal membrane protein. The gene products of these clones were characterized by RNA transcript sizing, immunoprecipitation of maxicell-directed protein synthesis, and hybrid-arrested translation.
Chloroplast DNA, isolated from a synchronized culture of Chlamydomonas reinhardii , was digested with restriction endonucleases and examined in the electron microscope. Restriction fragments containing displacement
loops (D-loop) were photographed and measured to determine the position of replicated sequences in relation to the restriction
enzyme sites. D-loops were located at two positions on the physical map of chloroplast DNA. One replication origin was mapped
at about 10 kb upstream of the 5′ end of a 16s rRNA gene. The second origin was spaced 6.5kb apart from the first origin and
was about 16.5 kb upstream of the same 16s rRNA. Initiations at those two sites were not always synchronized. Replication
initiated with the formation of a D-loop resulting from the synthesis of one daughter strand. After a short initial lag phase,
corresponding to the synthesis of 350±130 bp of one daughter strand, DNA synthesis then proceeded in both directions. Both
D-loop regions were preferred binding sites of undetermined protein complexes.
Chloroplast DNA (cpDNA), containing 10% replicative molecules, was isolated 2 h after onset of the dark period from cultures of Euglena gracilis strain Z. The DNA was digested with the restriction enzymes PvuII, SalI, BamHI, or EcoRI. Fragments that contained intact replicative loops were measured to determine the position of replicated sequences in relation to the restriction enzyme sites. It was found that replication starts at a unique position near one of the palindromic sequences I(2) (Koller and Delius, 1982a) which is located upstream (with respect to the direction of rRNA transcription) of the AT-rich region of variable size (Jenni et al., 1981; Schlunegger et al., in preparation). In the majority of cases DNA synthesis proceeds unidirectionally away from this region for 5000 nucleotides before it starts in the other direction (in the same sense as the rRNA transcription) through the Z-region and the second palindromic sequence.
Highly chlorophyllous photomixotrophic callus was visually selected from callus originating from soybean (Glycine max (L.) Merr. var. Corsoy) cotyledon. Suspension cultures initiated from this callus became photoautotrophic under continuous light with an atmosphere of 5% CO2 (balance air). Dry weight increases of 1000 to 1400% in the 2-week subculture period have been observed. The cellular Chl content ranged from 4.4 to 5.9 micrograms per milligram dry weight which is about 75 to 90% of the Chl content in soybean leaves under equivalent illumination (300 micro-Einsteins per square meter per second).
No growth can be observed in the dark in sucrose-lacking medium or in the presence of 0.5 micromolar 3-(3,4-dichlorophenyl)-1,1-dimethylurea, a concentration which does not inhibit heterotrophic growth (on sucrose). Photoautotrophic growth has an absolute requirement for elevated CO2 concentrations (>1%). During the 14-day subculture period, growth (fresh weight and dry weight) is logarithmic. Photosynthesis quickly increases after day 4, reaching a peak of 83 micromoles CO2 incorporated per milligram Chl per hour while dark respiration decreases 90% from day 2 to day 6. The pH of the growth medium quickly drops from 7.0 to 4.5 before slowly increasing to 5.0 by day 14. At this pH range and light intensity (200-300 microEinsteins per square meter per second), no O2 evolution could be detected although at high pH and light intensity O2 evolution was recorded.
Absolute DNA amounts of individual chloroplasts from mesophyll and epidermal cells of developing spinach leaves were measured by microspectrofluorometry using the DNA-specific stain, 4,6-diamidino-2-phenyl indole, and the bacterium, Pediococcus damnosus, as an internal standard. Values obtained by this method showed that DNA amounts of individual chloroplasts from mesophyll cells fell within a normal distribution curve, although mean DNA amounts changed during leaf development and also differed from the levels in epidermal chloroplasts. There was no evidence in the data of plastids containing either the high or low levels of DNA which would be indicative of discontinuous polyploidy of plastids, or of division occurring in only a small subpopulation of chloroplasts. By contrast, the distribution of nuclear DNA amounts in the same leaf tissues in which cell division was known to be occurring showed a clear bimodal distribution. We consider that the distribution of chloroplast DNA in the plastid population shows that there is no S-phase of chloroplast DNA synthesis, all chloroplasts in the population in young leaf cells synthesize DNA, and all chloroplasts divide.
CuticchiaAJ: Localization of a R-protein gene within the chloroplast DNA replica-tion origin of Chlamydomonas
- Lou Jk M Wu
- Chang
- Ch
Lou JK, Wu M, Chang CH, CuticchiaAJ: Localization of a R-protein gene within the chloroplast DNA replica-tion origin of Chlamydomonas. Curr Genet 11:537-541 (1987).
JM: Photoautotrophic growth of soybean cells in suspension culture
- M E Horn
- J J Sherrard
- ME Horn
S: Partial purification and characterization of a DNA helicase from chloroplasts of Glycine max
- G C Cannon
- GC Cannon