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© 1995 Oxford University Press 3681-3686

Fine mapping of replication origins (oriA and oriB) in Nicotiana tabacum chloroplast DNA

Fine mapping of replication origins ( ori A and ori B) in Nicotiana tabacum chloroplast DNA Muthusamy Kunnimalaiyaan+ and Brent L. Nielsen*

Department of Botany and Microbiology, Auburn University, Auburn, AL 36849, USA

Received May 12, 1997; Revised and Accepted July 25, 1997DDBJ/EMBL/GenBank accession no. Z00044

ABSTRACT

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 oriA and oriB 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 oris (pKN9) was tested. DpnI 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 (oriA or oriB), whereas D loops were observed for a clone (pKN9) containing both oris. 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.

INTRODUCTION

Photosynthesis and the biosynthesis of many important metabolites occur in chloroplasts. Some of the genes for these functions are encoded in chloroplast DNA (ctDNA). Although many aspects of chloroplast gene sequence, structure and gene expression are very similar to bacterial genes, ctDNA replication is unique as compared with replication in other systems. Relatively little is known about the molecular mechanism of ctDNA replication, although a general mechanism for ctDNA replication is known (1 ). According to the existing model ctDNA replication begins by the introduction of two displacement loops (D loops) 6-7 kbp apart in the ctDNA (1 ,2 ). Each D loop is generated by unidirectional replication from an origin, resulting in displacement of a single-stranded region. The D loops expand toward each other and fuse to form a Cairns structure, with replication proceeding bidirectionally around the ctDNA. After the daughter molecules are synthesized replication may continue by a rolling circle mechanism. D loops of 800-900 bp were found in 15-30% of the total number of intact supercoiled ctDNA molecules examined by electron microscopy (for reviews on ctDNA replication see 2 -4 ). It has been reported that the number and location of ctDNA replication origins may vary in different species (2 ). Differences in location of ctDNA replication origins may be expected as the overall size and organization of the genome varies from lower organisms to higher plants. Most plant species show low sequence conservation in some intergenic regions and most reports on ctDNA replication origins place them in intergenic regions.

Two-dimensional (2D) agarose gel electrophoresis has been successfully employed to examine in vivo and in vitro replication intermediates in several systems (5 -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 ). Four oris have been reported for Oenothera due to their location as identical pairs in each IR (12 ). The location of oriA may be conserved, as the sequence of this region shows a high degree of homology among species (2 ,13 ), whereas oriB shows homology only between tobacco and petunia among the sequenced genomes. The identification of minimal ori sequences and controlling elements may provide the basis for producing a replicating vector for chloroplast genetic engineering. Although such vectors may not be stable after introduction into chloroplasts, their copy number may increase by replication to facilitate homologous recombination into the chloroplast genome. Hence, an understanding of the molecular biology of ctDNA replication may help to address problems related to stable transformation of the chloroplast organelle. In order to achieve this it is essential to determine the minimal sequence elements required for replication. In addition, the development of a reliable in vitro ctDNA replication system will allow study of the role of individual proteins in replication. In this study we report identification of the minimal sequences of oriA (82 bp) and oriB (243 bp) required for in vitro replication of tobacco ctDNA by2D gel electrophoresis using a partially purified chloroplast replication fraction. We demonstrate herein the specificity of the replication fraction by DpnI and other control assays. Electron microscopy has been used to examine the mode of replication in vitro.

MATERIALS AND METHODS

Purification of the replication fraction

Two to three week old tobacco leaves were used to isolate chloroplasts. A Triton lysate mixture was prepared and the replication fraction was purified as described (14 -16 ), except for addition of a phosphocellulose column after the DEAE-cellulose column and bound proteins were step eluted for all the columns used. Briefly, the Triton lysate was loaded onto a 60 ml pre-equilibrated DEAE-cellulose (DE-52; Whatman) column. Equlibration was with buffer A (50 mM Tris-HCl, pH 8.0, 25% glycerol, 10 mM B-mercaptoethanol and protease inhibitors: 10 mM benzamide, 1 mM PMSF, 10 mM sodium metabisulfite) containing 50 mM KCl. After loading the column was washed extensively with ~10 column vol. buffer A + 50 mM KCl. 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. The dialyzed fraction was loaded onto a 20 ml activated phosphocellulose (Whatman) column equilibrated with buffer B. The column was washed extensively with buffer B and the bound proteins were collected as 8 ml fractions with a step elution of 400 mM phosphate buffer B. Fractions containing DNA polymerase activity were dialyzed against buffer C (50 mM Tris-HCl, pH 8.0, 25% glycerol, 1 mM MgCl2, 1 mM EDTA, pH 8.0, 1 mM B-mercaptoethanol and protease inhibitors: 10 mM benzamide, 1 mM PMSF, 10 mM sodium metabisulfite) containing 100 mM KCl. The dialyzed fraction was loaded onto a 10 ml column of heparin-Sepharose (Pharmacia) equilibrated with the same buffer. After thorough washing with buffer B containing 100 mM KCl the bound proteins were collected as 5 ml fractions by step elution with buffer B containing 600 mM KCl. DNA polymerase activity was assayed for each fraction. Active fractions were tested further for replication activity with specific ctDNA clones as described (10 ). Fractions which showed high DNA polymerase activity and low nuclease activity were used for further studies.

In vitro DNA replication assay

In vitro DNA replication reactions were carried out as described (13 ) with various tobacco ctDNA clones (Fig. 2 ). Smaller clones were made by PCR using specific primers, with cloning into the SmaI site of pUC19. These clones were confirmed by standard sequence analysis to ensure the correct sequence. After initial comparison by [3H]TTP incorporation in vitro replication products labeled with [32P]dCTP were prepared and analyzed by 2D gel electrophoresis as described (11 ).

DpnI assay

Radiolabeled in vitro replication products of different clones were restricted with DpnI, which requires methylation of the A nucleotide in the recognition sequence for restriction. Reaction products were separated in a 1% agarose gel. After electrophoresis the gel was soaked in 5% TCA for 30 min, dried and exposed to X-ray film.

Electron microscopy

DNA spreads were done according to the Davis and Davidson method for electron microscopy (17 ). One hundred nanograms of purified in vitro replication product were mixed in a solution containing 0.2 M ammonium acetate, pH 5.5, 0.001 M EDTA, pH 7.5, and 0.0003% (w/v) cytochrome c (type VI; Sigma). A droplet of 250 [mu]l was formed and spread DNA was picked up on a Parlodion support film on 400 mesh copper grids. DNA was stained with uranyl acetate (0.1 mM uranyl acetate in 90% ethanol) by adding 5 [mu]l to the grid for 30 s. Grids were then dipped in 95% ethanol for 5 s, air dried and rotary shadowed at an angle of 5o with gold/palladium (60%/40%). The grids were examined with a Zeiss transmission electron microscope. Photographs were taken at various magnifications and lengths were measured using an Optimas image analyzer system.

RESULTS

Purification of the chloroplast replication complex

The progress of purification of the replication complex as measured by DNA polymerase activity through various chromatographic steps is shown in Table 1 . Since the activity of the protein was underestimated in the Triton lysate due to the presence of high amounts of nucleases, the specific activity has been calculated starting from the DEAE-cellulose (DE-52) column. Though the DE-52 fraction showed DNA polymerase activity, the fraction showed some nuclease activity when incubated with DNA (data not shown). Most of the nucleases were apparently removed in later column steps. When the heparin-Sepharose column was eluted with a linear gradient of 100 mM-1 M KCl in buffer C, DNA polymerase activity was found in the latter fractions, whereas DNA binding proteins eluted in earlier fractions (data not shown). Hence, in these experiments we used a step elution for each column in order to retain necessary replication activities in the same fraction.

Table 1 . Purification steps for the replication complex from tobacco chloroplasts
Source Protein (mg) Volume (ml) Specific activitya
Triton lysate 402 150 Not determined
DEAE-cellulose 26.8 70 60.38
Phosphocellulose 10.5 32 805
Heparin-Sepharose 0.73 15 7647.8
aOne unit of DNA polymerase activity is defined as the amount of enzyme that will catalyze the polymerization of 1 [mu]mol [3H]TTP using activated calf thymus DNA as template at 37oC in 30 min. Specific activity is expressed as units DNA polymerase/mg protein.

2D gel analysis of replication intermediates


Figure 1. Schematic representation of replication intermediate patterns observed by 2D gel electrophoresis (based on refs 5,8). Linear monomer and dimer spots, the connecting diagonal of broken linear molecules and replication intermediate patterns are indicated. The E arc represents atypical patterns expected for D loops due to the inability of restriction enzymes to digest single-stranded regions.

Neutral/neutral 2D agarose gel electrophoresis (5 ,11 ,18 ) was used to detect and analyze replication intermediates from in vitro reactions. A schematic diagram for established patterns generated by this technique is shown in Figure 1 . A theoretical discussion of the type of patterns which may be observed by 2D gel electrophoresis of D loop-containing molecules has recently been reported (8 ) and includes a proposed extended arc (E arc) pattern (Fig. 1 ). E arcs show atypical patterns as a result of the inability of restriction enzymes to cleave single strands and contain long single-stranded regions joined to double-stranded DNA at different locations relative to the ends of the linearized fragments, making it difficult to predict the pattern which would be generated (8 ). Specific tobacco subclones (Fig. 2 ) were tested as templates for in vitro DNA replication using the partially purified tobacco chloroplast fraction and the results are shown in Figures 3 -5 . Only simple Y replication patterns along with X-shaped recombination patterns are expected for small clones containing a single D loop region (see Discussion), as seen in Figures 3 and 4 . For most templates a continuous diagonal signal representing broken linear molecules was also observed. The 507 bp template pKN1 from oriB exhibited a high level of in vitro DNA replication activity as detected by the generation of a strong simple Y pattern from the linearized product (Fig. 3 A). However, the 353 bp pKN2 clone, which lacks 154 bp containing upstream sequences and most of the stem-loop structure at one end of pKN1, was found to support little or no replication activity (Fig. 3 B), whereas the 243 bp PCR clone pKN3, which contains the complete stem-loop structure, showed a strong simple Y pattern (Fig. 3 C). Similarly, the pKN5 template from oriA, which contains a 1.3 kb insert, showed high replication activity and showed the expected simple Y pattern in the 2D gel (Fig. 4 A). However, for smaller subclones from pKN5, such as pKN6, pKN7 and pKN8 (Fig. 4 B, C and D respectively), there is some variation in the intensity of the signal though the input amount of template DNA in each reaction was identical. The exposure time for pKN7 was 4 days longer than for pKN6 and pKN8. The 82 bp pKN8 clone, which spans only the stem-loop structure and minimal flanking sequences, showed very high activity as compared with the other templates. No replication intermediates were detected for a clone (pKN4) from a region between the two oris (Fig. 3 D) or for pKN1 incubated with either Klenow fragment (Fig. 3 E) or with DNA polymerase I (Fig. 3 F) from Escherichia coli instead of the chloroplast replication fraction complex. These results suggest that the stem-loop sequences in oriA and oriB (10 ,11 ) play a necessary role for replication in vitro and that the patterns observed are not due to repair synthesis.


Figure 2. Map of the 23S rRNA gene region including flanking regions and clones used in this study. Coordinates indicate tobacco ctDNA sequences from IRA (28); these sequences are duplicated in IRB.


Figure 3. In vitro replication products were analyzed by 2D gel electrophoresis as described in Materials and Methods. For each template products were linearized with PstI. (A) pKN1, (B) pKN2, (C) pKN3 and (D) pKN4. As controls in vitro reactions were performed with pKN1 using E.coli Klenow fragment (E) or DNA polymerase I (F), instead of the chloroplast replication fraction. Electrophoresis in the first dimension is from left to right and the second dimension is from top to bottom. Each gel was exposed to X-ray film for 3 days.


Figure 4. 2D gel analysis of in vitro replication products for oriA templates. (A) pKN5, (B) pKN6, (C) pKN7 and (D) pKN8. Conditions were the same as in Figure 3, except that the gel in (C) was exposed to X-ray film for 7 days.

In order to determine what type of 2D gel pattern would be generated for a clone containing both oris together, template pKN9 was constructed. This was done by inserting the 4.5 kb EcoRI fragment from oriA into the EcoRI site of pKN1. The resultant pKN9 template was restricted with SacI after the in vitro reaction for 2D gel analysis. This clone showed a high level of in vitro replication activity (Fig. 5 ). A double Y pattern will be generated if two replication forks are converging from both ends of the fragment, such as may occur after the two expanding D loops have fused to form a Cairns intermediate (18 ). SacI restriction of pKN9 gives two bands (3.1 kb containing oriB and 4.5 kb containing oriA) and two steep straight diagonal lines similar to double Y patterns are observed by 2D gel analysis (small arrowhead in Fig. 5 ). In addition to the double Y, a pattern somewhat similar to replication bubbles but extending past dimer size was observed (large arrowhead in Fig. 5 ). This atypical pattern may include an E arc, as suggested by Han and Stachow (8 ), representing a large bubble structure not restricted by the enzyme, which may occur prior to fusion of the D loops.

Specificity of the in vitro reaction

To ensure that the purified replication complex is specific to the presence of ori sequences DpnI assays were carried out after the in vitro reactions (Fig. 6 ). The restriction endonuclease DpnI recognizes the sequence GATC and restricts between the A and T nucleotide positions only if the A is methylated in both DNA strands. During in vitro replication the newly synthesized strand is not methylated. Hence, DpnI cannot restrict the newly synthesized strand. From Figure 6 it is clear that in vitro replication products from the pKN1, pKN6, pKN7 and pKN8 (lanes 1, 4, 7 and 9 of Fig. 6 A and B) templates showed resistance to DpnI, whereas pUC19 and pKN1 incubated with Klenow fragment (data not shown) and the non-ori clones (Fig. 6 , lane 5) were completely restricted. For oriB pKN1 showed complete resistance to DpnI, while pKN2 showed complete digestion by DpnI, reflecting the same specificity as observed by 2D gel analysis. For oriA clones some DNA is completely restricted, suggesting that only a portion of the template molecules are replicated in vitro. Some radioactive incorporation is observed with all clones, suggesting a certain level of random incorporation. This assay, coupled with assays using Klenow fragment or DNA polymerase I as discussed in the previous section, is a powerful tool to detect specific replication activity.


Figure 5. In vitro replication products of clone pKN9 containing both oris were analyzed by 2D gel electrophoresis after SacI restriction. The small arrowhead points to the double Y pattern, whereas the large arrowhead points to the bubble-like pattern. Conditions are as in Figure 3.


Figure 6. DpnI assays of in vitro replication products to distinguish replication from repair activity. (A) Ethidium bromide stained agarose gel of DpnI- restricted reaction products. (B) Autoradiograph of the same gel. Lane 1, pKN1; lane 2, pKN2; lane 3, cold pKN1; lane 4, pKN6; lane 5, pKN4; lane 6, cold pKN6; lane 7, pKN7; lane 8, cold pKN8; lane 9, pKN8. Clones in lanes 3, 6 and 8 were not subjected to the in vitro reactions, as controls, and show complete digestion. Lane m, 1 kb ladder as a molecular weight marker.

Electron microscopic analysis

In vitro replication products of different clones with unlabeled dNTPS were processed for electron microscopic observation. Clones containing a single ori (either oriA or oriB) formed [sigma]-like structures (Fig. 7 A and B), indicative of rolling circle replication. The length of the tail from the [sigma] structure reflects the extent of replication, with some longer than unit length. Reaction products with templates carrying non-ori sequences (Fig. 7 C) or clones having an ori sequence but treated with no protein (data not shown) showed only circular molecules. Clones containing both oris (pKN9) showed the presence of D loop structures (Fig. 7 D). Observation of a D loop structure suggests that the mode of replication of pKN9 is different from other clones containing only one ori and having tails, as suggested by 2D gel analysis.

DISCUSSION

Two-dimensional agarose gel electrophoresis is a powerful technique to identify replication intermediates and has been utilized in various systems (see Introduction). 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. We report here for the first time a reliable and reproducible method to isolate a chloroplast replication complex and show the capability of this complex to distinguish between true replication and non-specific or repair DNA synthesis in vitro. The availability of a faithful in vitro ctDNA replication system will facilitate further study of the role of individual proteins in replication.


Figure 7. Electron microscopy analysis of in vitro ctDNA replication products. (A) Clone pKN1 (oriB) shows a rolling circle replication intermediate. (B) Clone pKN8 (oriA) shows a rolling circle replication intermediate. (C) A control template (pKN2) shows only circular DNA. (D) Clone pKN9 containing both oris shows a D loop intermediate. Open arrows indicate tails whereas the filled arrow shows the D loop. Bar 1 kb.


Figure 8. Stem-loop structure-forming regions of oriA (A) and oriB (B) from tobacco chloroplast DNA. The long horizontal arrows on top of the sequence indicate the stem-loop-forming structures. The large open arrows indicate the direction of replication from oriA and oriB. Pertinent restriction sites are shown and direct repeat elements are underlined. The ends of the minimal ori sequences from in vitro replication experiments are indicated by small vertical arrowheads (82 bp for oriA and 243 bp for oriB). Sequence coordinates for IRA are shown at each end of the ori sequences from the published tobacco ctDNA sequence (28; EMBL accession no. Z00044). These sequences are also present in IRB.

We have earlier reported the identification and localization of ori regions in the IR of tobacco ctDNA (10 ,11 ). In this study we describe detailed in vitro 2D gel analysis of ori regions of tobacco ctDNA using a partially purified tobacco chloroplast DNA replication complex. This fraction contains DNA polymerase, topoisomerases, DNA primase, single-stranded DNA endonuclease and helicase, which have been identified and purified to homogeneity from pea chloroplasts (16 ,19 -23 ). Recently a 43 kDa DNA binding protein from pea chloroplasts has been purified which exhibits non-specific DNA binding and stimulates ctDNA polymerase activity (24 ). Analysis by 2D gel electrophoresis of in vitro replication products from oriA or oriB clones (pKN1, pKN2, pKN3, pKN5, pKN6, pKN7 and pKN8) showed simple Y patterns (Fig. 4 ). However, clones containing non-ori sequences (pKN4) did not produce any simple Y pattern (Fig. 3 D). Only simple Y patterns are expected for clones containing a single ctDNA replication origin. As shown earlier (10 ,11 ), such clones replicate by a rolling circle mechanism (25 ).

Initial knowledge about the mechanism of ctDNA replication was based on electron microscopic examination of replication intermediates and the occurrence of D loops. We were interested to know the mechanism of in vitro replication of the clones containing one or both oris by electron microscopic studies. Electron microscopic analysis of in vitro replication products showed circular molecules with tails for single ori sequence- containing clones, whereas only circular molecules were seen for non-ori sequence clones (Fig. 7 A-C). The observation of D loops in pKN9 (Fig. 7 D) and rolling circle intermediates with clones containing either ori (oriA or oriB) alone suggest that the mechanism of ctDNA replication in vitro and in vivo are the same. However, it is not known what causes these changes in the mode of replication. Apparently the distance between the two oris is important to determine the mode of replication used. Further experiments are in progress to examine this.

In order to demonstrate the specificity of the replication fraction we carried out DpnI resistance assays. A DpnI resistance assay has recently been used to characterize autonomous replication of plasmids in chicken cells (26 ). DpnI restricts pUC19, which is our basic vector for cloning different ctDNA inserts, into 16 fragments, of which the largest is 955 bp. In addition, the insert from pKN1 (oriB) contains six recognition sites, whereas the insert in pKN8 (oriA) contains three sites for DpnI. If the newly synthesized DNA is not methylated during the in vitro replication reaction, the template will not be completely restricted by DpnI. Specific ori sequence clones did not show complete restriction (Fig. 6 , lanes 1, 4, 7 and 9). However, the non-ori clone pKN4, which contains two DpnI sites in the insert in addition to the sites in the vector, showed complete restriction (Fig. 6 , lane 5), although some radioactive label was incorporated. As a control experiment we used template DNA not subjected to the in vitro reaction, which showed complete restriction (Fig. 6 A, lanes 3, 6 and 8). Thus it is clear that in vitro DNA synthesis by the chloroplast fraction represents true replication rather than DNA repair synthesis. Although we cannot rule out the possibility that the fraction contains some DNA repair synthesis activity (i.e. the monomer spot in Fig. 3 D and incorporation of radioactivity in the non-ori sequence template in Fig. 6 B, lane 5), it is clear that the replication fraction contains specific replication activity for the ori sequences, for the following reasons. (i) In vitro replication reactions using either E.coli Klenow fragment or DNA polymerase I in place of the chloroplast replication fraction did not show any simple Y structure by 2D gel analysis (Fig. 3 E and F) though the template pKN1 contains oriB sequences. Similarly, the oriA clone pKN8 also did not show any simple Y pattern with either of the E.coli enzymes (data not shown). (ii) In vitro replication reactions using the chloroplast replication fraction with non-ori sequence clones showed only a monomer spot by 2D gel analysis (Fig. 3 D) and these products were restricted completely by DpnI (Fig. 6 , lane 5), reflecting random repair incorporation.

The smallest oriA clone which showed high in vitro replication activity, pKN8, contains 82 bp (137 683-137 764) and is ~1.2 kb from the reported autonomously replicating NICE1 sequence (27 ). Interestingly, the 82 bp sequence reported here contains the entire stem-loop structure and the two 8 bp direct repeats identified earlier (Fig. 8 A; see 10 ). It has been observed that some protein(s) from the chloroplast fraction binds specifically to this region (Kunnimalaiyaan et al., unpublished observation). Analysis of in vitro DNA replication products from a set of tobacco ctDNA subclones (Fig. 3 A-C) by 2D gel electrophoresis indicated that the 154 bp region from the SspI site at the end of the IR to the second BamHI site (positions 130 502-130 656) is essential for replication activity. For oriB minimal required sequences were localized to 243 bp (Fig. 8 B). Though the oriBclones pKN1 and pKN3 showed high levels of in vitro replication activity, the level of activity was less than that obtained with a clone from the oriA region (pKN7, compare Fig. 3 A and C with 4 D). This may suggest that oriA is more active than oriB for initiation of replication. Further studies are needed to determine what activates replication from each of these two oris. Multiple binding sites for sequence-specific ctDNA binding proteins in pKN1 and pKN3 have been reported (11 ). In earlier studies we found that this region has a relatively strong stem-loop structure, four nearly identical 9 bp AT-rich direct repeats and two exact copies of a different 9 bp sequence (Fig. 8 B; see 11 ). These sequences are not found elsewhere in the IR. Our next step in this area of research is to identify and purify the ori-specific binding protein(s) and preliminary experiments are in progress in our laboratory. In addition, the minimal ori sequences are potential candidates for construction of vectors for chloroplast transformation.

ACKNOWLEDGEMENTS

The authors are thankful to Selvi Kunnimalaiyaan for technical assistance and Medha Gandhi and Lance Eckerle for their help in EM analysis. This work was supported by a grant from the USDA (95-37301-2079) and by the Alabama Agricultural Experiment Station.

REFERENCES

1 Kolodner,R.D. and Tewari,K.K. (1975) Nature (Lond.), 256, 708-711 MEDLINE Abstract

2 Kunnimalaiyaan,M. and Nielsen,B.L. (1997) J. Plant Biochem. Biotechnol., 6, 1-7.

3 Tewari,K.K. (1988) In Bryant,J.A and Dunham,V.L. (eds), DNA Replication in Plants. CRC Press, Boca Raton, FL, pp. 69-116.

4 Heinhorst,S. and Cannon,G.C. (1993) J. Cell Sci., 104, 1-9.

5 Brewer,B.J., Sena,E.P. and Fangman,W.L. (1988) Cancer Cells, 6, 229-234.

6 Dijkwel,P.A., Vaughn,J.P. and Hamlin,J.L. (1991) Mol. Cell. Biol., 11, 3850-3859. MEDLINE Abstract

7 Martin-Parras,L., Hernandez,P., Martinez-Robles,M.L. and Schvartzman,J.B. (1992) J. Biol. Chem., 267, 22496-22505. MEDLINE Abstract

8 Han,Z. and Stachow,C. (1994) Chromosoma, 103, 162-170. MEDLINE Abstract

9 Hedrick,L.A., Heinhorst,S., White,M.A. and Cannon,G.C. (1993) Plant Mol. Biol., 23, 779-792. MEDLINE Abstract

10 Lu,Z., Kunnimalaiyaan,M. and Nielsen,B.L. (1996) Plant Mol. Biol., 32, 693-706. MEDLINE Abstract

11 Kunnimalaiyaan,M., Shi.,F. and Nielsen,B.L. (1997) J. Mol. Biol.,268, 273-283 MEDLINE Abstract

12 Chiu,W.-L. and Sears,B.B. (1992) Mol. Gen. Genet., 232, 33-39.

13 Nielsen,B.L., Lu,Z. and Tewari,K.K. (1993) Plasmid, 30, 197-211. MEDLINE Abstract

14 Meeker,R., Nielsen,B.L. and Tewari,K.K. (1988) Mol. Cell. Biol., 8, 1216-1223.

15 Carrillo,N. and Bogorad,L. (1988) Nucleic Acids Res.,16, 5603-5620. MEDLINE Abstract

16 Nielsen,B.L., Rajasekher,V.K. and Tewari,K.K. (1991) Plant Mol. Biol., 16, 1019-1034. MEDLINE Abstract

17 Davis,R.W. and Davidson,N. (1968) Proc. Natl. Acad. Sci. USA, 60, 243-250. MEDLINE Abstract

18 Brewer,B.J. and Fangman,W.L. (1988) Cell, 55, 637-643. MEDLINE Abstract

19 McKown,R.L. and Tewari,K.K. (1984) Proc. Natl. Acad. Sci. USA, 81, 2354-2358.

20 Nielsen,B.L. and Tewari,K.K. (1988) Plant Mol. Biol., 11, 3-14.

21 Mukherjee,S.K., Reddy,M.K., Kumar,D. and Tewari,K.K. (1994) J. Biol. Chem., 269, 3793-3801. MEDLINE Abstract

22 Kumar,D., Mukherjee,S.K., Reddy,M.K. and Tewari,K.K. (1995) J. Exp. Bot., 46, 767-776.

23 Tuteja,N., Phan,T.N. and Tewari,K.K. (1996) Eur. J.Biochem., 238, 54-63.

24 Chen,W., Gaikwad,A., Mukherjee,S.K., Choudhury,N.R., Kumar,D. and Tewari,K.K. (1996) Nucleic Acids Res., 24, 3953-3961. MEDLINE Abstract

25 Reddy,M.K., Choudhury,N.R., Kumar,D., Mukherjee,S.K. and Tewari,K.K. (1994) Eur. J. Biochem., 220, 933-941.

26 Bogdanova,A.N., Razin,S.V. and Vassetzky,Y.S. (1995) Biochemie, 77, 880-887.

27 Staub,J. and Maliga,P. (1994) Proc. Natl. Acad. Sci. USA, 91, 7468-7472.

28 Shinozaki,K., Ohme,M., Tanaka,M., Wakasugi,T., Hayashida,N., Matsubayashi,T., Zaita,N., Chunwongse,J., Obokata,J., Yamaguchi-Shinozaki,K., Ohto,C., Torazawa,K., Meng,B.Y., Sugita,M., Deno,H., Kamogashira,T., Yamada,K., Kusuda,J., Takaiwa,F., Kato,A., Tohdoh,N., Shimada,H. and Suguira,M. (1986) EMBO J., 5, 2043-2049.

*To whom correspondence should be addressed. Tel: +1 334 844 1671; Fax: +1 334 844 1645; Email: bnielsen@acesag.auburn.edu

+Present address: 307 Montgomery Hall, Department of Biology, Northern Illinois University, DeKalb, IL 60115, USA
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D. Wang, Y.-W. Wu, A. C.-C. Shih, C.-S. Wu, Y.-N. Wang, and S.-M. Chaw
Transfer of Chloroplast Genomic DNA to Mitochondrial Genome Occurred At Least 300 MYA
Mol. Biol. Evol., September 1, 2007; 24(9): 2040 - 2048.
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 Nucleic Acids ResHome page
S. Y. Jeong, A. Rose, and I. Meier
MFP1 is a thylakoid-associated, nucleoid-binding protein with a coiled-coil structure
Nucleic Acids Res., September 1, 2003; 31(17): 5175 - 5185.
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 Mol Biol EvolHome page
C. Schmitz-Linneweber, R. Regel, T. G. Du, H. Hupfer, R. G. Herrmann, and R. M. Maier
The Plastid Chromosome of Atropa belladonna and its Comparison with that of Nicotiana tabacum: The Role of RNA Editing in Generating Divergence in the Process of Plant Speciation
Mol. Biol. Evol., September 1, 2002; 19(9): 1602 - 1612.
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 Plant CellHome page
J. W. Lilly, M. J. Havey, S. A. Jackson, and J. Jiang
Cytogenomic Analyses Reveal the Structural Plasticity of the Chloroplast Genome in Higher Plants
PLANT CELL, February 1, 2001; 13(2): 245 - 254.
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