27 Original Paper Plant Biotechnotogy, 19 (1), 27- 35 (2002) Mapping Major Replication Origins on the Rice Plastid 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 gene. 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 Plastids 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 chloroplast, chromoplast, (origins) of unidirectional replication of ptDNA. amyloplast and leucoplast 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 cell 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 plasmid 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.