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Original Paper Biotechnotogy, 19 (1), 27- 35 (2002) Mapping Major Replication Origins on the Rice DNA

Ying WANG1, Kohya TAMURA2, Yasushi SAITOHl'2, Tadashi SAT03 Soh HIDAKA4 and Ken- ichi TSUTSUM11,2,*

lUnited Graduate School ofAgricultural Sciences and -~Cryobiosystem Research Center, lwate University, Ueda, Morioka. Iwate 020- 8550, Japan 3Department of Ecology and Evolutionary Biology, Graduate School ofLlfe Science, Tohoku University, Katahira. Sendai, Miyagi 980- 8577, Japan dDepartment of Crop Breeding, National Agricultural Research Center for Tohoku Region, Shimokuriyagawa, Morioka, Iwate 020-0123, Japan. *Corresponding author E-mail address: kentsu@iwate- u.ac.jp Received 5september 2001; accepted 15 october 2001

Abstract

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 - Iabeled with fractionated. of these strands the bromodeoxyuridine were isolated and size - Locations nascent on ptDNA determined the two major origin regions around the 3' region of each 23S rDNA in the inverted gel electrophoresis of the replication intermediates repeats (IRA and IRB). Two - dimensional agarose suggested that replication from each origin proceeds bidirectionally. This contrasted to replication by the

double D - Ioop mechanism.

gel Keywords: plastid DNA, replication, replication origin, rice, two - dimensional agarose electrophoresis.

understood. Abbreviations As to initiation of ptDNA, several experimental BND, benzoylated-naphthoylated DEAE; BrdU, approaches have been made. Analysis of electron 5-brom0-2-deoxyuridine; D-loop, displacement microscopic (EM) images of pea ptDNA revealed 100p; EM, electron microscopy; EtBr, ethidium two regions with displacement loop (D- Ioop) Iocat- bromide; IR.~, inverted repeat A; IR~, inverted ing on opposite strand, which was thought to be a Tewari, repeat B; LSC, Iarge single copy; SSC, small single replication intermediate (Kolodner and 1975). extended toward each copy; ptDNA, plastid DNA; rDNA, rRNA . These two D-loops other until replication reaches the initiation sites of Introduction the D-loop on opposite strands, then Cairns-type (theta structure) replication starts (Kolodner and Therefore, initiation sites of are plant organelles derived from a pro- Tewari, 1975). D- genitor proplastid, and give rise to various orga- loops have been considered as initiation sites nelles including , , (origins) of unidirectional replication of ptDNA. amyloplast and under control of the host Origins of this type of replication have been re- cells. To maintain and to differentiate into various ported for several plant and algal species (Waddell plastid lineages, replication of the plastid DNA et al., 1984; Chiu and Sears, 1992; Kunnimalai),aan (ptDNA) and division of the plastids must take and Nielsen, 1997b). for place. Recent reports (Osteryoung et al., 1998; Different approaches have also been made vitro repli- Colletti et aL, 2000) showed that division of the mapping origins of ptDNA, including in plastid chloroplast extracts (Gold et plastids occurs, in principle, Iike that of E. coli, in cation using or that plant homologues of bacterial division al., 1987; Carrillo and Bogorad, 1988; Hedrick et proteins FtsZ and MinD play important roles. al., 1993; Reddy et al., 1994), two-dimensional Replication of ptDNA, however, has been less agarose gel (2D gel) analysis of replication interme- 28

diates (Hedrick et al. 1993; Nielsen et al. 1993; Lu Heinhorst et al. (1990), and extracted , , ptDNA was et al., 1996; Kunnimalaiyaan et al., 1997), and according to the method described by Hirai et al. functional analysis of mutant ptDNA (Day and (1985). In some cases, ptDNA was further purified E1lis, 1984). However, different experimental ap- through cesium chloride density-gradient centrif- proaches did not always result in the location ugation (Zhao et al. 1997). same , of the origins. For example, soybean origin regions determined by in vitro replication did not all corre- Labeling with BrdU and immuno-detection of the spond to the origins determined by 2D gel analysis BrdU- Iabeled DNA (Hedrick et al., 1993). Considering together with Rice cells cultured in the of were presence 22 l! M other examples (e.g., Takeda et al., 1992), it seems BrdU for the indicated time periods. Plastid DNA necessary to employ several different experimental was then isolated as described above. Appropriate approaches for determination of the origin of amounts of the BrdU-labeled ptDNA were blotted replication. ptDNA In this respect, detailed analysis directly or after agarose gel electrophoresis onto a of in vivo replication intermediates might be of nylon membrane (Hybond-N+, Amersham Phar- particular importance. macia Biotech). The membrane was treated with 0.4 In the present study, we aimed to identify and M NaOH containing 1.5 M NaCl for 10 min, washed characterize the replication origin of ptDNA of the two times with 2 x SSC, and baked at 80 'C for 10 rice suspension- cultured cells. For this purpose, we min. Immuno-detection of the BrdU-containing used two different experimental procedures that DNA with anti-BrdU antibody (MBL Co. Ltd., have not commonly been employed for plant stu- Japan) was carried out by using ECL Western dies. First, we characterized RNA-primed nascent Blotting Detection System (Amersham Pharmacia DNA strands derived from the origin region. The Biotech). bromodeoxyuridine (BrdU)-labeled nascent DNA chain was isolated using anti-BrdU antibody, di- [solation of BrdU-labeled DNA using anti-BrdU gested with ~ -exonuclease to remove nicked and antibody degraded DNA, and then size-fractionated. These BrdU-labeled ptDNA (50 l!g) dissolved in 200 procedures could result in ,ul enrichment of intact RNA TBSE buffer (10 mM Tris-HCl, pH 9.5, con- -primed replicating DNA strands. Second, the taining 150 mM NaCI and O.1 mM EDTA), was heat putative origin region was subjected to neutral (for -denatured, and subjected to immuno-adsorption the first dimension)/alkaline (second dimension) 2D using anti- BrdU antibody and protein A+G agarose gel electrophoresis size distribution agarose to see beads (Oncogene Research Products) as follows. of the replication intermediates and direction of BrdU-1abeled ptDNA was mixed with 4.5 !lg anti- replication. These experiments revealed a major BrdU antibody and incubated at 4~, for 3h with origin region at around 3' the in end of 23S rDNA occasional mixing. Protein A+G agarose beads (50 each of IR.~ and IRB. /ll), Previously washed with 10 volume of TBSE buffer, was then added, and incubated for further 3 Materials and Methods h. The beads were spun down, washed 3 times with 10 volume of TBSE buffer, and resuspended in 400 Maintenance of suspension cultured cells of fll - rice proteinase K buffer (50 mM Tris-HCl, pH 7.5, (Oryza L.) sativa containing 10mM EDTA and 0.59~~] SDS). Protei- Suspension-cultured cells established from the nase K was then added to a final concentration of rice sativa L. Oryza var. Nipponbare were main- 0.5 ml-1, and incubated overnight at 37 'C BrdU mg . tained at 25 'C in AA medium (Mtiller and Grafe, -DNA was recovered by centrifugation. The DNA 1978) containing 3% (w/v) sucrose. An aliquot of in the supernatant was extracted twice with phe- cultured cells was maintained by placing in the 90 nol/chloroform (1 :1), once with chloroform/isoamyl ml fresh medium once a week. Lump cells were alcohol (24:1), and ethanol precipitated. The BrdU- filtration removed by through nylon net (200 preparation further digested ). a /Im DNA was with - pore size) every two weeks. Four-day-old cells exonuclease to eliminate nicked and degraded afier the routinely passage were used in this study. DNA. Prior to this digestion, 5' end of DNA was phosphorylated using T4 polynucleotide kinase and Isolation of DNA ATP. After phenol/chloroform (1:1) extraction and Cells were harvested at the 4th day after transfer ethanol precipitation. DNA was dissolved in 67 mM into fresh medium. Total isolated DNA was as Glycine-KOH, pH 9.4, containing '-.5 mM MgCl, described by Lodhi et al. (1994). Plastid pre- and 50 /lgml-1 BSA, and digested with was 15 U ~ - pared according to the procedure described by exonuclease (New England Biolab) at 37'C over- 29 night. 32P-labeled linearized DNA (pBS) was included in the reaction mixture as an intemal ptDNA (ng) lOOO lOOO 1000 1OOO 1000 control to monitor the digestion. RNA-primed BrdU-labeled BrdU-containing ptDNA thus obtained was further 25 50 100 ptDNA(ng) o 5 size-fractionated by 1% agarose gel electro- phoresis. , , , , Enrichment of replication intermediates by benzoy- Fig. I Incorporation of BrdU into rice ptDNA. Rice lated-naphthoy'lated (BND) cellulose chro- DEAE - cells were labeled with BrdU for 24 h, and the tograph ma y ptDNA was prepared. Indicated amounts of the Enrichment of replication intermediates con- BrdU - Iabeled ptDNA were mixed with 1000 ng taining single-stranded DNA regions was per- nylon of non - Iabeled ptDNA, spotted onto a cellulose chromatography formed by BND- column filter, and incubated with anti- BrdU antibody. essentially described by Huberman (1993). Total as BrdU-bound antibodies were detected using the rice cells digested overnight DNA from was HRP- Iinked anti- IgG antibody and ECL West- appropriate restriction After di- with an enzyme. em Blotting Detection System (Amersham Pha- precipitated with ethanol and gestion, DNA was macia Biotech). dissolved in NET buffer (10 mM Tris- HCl, pH 8.0, containing 800 mM NaCl and ImM EDTA). The DNA. To our knowledge, however, studies showing digested DNA was adsorbed to BND-cellulose incorporation of BrdU into ptDNA have not been column (Sigma), which was pre-equilibrated with reported, except for a unicellular chrysophyte the same buffer. The column was washed with 10 (Nerozzi and Coleman, 1997). Therefore, we ex- volume of NET buffer and then DNA was eluted amined incorporation of BrdU into ptDNA in the with NET buffer containing 1.8% (w/v) caffeine. rice cultured cells. Eluted DNA was ethanol- precipitated, and stored at Rice cells were cultured for 24 h in the presence -80 ̃C of 22 !lM BrdU, and ptDNA was prepared. Incorpo- . ration of BrdU into ptDNA was examined by using Two dimensional (2D) agarose gel electrophoresis anti-BrdU antibody. Various amounts of BrdU- of replication intermediates labeled and non- Iabeled ptDNA were spotted onto DNA purified by BND- cellulose column chroma- a nylon filter and incubated with the antibody. BrdU tography (25 to 30 flg) was loaded onto 0.4% -DNA-bound antibody on the filter was detected agarose gel for the first dimension. Electrophoresis by using HRP- Iinked anti- IgG antibody. As shown was performed in 40 mM Tris-acetate containing 1 in Fig. 1, anti-BrdU antibody bound to BrdU- cm̃] intensities in mM EDTA and 0.1 !lg ml-1 EtBr at 0.72 V for labeled ptDNA with increasing pro- about 30 h, as described by Little et al. (1993) and portion to the amount of DNA spotted, while no Huberman (1993). For electrophoresis in the second binding to the nonlabeled DNA was detected. Thus, dimension, the gel strip of the first dimension was BrdU was incorporated into ptDNA in the rice placed on top of 1%, agarose gel. The agarose gel cultured cells. We next performed similar immuno- was placed in circulating alkaline electrophoresis detection using ptDNA Iabeled with BrdU for 30 buffer (40 mM NaOH containing 2mM EDTA) and min, 60 min and 24 h. Equal amounts of the labeled incubated at room temperature for Ih. Then electro- ptDNAS Were digested with Sall, separated on an phoresis was started at 0.56V cm̃1 for 37h. After agarose gel and blotted onto a nylon filter. BrdU- electrophoresis, DNA was transferred onto a nylon containing DNA fragments were detected by bind- membrane and hybridized with various probes. ing of anti-BrdU antibody as described above. As Probe labeling, hybridization, and detection were shown in Fig. 2, incorporation of BrdU into ptDNA performed using Gene Images Labeling and Detec- increased with the period of labeling. tion Kit (Amersham Pharmacia Biotech). Mapping initiation region of ptDNA replication by Results labeling with BrdU To determine origin region of replication, rice Incorporation of Brd U into replicating ptDNA cells were pulse- Iabeled for 30 min with BrdU. The One way to determine origin of DNA replication BrdU-labeled nascent strands were purified by is to identify newly replicated short DNA fragment. binding to anti-BrdU antibody. As described in Incorporation of BrdU into DNA during replication Materials and Methods, ̃-exonuclease was used to molecules is a useful method for marking newly replicated eliminate nicked and degraded DNA 30

expected to be larger than unligated Okazaki frag- A ment of the lagging strand, although it was uncer- Immuno-detection tain if it existed. In addition, for detennination of replication origin, the nascent should be short l: ĩ DNA : '' '* and origin proximal. '* ̃ ̃ - S . To delimit the ptDNA region from which the e'ce)e¥ẽer̃ nascent DNA derived, the 0.7-0.9 kb nascent BrdU total - DNA and BrdU- DNA without size- fraction- ation were hybridized with ptDNA digested with Bglll, Sacl or Sall. As shown in Fig. 3A, the 0.7- 0.9 kb nascent DNA probe preferentially hybridized with 5.3 kb fragment in the Bglll digest, with 8.7 and 11.4 kb fragments in the Sacl digest, and with 7.5 and 14.7kb fragments in the Sall digest. In contrast, the total BrdU-DNA probe hybridized with much more fragments, indicating that BrdU was incorporated randomly into replicating DNA over the entire ptDNA region. Since the size-frac- tionated 0.7-0.9 kb probe represents RNA-primed B short nascent strands synthesized from origin, the EtBr-staining fragments that the probe hybridized must contain . However, it should be noted that the 0.7-0.9 kb probe hybridized, although to a lesser extent, with several fragments in addition to those described above (marked by asterisks in Fig. 3A and B). They distributed randomly over the ptDNA and did not overlap. It is not clear at present whether these signals represent multiple replication origins or contamination of nuclear or mitochondrial DNAs. 1 2 3 Considering together, these results indicated that 4M within or in the very vicinity of the overlapping Fig. 2 Immuno-detection of BrdU-labeled ptDNA region of the DNA fragments that the 0.7-0.9kb fragments. (A) ptDNAS prepared from rice Were DNA probe preferentially hybridized, the preferred ce]Is labeled with BrdU for min (lane l), 30. O or major origin of ptDNA replication locates (Fig. min (lane 2), 60 min (lane 3) and 24 h (1ane 4). 3B and C). According to the registered sequence of The BrdU-labeled ptDNA (3 each) flg was the rice ptDNA (GenBank/EMBL/DDBJ Accession digested with Sall, separated on a 0.8% agarose no. NC O01320), the overlapped region corre- gel, blotted, and detected using anti-BrdU sponded to 2.3 kb DNA region including 3' end of antibody as described in Fig. 1. (B) shows EtBr - the 23S rDNA in each of IR,¥ and IRB. staining of the gel. Lane M. Hindl[1 digested - A In tobacco, a pair of D-loop type replication as a size marker. - DNA origins has been mapped in each IR; oriA between the 16S rDNA and the 23S rDNA, and oriB down- possibly generated during DNA preparation. Com- stream of the 23S rDNA (Kunnimalaiyaan and plete digestion 32P- was monitored by including Nielsen, 1997b). By contrast, there seems to be only labeled plasmid DNA in the reaction mixture (data one major origin region in each IR in the rice shown). not The nuclease does not attack RNA-- ptDNA, the position of which corresponds to that primed DNA molecules (Abdurashidova et al., between oriA and oriB in tobacco. Therefore, we 2000). Thus, RNA-primed nascent DNA strand next intended to know how replication begins and remains undigested. proceeds by 2D agarose gel electrophoresis. RNA-primed BrdU-containing DNA strands further size-fractionated were by agarose gel elec- 2D agarose gel electrophoresis of replication inter- trophoresis, and the BrdU-DNA ranging from O.7 mediates to 0.9kb extracted. Because this size Fig. was was 4 shows schematic representation of neu- 31 A ̃, ̃J Ĩ en eS ẽ Ee Cl: V)

̃ '̃::iij!̃;- li.4 kb ̃ =' 14'7kb̃ -_i'_i' ̃ ̃̃ ̃ :S'̃ 8.7 kb 7'5 kh̃*

5'3kh)F '

;P]õ 0.7 ;̃o ̃7 f̃l ̃7 i:õE Probe al *es ,l *09 *e9 *̃/ '4h 'kh '4h

B LSC IRB SSC IRA 134'25 LSC * Bglll [̃̃l 5.3 kh kb 7.4 kh I Ĩi [̃] Sacl S_7 kb Il1.4Ikb 4.5 kb Sall [Il 14 7kb ̃7.5 kb 21 kb 6.9 kb

c IR

5_ .3 kb Bglll

IRA Sacl

SalT IRB

̃̃.3 kb Bglll .s

Sall

Fig. 3 Loc.atioT] of the BrdU Iabeled newly replic.ated nascent DNA strands on the ptDNA. (A) BrdU- Iabeled RNA- primed nascent strands (see text) were size - fractionated, and those with sizes of 0.7- O.9 kb and total BrdU - Iabeled nascent strands (total) were used as probes for hybridization with ptDNA (5 !1g each) digested with BgIII. Sacl and Sall. Probe labeling and detection were carried out using Gene Images Labeling and Detection Kit (Amersham Pharmacia). (B) Summary of hybridization of the BrdU- Iabeled nascent strands. Positions of the preferentially hybridized fragments are depicted by filled boxes. The fragments that the probe hybridized to a lesser extent (marked by asterisks in A) are also depicted b), open boxes with asterisks. (C) Restriction map around the IR,̃ aT]d IRr, regions and the fragments to which the 0.7- 0.9 kb probe preferentially hybridized. Filled boxes with their sizes Indicate the hybridi7.ed fragments Overlapping region (about 2.3 kb) of the hybridized fragments is indicated by gray zone. tral/alkaline (N/A) 2D gel electrophoresis. N/A 2D condition to denature DNA. Because template DNA gel method detects the direction of replication fork strands have a constant size regardless of the extent movement through a particular restriction fragment of replication, they generate horizontal signal after (Huberman et al., 1987; Huberman, 1993), In this hybridization with a specific probe. However, repli- approach, restriction enzyme -digested DNA is sep- cating nascent strands vary in their length, and thus arated on a first dimension gel according to their generate a diagonal signal. If DNAS from different molecular weight, that is, the extcnt of replicatlon. positions of a restriction fragment are used as The second dimension gel is run under alkaline probes (e.g., probes 1, 2 and 3 in Fig. 4), thc 32

R R Origin moves in the opposite direction of 23S rDNA tran- scription. However, nascent strands extending in A both directions were detected in the Dral 4.8 kb and probe 1 2 3 Dral 4.4 kb regions, because full range (from short to long) of nascent strands was detected using probes corresponding to either end of each frag- / B ment. This means that in these regions replication proceeds from both ends. These results, together Digestion with with the data obtained by nascent strand analysis restriction enzyme (Fig. 3) suggested the replication fork movement BND-cellulose shown in Fig. 6. Two replication origins locate chromatography around the 3' of the in the Neutral agarose gel end 23S rDNA two IRs, electrophoresis and replication from each origin proceeds in both O e directions. C Discussion

In the present study, two major replication origins Alkaline agarose gel electrophoresis of the rice ptDNA were mapped around the 3' region of each 23S rDNA in IRs, based on local- D ization of RNA-primed, BrdU-labeled nascent DNA strands. It seems that ptDNA replication origins may have a general characteristic of prox- imity to the rDNAS but a flexible positioning rela- tive to them. In ptDNA from tobacco seedlings (Fig. 6), two D-loop-started origins, oriA and oriB, were mapped around 16S and 23S rDNA probe 1 2 3 (Kunnimalaiyaan and Nielsen, 1997b). Nucleotide sequence of the oriA region is highly conserved Fig. 4 Schematic representation of N/A 2D agarose among plant species (Hiratsuka et al., 1989; Kunni- gel electrophoresis for determination of repli- malaiyaan and Nielsen, 1997a), and oriA was cation direction. At the top (A), the positions of mapped in the same region in pea and tobacco. restriction sites (R), probes 1, and 3, and origin 2 However, no replication origin was found in the are shown. Replication intermediates (B), are corresponding region in soybean (Hedrick et al., digested with the restriction enriched by enzyme, 1993) and rice (this study). Initiation of ptDNA BND cellulose chromatography and subjected - replication does, therefore, not primarily depend on to neutral (C, for the first dimension) and alkaline nucleotide sequence itself. (D, for the second dimension) gel agarose Analysis by N/A 2D agarose gel electrophoresis electrophoresis. Different hybridization patterns suggested that replication extends bidirectionally will be detected with different probes 1, 2, and 3. from each of the two origins (see Fig. 6 for sum- Origin-distal probe I detects only the long mary). However, this does not simply mean that nascent strands, whereas origin proximal DNA - both parental strands are simultaneously replicated. probe detects the full of strands. 3 range nascent An alternative is also possible that two opposite direction replication of can be deduced. The origin- strands, which are switched at the origin, are used as distal probe (probe 1) detects only the long nascent templates. In any case, the mode of ptDNA repli- strands, whereas the origin-proximal probe (probe cation in the rice cultured cells apparently differs 3) detects the full range of nascent strands. from the double D-100p mechanism (Kolodoner Replication intermediates around the origin re- and Tewari, 1975), in which the two D-loops gions were analyzed by N/A 2D gel electrophoresis. expand toward each other and only one parental Direction of replication was examined for the 5.3 kb strand serves as a template. However, replication of Bglll fragment, 4.8kb, 4.1 kb and 4.4 kb Dral the far reported is restricted ptDNA so not to the D - fragments as shown in Fig. 5. In the 5.3 kb Bglll loop mechanism. EM studies of Euglena gracilis fragment, only long nascent strands were detected ptDNA indicated that replication from a single with the 593 bp probe, suggesting the probe region origin near the 5'-end of the supplementary 16S origin- distal. to be This shows that replication fork rDNA proceeds bidirectionally and that both paren- 33

Origin Origin t̃ e IRA 23s 23s

_--11 ' L lr-* 1 l +, L l L l h L L I 1 l 1"':""""";:,:,,,,1,,̃

:: ̃ ̃ ̃ e,, 1' BgIII 5.3 4.8 I,) 4.l ̃ 4.4 ;̃ ul BgIII 5.3 ,:̃ er̃ ̃ Dral ̃ Dral ̃ Dral ̃ _ - : ̃] [l [̃] [̃l Cll [̃l [̃] C̃̃] [̃l [] 593 bp 1166 bp 711 bp 585 bp 674 bp 653 bp 615 bp 711 bp 1166 bp 593 bp T-- ̃- ̃.. r-

Fig. 5 N/A 2D agarose gel electrophoresis of the replication intermediates around the origin regions. Structure of the rice ptDNA is shown at the top. Large single copy (LSC), small single copy (SSC), inverted repeat regions (IR,̃ and IRẼ, filled boxes) and the replication origins are indicated. The 23S rRNA gene is depicted by open arrows. 2D agarose gel pattems for the 5.3 kb BgIII, 4.8 kb Drar, 4.1 kb Dral and 4.4 kb Dral fragments are shown in the middle panel. In each 2D gel, 25 to 30 /lg of the BND- cellulose- enriched replication intermediates was subjected to the electrophoresis. Arrows indicate replicating nascent strands. Cartoon forms of each result and the position of the probes (open box) are shown at the bottom.

origi* o*igi*

Rice 16S 23s I1̃s l1 23s 16S 4,ss - Ssc 5s 4.5s IRB IRA

Tobacco .,i A "i B ori B ori A c-'- 'F;･ ̃-,t 4.Lli: :i:･;--̃ Fig. 6 Replication origins and the direction of replication of ptDNA in the rice cultured cells and the tobacco plant. Origins are indicated by filled circles, and the direction of replication by arrows. SSC and IRs, and rRNA are also shown. Dotted arrows indicate unidirectional replication according to the double D - Ioop mechanism (see text). tal strands are simultaneously replicated (Ravel- while coupled leading- and lagging-strand repli- Chapuis et al., 1982). In vitro replication experi- cation for rapid increase of mtDNA copies. Repli- ments using maize ptDNA also showed that both cation of ptDNA might be fundamentally similar to DNA strands were simultaneously used for tem- that of mtDNA. However, we do not know at plates (Carrillo and Bogorad, 1988). Moreover, an present whether coupled leading- and lagging- interesting observation has recently been reported strand synthesis occurs. for human mitochondrial DNA (mtDNA) that repli- Whether the two origins in IRA and IRB are both cation occurs by coupled leading- and lagging- fired on the same ptDNA molecule is not clear at strand synthesis in addition to D-loop-started present. A possibility still remains to be elucidated unidirectional replication (Holt et al., 2000). It is that replication of one ptDNA molecule starts from considered that D-100p-started replication is the origin in one of the IRS While the other molecule employed for maintenance of mtDNA copy number, from the other origin. 34

Finally, it should be noted that immuno- detection inversion during the evolution of the cereals. Mol. Gen. of BrdU-labeled in Fig. resulted in C.enet 217: 185- 194. DNA shown 2 , the discrete of the labeled Holt, l. J., Lorimer, Jacobs, H_ T., 2000. Coupled bands DNA fragments H E , that contain replication origins, i,e., 14.7 and 7.5 kb leading- and lagging-strand synthesis of mammalian Sall fragments (not indicated in Fig. 2). BrdU- m. itochondrial DNA Cell, 100: 515 - 524. Huberman, J. A 1993 Analysis of DNA replication origins labeling was performed using randomly growing , and directions by two- dimellsional gel electropharesis. rice cells, so that incorporation of BrdU should In: Fantes, P Brooks, R. (Eds): The , a occur randomly and uniformly into the ptDNA. , practical approach, pp. 213-234. Oxford University Therefore, the results indicate paused repli- may Press, New York. cation intermediates. Indeed, replication has pause Huberman, J A., Spotila, L. D., Nawotka, K. A., El- been found in tobacco ptDNA (Kunnimalaiyaan and Assouli, S. Davis, L. R., 1987 The In vivo M , Nielsen, 1997a). In tobacco, paused nascent strands replication origin of the yeast 2 /~m pl~smid. Cell, 51: reported to be 0.8 to 2.5 kb- Iong. 473 48 1 were - . Kolodner, R. D., Tewari, 1.975. Ch[oroplast K K , DNA References from higher replicates by both the Cairns and the rolling circle mechanism, Nature, 256: 708 71 Abdurashidova, G., Deganuto, M Klima, R., Riva, S., - 1 , Kunnimalaiyaan, M., Nielsen, B. L 1997a. Chl.oroplast Biamonti, G Giacca, M Falaschi, A., 2000. Start , , , DNA replication: mechanism, and replication sites of bidirectional DNA synthesis at the human lamin enzymes origins. J. Plant Biocbcm- Biotechnol., 6: 7. B.2origin. Science, 287: 2023-2026 I- Kunnimalaiyaan, M., Nielsen, B L 1997b Fine mapping of Canillo, N., Bogorad, L., 1988_ Chloropl.ast DNA repli- , replication origins (oriA and oriB) in Nicotiana cation in vitro: site-specific initiation from preferred tabacum chlorop]ast DNA. Nucleic Acids Res 25: templates Nucleic Acids Res., 16: 5603- 562C. , 3681 3686. Chiu, W. L., Sears, B. B., 1992 Electron microscopic - Kunnimalaiyaan, Shi, F., Nielsen, B. L., 1997. Analysis localization of replic.ation origins in Oenothera chloro- M , of the tobacco chloroplast replication origin plast DNA. Mol. Gen. Genet., 232: 33- 39. DNA (oriB) downstream of the 23S J. Colletti, K. S., Tattersall, E. A., Pyke, K. A., Froelich, J. E., rRNA gene. Mol Biol., 268: 273-283. Stokes, K. D., Oster)'oung, 2000. homologue K W = A Little, R. D., Platt, H. K., Schildkraut, C. 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