Genetics: Correction

Correction. In the article "Nucleotide sequence of a pre-ferred maize chloroplast genome template for in vitro DNAsynthesis" by Bert Gold, Nestor Carrillo, Krishna K.Tewari, and Lawrence Bogorad, which appeared in number1, January 1987, ofProc. Natl. Acad. Sci. USA (84, 194-198),

AARS 70

ATCGTAACTT GACTTTTGAA ACAACAAATA AAAATATAAA GCAATAAATA AAGAGCAGCC GGGTTAATAA

140AACTGAGAAA ATTGACTCGG AAAAAAAAAT TGGAAGCTCC ATTGCAGGAT TCAAACCTAA CCATTAAAGG

210AGAAGCTGTG GGAACGACAA AACCTATGAC TACATAGGAT TTATTGAAAG AATCCTAACA CTTCATTGGT

I 280GGATGGCGGA CAAACCACAA AATTGCTTTA TTTGATAAGG TTCTAAATTA ACAAATAAGA CAGGAAAGAG

350TAAATATTCG CCCGCGAAAT CCTTATTGGA TTAGAATACT TTACCACGAT TCAATAAGAA TAAAAAAAAA

U1 420GGAGATTCCA AAAAAGGAAG GATTACATTT TATATAAATA TTGAATTTGG ATATATTCTA CATCTTCTTT

U 490CTTGACTATA TATTTAATTT TTCT= A AGTTTAAGAA AGTCAAAATC AAAAAAACCT AACTTTTTCT

I I 560ATTATATATT GATGTCTATC TATCTATAAT ATTTTTGAAT ATTATGGAAT TAGAGAAATG ACATTCGCAC

630GCTTCTCATT TCATTCGCGA GGAGCTGGAT GAGAAGAAAC TCTCATGTCC AGTTTTGTAG TAGAGATGGA

ARC ORF1ACTAAGAAGG AACCATCGAC TATAACCCCA AAAGAACTAG ATTTCGTAAA CAACATAGAG GAAGAATGAAAaattatAaa tAaaAaCatg TtaAgtCCaA AAAGAACaAa ATTcCGTAAA CcACAccGtG GtiattTaAg

I 770GGGAAAATCC TGCCGAGGCA ATCATATTTG TTTTGGTAGA TATGCTCTTC AAGTACTTGA ACCCGCTTGGaGGAAAAgCa acaCGtGGtA ATaAaATTgt aTTTGGTgat TtTGCatTaC AAGcACaaGA ACCttgTTGG

840ATCACCGCGA GACAGATAGA AGCAGGACGA AGAGCAATGA CACGATATGC ACGTCGTGGT GGAAAAATATATtACatCac GtCAaATtGA AGCcGGACGt cGtGtttTaA CACGtTATGt tCGTCGTGGT GGtAAAtTAT

910GGGTGCGTAT ATTTCCCGAC AAACCGGTTA CAATAAGACC CACAGAAACA CGTATGGGCT CGGGAAAGGGGGaTtCGTAT tTTcCCaGAt AAAgCtGTTA CtATgcGtCC tgCtGqtACt CGTATGGGtT CtGGtAAaGG

I 980ATCCCCCGAA TATTGGGTAG CCGTTGTTAA ACCACGTCGA ATACTTTATG AAATGAGCGG AGTATCCGAAtgCaCCtGAt TATTGGGTAG CtGTTGTacA tCCtGGTaaA ATttTaTATG AAATGcaaGG tGTATCtGAA

1050ACTGTAGCTA GAGCAGCTAT CTCTATAGCT GCCAGTAAAA TGCCCATACG AAGTCAATTT CTTCGATTAGACaaTtGCTA GAcaAGCaAT gcgcATtGCa GCttaTAAAA TGCCagTAaa AAcaaAATTT tTaacAaaAa

1120AGATATAGAA CCCCAAAAAG GAATATTGAA GATAAAAAAC CCCTAGTTTT TCTTTCTGGA AAGACAATAT

ORF2 1190TTCTTTCATC CTTTTGCATT TGAAATAACA AATGGAAACC AAATAATAT OATTCAACCTC AGACCCTTTT

1260AAATGTAGCA GATAACAGTG GAGCTCGAAA ATTGATGTGT ATTCGAGTCA TAGGAGCCGC TrGTAATCAC:

1330CGATATGCTC GTATTGGTGA TGTTATTATT GCTGTAATCA AAGACGCAGT GCCCCAAATG CCTCTAGAAA

1368GATCCGAAGT AATTCGAGCT GTAATTGTAC GTACACGT

Proc. Natl. Acad. Sci. USA 84 (1987) 1643

a portion of the nucleotide sequence (nucleotides 1200-1259)of the maize chloroplast DNA fragment EcoRI x (Eco x) inFig. 4A was omitted; instead the sequence from nucleotide1260 to 1319 was typed twice. The corrected figure and itslegend are shown below.

B-20 30

GGAACTAAG AAGGAACCAT CGACTATAAC CCC AAA AGA ACT AGA TTT CGT AAA CAA CAT AGAZea mays Y P K R T R F R K Q H RNicotiana tabacun MLS P K R T R F R K H RCntamydomonas reinhardii MLS P K R T K F R K P H REscherichia coZi MLQ P K R T K F R K M H K

60 90GGA AGA ATG AAG GGA AAA TCC TGC CGA GGC AAT CAT ATT TGT TTT GGT AGA TAT GCTG R M K G K S C R G N H I C F G RG R M K G H R G N H I S F G K AG H L R 0 K A T R GH v F G DFG RI N R G L A Q - 0 T D V S F G S F G

120CTT CAA GTA CTT GAA CCC GCT TGG ATC ACC GCG AGA CAG ATA GAA GCA GGA CGA AGAL Q V L E P A W I T A R Q I E A G R R

L Q A L E P A W I T S R Q I E A G R RL Q A TLS R Q I E A C R R

LJKA V G R G R L JT A R Q I E A A R

150 180GTA ATG ACA CGA TAT GCA CGT CGT GGT GGA AAA ATA TGG GTG CGT ATA TTT CCC GACA M T R Y A R R G G K I W V R I F P DA M T R N A R R G G K I W V R I F P DV L I T R N V R 0 G K Lw I R I F P D|A M T R A R I V F P

210 240AAA CCG GTT ACA ATA AGA CCC ACA GAA ACA CGT ATG GGC TCG GGA AAG GGA TCC CCCK P V T I R P T E T R M G S G K G S PK P V T L R P A E T R M G S G K G S PK L 1 T M R P A 0 T R M G S G K 0 A

IK P L A V R V

270 300(-AP. ACA TC(; GTA GCC OTT GTT AAA CCA GOT CGA ATA CTT TAT GAA ATG AGC GGA GTAE Y W V A V V K P G R I L Y E M S G VE Y W V A V V K P G R I L Y E M G G V0 Y W V A V Vi L Y E M Q G VK Y W V Af L I Q P G K V L Y E M D G V

330 360TCC GAA ACT GTA OCT AGA GCA GCT ATC TCT ATA GCT GCC AGT AAA ATG CCC ATA CGA|S E T V A R A A I S I A A S K M P I K

E N I A R R A I S L A A S K M P I RE N I A R Q A H R IA A Y K M P V KE L |A R E A F K A K

390 432ACT CAA TTT CTT CGA TTA GAG ATA TAG AACCCCAAAA AGGAATATTG AAGATAAAAA ACCCCTS iQRFL R L E I -a-T IQ Fi I I ST F J T K T VT TLJV T K T V M

FIG. 4. (A) Nucleotide sequence of the 1368-bp Eco x of maize chloroplast DNA. Horizontal lines are drawn above regions related to theyeast ARS consensus sequence, to the conserved elements I and II found in C. reinhardtii ARS fragments, and to the 19-bp consensus sequence(ARC) found in C. reinhardtii cpDNA fragments that promote autonomous replication in the alga. Underlined regions represent sequences thatcan form stable stem-loop structures. Starts and reading directions of the two ORFs (ORF 1 and ORF 2) are marked. The sequence of C.reinhardtii cpDNA D-loop region (nucleotides -17 to +403 in ref. 22) is aligned below its homologous region in maize cpDNA. Conservednucleotides are indicated by capital letters and differences by small letters in the C. reinhardtii sequence. No gaps were introduced for thealignment. (B) Nucleotide sequence of ORF 1 and flanking regions (nucleotides 628-1094 in A). The sequence of the nontranscribed strand ofORF 1 is arranged in codons, and the corresponding amino acid sequence is shown below. Translation was started upstream of the first ATGcodon (indicated by a double line above the triplet) to maximize homology with the primary structures of the ribosomal protein L16 from tobacco(23), C. reinhardtii (22), and E. coli, which are aligned below the maize gene. Conserved amino acids are boxed. Numbering starts at the firsttranslated triplet (CCC). The arrow indicates the position of the 1020-bp intron site in tobacco L16 gene.

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Proc. Natl. Acad. Sci. USAVol. 84, pp. 194-198, January 1987Genetics

Nucleotide sequence of a preferred maize chloroplast genometemplate for in vitro DNA synthesisBERT GOLD*t, NESTOR CARRILLO*, KRISHNA K. TEWARIt, AND LAWRENCE BOGORAD*§*Department of Cellular and Developmental Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138; and tDepartment of Molecular Biologyand Biochemistry, University of California, Irvine, CA 92717

Contributed by Lawrence Bogorad, September 23, 1986

ABSTRACT Maize chloroplast DNA sequences represent-ing 94% of the chromosome have been surveyed for theiractivity as autonomously replicating sequences in yeast and astemplates for DNA synthesis in vitro by a partially purifiedchloroplast DNA polymerase. A maize chloroplast DNA regionextending over about 9 kilobase pairs is especially active as atemplate for the DNA synthesis reaction. Fragments fromwithin this region are much more active than DNA fromelsewhere in the chromosome and 50- to 100-fold more activethan DNA of the cloning vector pBR322. The smallest of thestrongly active subfragments that we have studied, the 1368-base-pair EcoRI fragment x, has been sequenced and found tocontain the coding region of chloroplast ribosomal protein L16.EcoRI fragment x shows sequence homology with a portion ofthe Chlamydomonas reinhardtii chloroplast chromosome thatforms 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 autono-mous replication ofDNA in yeast are not active as templates forDNA synthesis in the in vitro assay. The template active regionwe have identified may represent one of the origins of repli-cation of maize chloroplast DNA.

Replicative intermediates have been located at two sitesapproximately 7.06 kilobase pairs (kb) apart on the maizechloroplast chromosome (1). A 6-form replicative interme-diate is formed by the expansion of these two displacementloops (D-loops) toward one another. Kolodner and Tewari (2)have proposed that rolling circle DNA synthesis initiates atthe site of termination of the Cairns replication round about1800 across the 140-kb circle from the bipartite initiation sites.Wu and coworkers (3, 4) have located a D-loop on Chlamy-domonas reinhardtii chloroplast DNA. This has beenmapped to a 1055-base-pair (bp) restriction fragment that hasbeen cloned and shown to form a denaturation loop in vitroat the site of the D-loop (4).

Efforts to understand chloroplast DNA replication havealso included studies of DNA synthesis in vitro using chlo-roplast extracts from maize (5) and Marchantia polymorpha(6) as the sources of DNA polymerase activity and chloro-plast DNA templates. No specific origins of replication wereobserved in the former study (5), whereas in the latter, somefragments were shown to be preferred templates (6). In anattempt to map the replication origin, Rochaix and coworkers(7-9) identified regions of the Chlamydomonas chloroplastchromosome that promote autonomous replication in yeast.Comparable work was undertaken by Schlunegger and co-workers (10, 11) with Euglena and by de Haas et al. (12) withpetunia.

In the present paper we describe the analysis of clonedfragments representing 94% of the maize chloroplast chro-mosome for their DNA synthesis template activity using

partially purified pea chloroplast DNA polymerase (13). Wehave identified two regions of the maize chloroplast chro-mosome that are especially active as templates. One of theseregions has been studied in detail and sequenced.

MATERIALS AND METHODSDNA Preparation. Chloroplast DNA (cpDNA) was pre-

pared by the lithium chloride method according to Bogoradet al. (14). Plasmids were purified using chloramphenicolamplification followed by the preparation of a Hirt (15)supernatant that was then banded in CsCl. Phage DNA wasisolated using a scaled-up liquid lysis procedure (16).

Plasmid Constructions. Vectors were bacterial or calfalkaline phosphatase-treated BamHI- and/or EcoRI-digestedpBR322, YIpS, or pCCATV10 (17). Fragments were derivedfrom plasmids as follows: Inserts in pZmcBam 19, pZmcBam870, pZmcBam 500, and pZmcBam 9' were derived fromBamHI digests of the Pst I fragment 1 containing plasmidpZmc 635. Plasmids pZmcBam 17' and pZmcBam 22' weremade by ligating subfragments of X Charon 4A clone 2 (18) tovector pBR322. pZmcEco x was formed by subcloning a1368-bp EcoRI subfragment from X Charon 4A clone 2 intopBR322. pBE 950 was formed by subcloning a 950-bpBamHI-EcoRI fragment of pZmc 504. pZmcEco n was alsosubcloned from X Charon 4A clone 2. pZmcEco s' andpZmcEco u were subcloned from pZmcBam 9'. pBE 400,pEco 600, and pBE 1470 were subcloned from pZmcBam 17'.pBE 680 and pBE 750 were subcloned from pZmcBam 22';pBE 700 was subcloned from pZmcBam 9'. pPGA35 and pDR1 are subclones of pZmc 427 made by Lyle Crossland andDavid Russell (Harvard University), respectively. pSC3-1,pEgcH4, and pCA2 were kindly supplied by Madeline Wu(University of Maryland), Erhard Stutz (University ofNeuchatel, Switzerland), and Jean Rochaix (University ofGeneva), respectively. YIp5 was a gift of Daniel Stinchcomb(Harvard University).DNA Synthesis in Soluble Extracts. Extracts containing

DNA polymerase activity were obtained by passing Triton-disrupted chloroplasts isolated from 500 g of 7-day-old pealeaves (19) through 100 ml of DEAE-cellulose equilibratedwith 0.1 M (NH4)2SO4 in buffer A [25% (vol/vol) glycerol/10mM Tris HCl, pH 8.0/50 mM 2-mercaptoethanol/2 mMphenazine methosulfate]. After extensive washings with theequilibration buffer, the column was eluted with 0.5 M(NH4)2SO4 in buffer A. Fractions containing DNA polymer-ase activity were pooled, dialyzed against 0.1 M (NH4)2SO4in buffer B [25% (vol/vol) glycerol/50 mM Tris HCl, pH8.0/0.1 mM EDTA/0.1% Triton X-100/2 mM phenazinemethosulfate/50 mM 2-mercaptoethanol], and adsorbed to a

Abbreviations: D-loop, displacement loop; ORF, open readingframe; ARS, autonomously replicating sequence in yeast; kb, kilo-base(s); bp, base pair(s); cp, chloroplast.tPresent address: BioTechnica International, Inc., 85 Bolton St.,Cambridge, MA 02140.§To whom reprint requests should be addressed.

194

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 84 (1987) 195

20-ml heparin-Sepharose column equilibrated with dialysisbuffer. This column was eluted with 0.5 M (NH4)2SO4 inbuffer B, and the DNA polymerase-containing fractions wereagain dialyzed against 0.1 M (NH4)2SO4 in buffer B. Thedialyzed enzyme was loaded onto a 5-ml phosphocellulosecolumn, washed with dialysis buffer, and eluted with 0.5 M(NH4)2SO4 in buffer B. As many as 18-24 protein bands werefound in the DNA polymerase-containing fractions using asilver staining procedure after NaDodSO4/polyacrylamidegel electrophoresis.

In the assay, 1 ug of template DNA was incubated for 30min at 370C in 100 A.l containing 50 mM Tris HCl (pH 7.0); 10,uM each ofdATP, dGTP, and dCTP; and 1 ,uCi [3H]dTTP (80Ci/mol; 1 Ci = 37 GBq); 120 mM KCI; 12 mM MgCl2; and 1-5x 10-2 units ofDNA polymerase (unit definition as in ref. 13).The acid insoluble radioactivity was assayed as described(13). The molecular size of the in vitro synthesized DNA wasdetermined by substituting 50 /.Ci of [32P]dTTP (3200 Ci/mol)for [3H]dTTP in the incubation mixture. This was followed byphenol extraction, ethanol precipitation, and agarose gelelectrophoresis of the resulting DNA product.DNA Blot Hybridization. Agarose minigels were blotted

using a procedure supplied by International Biotechnologies(New Haven, CT). After transfer to nitrocellulose filters andbaking, blots were prehybridized in 5x Denhardt's solution(1x Denhardt's = 0.02% Ficoll/0.02% bovine serumalbumin/0.02% polyvinylpyrrolidone) for 2 hr at 50°C. Theprehybridization solution was replaced with a mixture con-sisting of 5x SET/0.5% NaDodSO4/1x Denhardt's solu-tion/10% (wt/vol) dextran sulfate/1 x 107 cpm 32P-labeleddenatured (boiled) probe and incubated for 16 hr at 50°C. (1 xSET = 0.15 M NaCl/0.03 M Tris HCI, pH 8.0/1 mM EDTA.)The blots were then washed twice with 2x SET containing0.5% NaDodSO4 and twice with 0.1 x SET at room temper-ature, dried, and autoradiographed.The following DNA fragments (digested from the corre-

sponding plasmids, purified by agarose gel electrophoresisand electroeluted prior to nick-translation) were employed asprobes: (i) a 1055-bp BamrHI-Cla I fragment from pSC3-1 (4);(ii) a 2.6-kb HindIII-Bgl II fragment from pEgcH4 (10, 11);and (iii) a 600-bp EcoRV-Sal I fragment from pCA2 (7).

Yeast Transformation and ARS Assay. Yeast transforma-tion and assay for ARS (autonomously replicating sequencesin yeast) were carried out on EcoRI and BamHI banks ofmaize plastid DNA in YIp5. The methods used have beendescribed in detail (17) except that employed for subcloninginto pCCATV10 that is a pUC9 derived, ura(+) vectorgenerously provided by J. R. Scott and C. Cirks (Universityof Illinois). pYEco i and pYEco x' consist of maize cpDNAEcoRI fragments i and x' (Eco i and x') cloned in this vector.DNA Sequence Analysis. EcoRI fragment x (Eco x) was

digested with appropriate restriction endonucleases to obtainsmall secondary fragments for sequencing. These fragmentswere labeled at their 3' termini by fill-in synthesis using32P-labeled deoxyribonucleotide triphosphates and the largefragment ofDNA polymerase I. After digestion with a secondrestriction endonuclease, radiolabeled fragments were recov-ered from polyacrylamide gels and sequenced (20).

RESULTSMapping Autonomously Replicating Sequences on the Maize

Chloroplast Chromosome. As a start toward mapping regionsof the maize chloroplast chromosome that might representthe origin(s) of replication identified by electron microscopy(2, 21), a search was made for segments of the chromosomethat would facilitate autonomous replication of DNA (ARS)in Saccharomyces cerevisiae. Samples of maize cpDNAdigested with BamHI or EcoRI were ligated into the Esche-richia coli-yeast shuttle vector YIp5. If an ARS from any

source is inserted into this plasmid, it will convert the yeasturacil auxotroph in which it grows to the ura' phenotype athigh efficiency (17). From analyses of EcoRI and BamHImaize cpDNA libraries, BamHI fragment 10 and its subfrag-ment Eco x' (see locations in the map of Fig. 1) were foundto have ARS activity. pYEco i, which contains a maizecpDNA sequence adjacent to Eco x', does not show ARSactivity.DNA Replication Template Activity of Maize Chloroplast

DNA Fragments. The major part of this work involvedscanning =94% of the maize chloroplast chromosome forsequences with strong template activity in an assay using apartially purified pea chloroplast DNA polymerase. Theenzyme preparation used also contains RNA polymerase,topoisomerase, and DNA ligase activities as well as a DNAbinding protein (R. McKown and K.K.T., unpublished re-sults) that depresses the melting temperature ofDNA. It doesnot contain any detectable endo- or exonuclease activity. Thechloroplast DNA sequences to be tested as templates weresupplied as fragments cloned into pBR322. Data given in Fig.1 are from two experiments using a single enzyme prepara-tion to test most of the templates, but the most activefragments were tested with four to six different enzymepreparations with consistent results.The locations in the maize chloroplast chromosome of the

DNA sequences used as templates are shown in Fig. 1 (givenas the name of the cloned DNA supplied in the assay). Thetemplate activity (cpm x 10-3) is shown in parentheses afterthe name of each plasmid DNA used. In the absence ofDNA,about 1 x 103 cpm of [3H]dTTP were incorporated intoacid-precipitable material during the 30-min incubation peri-od. When pBR322, the cloning vector for all of the cpDNAused in these experiments, was used as a template, 2 x 103cpm were incorporated. For most of the chloroplast chro-mosome, values of 3-20 x 103 cpm were obtained. A regionof especially high activity was observed at about the 3 o'clockposition (bordered heavy line on the map given in Fig. 1). Asseen in Fig. 1, the DNA synthesis activity was not dependenton the size of the fragments provided. For instance, largecpDNA sequences such as those in pZmc 545 (9.8 kb), pZmc100 (12.5 kb), and pZmc 548 (12.5 kb) showed less than 20 X103 cpm in the in vitro assay whereas pZmcBam 17', whichis only 2.5 kb long, was 5.5 times more active.

Proceeding clockwise from a position at about 12 o'clock,there is very little template activity until BamHI fragment 17'(Bam 17') is reached, this activity continues through BamHIfragment 9' (Bam 9') (a distance of some 9 kb) and then dropsoff rapidly to the base-line level until about 180° across thecircle where BamHI fragment 8 (Bam 8) DNA has higheractivity (70 x 103 cpm). The remainder of the chromosome isinactive except for DNAs of pZr 36 and pZmc 573, whichsupport high activity.The high efficiency of the system (40%-80% of the tem-

plate DNA was copied when sequences of the most activeregions were provided in the assay) strongly suggests that theenzyme preparation used contains a true replicase and not arepair polymerase. Zimmermann and Weissbach (5) pro-posed that they were studying a repair enzyme.

Cross-Hybridization of Maize Chloroplast DNA Sequenceswith D-Loop DNA of Algae. Wang et al. (4) have identifiedChlamydomonas cpDNA EcoRI fragment 13 as the site of aD-loop. This fragment has been cloned in pBR322 (designatedpSC3-1) and sequenced (22). The plasmid pEgcH4 contains a6.1-kb HindIl fragment of Euglena cpDNA cloned intopBR322 (10, 11). The 2.6-kb HindIII-Bgl II fragment used asa probe here, contains a probable origin of replication in theEuglena chloroplast genome based on D-loop mapping (10,11). This 2.6-kb fragment, as well as DNA of pSC3-1, wereused to probe the maize chloroplast DNA for homologousregions by hybridization. Another DNA sequence used as a

Genetics: Gold et al.

Proc. Natl. Acad. Sci. USA 84 (1987)

FIG. 1. Locations on the maize cpDNA map (18) of cloned fragments used as templates for DNA synthesis in vitro. The template activityof each fragment, in cpm x 10-3, is shown in parentheses after the name of the clone. Bold lines denote areas of greatest template activity.Some of the plasmids are named on the basis ofEcoRI orBamHI fragments included in them. The designations of other pBR322 derived plasmidsand their cloned DNAs follow: pZmc 525 contains BamHI fragment 10 (Bam 10); pZmc 503 contains BamHI fragment 15' (Bam 15'); pZmc 460contains BamHI fragment 9 (Bam 9); pZmc 539 contains both BamHI fragments Bam 5 and Bam 15, distant fragments ligated during cloning;pZr 7 contains Eco d; pZmc 556 contains Bam 17: pZmc 566 contains Bam 24; pZmc 561 contains Bam 3; pZmc 573 contains Bam 25; pZmc565 contains Bam 12; pZmc 501 contains Bam 16; pZmc 545 contains Bam 2; pZmc 510 contains Bam 11; pZmc 427 contains Eco o'; pZmc 150contains Eco m in the vector pMB9; pDR 1 contains a BamrHI-Sma I fragment of pZmc 427 in pBR322; pPGA35 contains a promoter deletionfrom the photogene in pZmc 427; pZmc 533 contains Bam 7; pZmc 100 contains Eco a; pZr 36 contains Eco j; pZmc 630 contains Pst 9; pZmc569 contains Bam 20; pZmc 548 contains Bam 1; pZmc 504 contains Bam 6; the order of Eco s' and Eco u within the Bam 9' fragment has notbeen determined. Controls were activated calf thymus DNA (13); pSC3-1, Chlamydomonas chloroplast D-loop DNA sequence (3, 4); pEgcH4,Euglena chloroplast D-loop sequence (10, 11).

probe was pCA2, containing an autonomously replicatingsequence in Chlamydomonas cloned into pBR322 (7).No significant hybridization of maize cpDNA by either

pEgcH4 or pCA2 DNA was observed under relatively per-missive hybridization conditions. However, the BamrHI-ClaI fragment of pSC3-1, that contains a putative D-loop se-

quence from C. reinhardtii cpDNA, hybridized to BamHIfragments 6 (Bam 6) and 17' as well as to Eco x of maizecpDNA. Eco x contains some sequences present in Bam 6and the adjacent Bam 17' fragment and was highly active asa template for the DNA synthesis reaction (Fig. 1). Hybrid-ization to BamHI- and EcoRI-digested cpDNA fragmentsunder permissive hybridization-wash conditions was verifiedby hybridization with maize cpDNA cloned as an EcoRIpartial digest into Charon 4A (18). As shown in Fig. 2,cpDNA in X Charon 4A clones 1, 5, and 6 does not hybridizewith pSC3-1 DNA, while BamHI and EcoRI digests of X

Charon 4A phage 2 do.Despite hybridization of pSC3-1 DNA to the region of the

maize plastid chromosome that was active as a template (Ecox and Bam 17'), this Chlamydomonas cpDNA sequenceshowed lower activity in our assay than did either of the twomaize DNA fragments (Fig. 1). However, the DNAs synthe-sized from pSC3-1 and pZmcEco x as templates were the sizeof the entire plasmid when analyzed by neutral and alkalinegel electrophoresis (data not shown).

Sequence of EcoRI Fragment x. A restriction map of themaize chloroplast DNA region containing Eco x is shown inFig. 3 and the nucleotide sequence of the 1368-bp fragment is

1 2 3 4 5 6 7 8 9 10

FIG. 2. Hybridization of Chlamydomonas D-loop cpDNA tomaize cpDNA. Lane 1, BamHI-digested maize cpDNA; lane 2,EcoRI-digested maize cpDNA; lane 3, EcoRI-digested Charon 4A 1;lane 4, BamHI-digested Charon 4A 2; lane 5, EcoRI-digested Charon4A 2; lane 6, BamHI-digested Charon 4A 5; lane 7, EcoRI-digestedCharon 4A 5; lane 8, BamHI-digested Charon 4A 6; lane 9, EcoRI-digested Charon 4A 6; lane 10, equal mixture of HindIII-digested XDNA and Hae III-digested x175 DNA. The figure in the left-handportion of each lane is a photograph of the ethidium bromide-stainedgel; the autoradiogram of the hybridized DNA blot is at the right. Theprobe was 1 x 107 cpm of the 1055-bp fragment of pSC3-1. Theuppermost hybridizing band in lane 4 is a partial digestion product.

196 Genetics: Gold et al.

Genetics: Gold et al. Proc. Natl. Acad. Sci. USA 84 (1987)

BamHIEco RI 7T

6r x I600I

t17' 1 22'n

_-a

I I I I

s_,s _ z _

Z O-sc Z. Z. WmFxi I1.~~~~

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100 bp100 bp

FIG. 3. Detailed restrictionmap of the region of maizecpDNA containing Eco x (Up-per) and sequencing strategy(Lower). DNA fragments aredesignated in order of descend-ing size (18) or given in bp(EcoRI fragment 600). Arrowsindicate the direction and extentof the DNA regions sequencedby the Maxam and Gilbert (20)procedure.

presented in Fig. 4A. Eco x has an overall A+T content of region of C. reinhardtii cpDNA (22). This homology accounts64%, and several regions can be predicted to form stable for the hybridization patterns (Fig. 2). The homologousstem-loop structures. The sequence extending from nucleo- region in DNAs from both species could code for polypep-tide 650 to 1060 is 67% homologous to the D-loop containing tides that are 56% (C. reinhardtii) and 50% (Zea mays)

A ARS 70

ATCGTAACTT GACTTTTGAA ACAACAAATA AAAATATAAA GCAATAAATA AAGAGCAGCC GGGTTAATAA140

AACTGAGAAA ATTGACTCGG AAAAAAAAAT TGGAAGCTCC ATTGCAGGAT TCAAACCTAA CCATTAAAGG

210AGAAGCTGTG GGAACGACAA AACCTATGAC TACATAGGAT TTATTGAAAG AATCCTAACA CTTCATTGGT

I 280GGATGGCGGA CAAACCACAA AATTGCTTTA TTTGATAAGG TTCTAAATTA ACAAATAAGA CAGGAAAGAG

350TAAATATTCG CCCGCGAAAT CCTTATTGGA TTAGAATACT TTACCACGAT TCAATAAGAA TAAAAAAAAA

420GGAGATTCCA AAAAAGGAAG GATTACATTT TATATAAATA TTGAATTTGG ATATATTCTA GATCTTCTTT

II 490CTTGACTATA TATTTAATTT TTC'NTTTTA AGI1fAAGAA AGTCAAAATC AAAAAAACCT AACTTTTTCT

I I 560ATTATATATT GATGTGTATC TATCTATAAT ATTTTTGAAT ATTATGGAAT TAGAGAAATG ACATTCGCAC

630GCTTCTCATT TCATTCGCGA GGAGCTGGAT GAGAAGAAAC TCTCATGTCC AGTTTTGTAG TAGAGATGGA

ARC ORF1ACTAAGAAGG AACCATCGAC TATAACCCCA AAAGAACTAG ATTTCGTAAA CAACATAGAG GAAGAATGAAAaattatAaa tAaaAaCatg TtaAgtCCaA AAAGAACaAa ATTcCGTAAA CcACAccGtG GtcattTaAg

I 770GGGAAAATCC TGCCGAGGCA ATCATATTTG TTTTGGTAGA TATGCTCTTC AAGTACTTGA ACCCCCTTGGaGGAAAAgCa acaCGtGGtA ATaAaATTgt aTTTGGTgat TtTGCatTaC AAGcACaaGA ACCttqTTGG

840ATCACCGCGA GACAGATAGA AGCAGGACGA AGAGCAATGA CACGATATGC ACGTCGTGGT GGAAAAATATATtACatCac GtCAaATtGA AGCcGGACGt cGtGtttTaA CACGtTATGt tCGTCGTGGT GGtAAAtTAT

910GGGTGCGTAT ATTTCCCGAC AAACCGGTTA CAATAAGACC CACAGAAACA CGTATGGGCT CGGGAAAGGGGGaTtCGTAT tTTcCCaGAt AAAgCtGTTA CtATgcGtCC tgCtGgtACt CGTATGGGtT CtGGtAAaGG

I 980ATCCCCCGAA TATTGGGTAG CCGTTGTTAA ACCAGGTCGA ATACTTTATG AAATGAGCGG AGTATCCGAAtgCaCCtGAt TATTGGGTAG CtGTTGTacA tCCtGGTaaA ATttTaTATG AAATGcaaGG tGTATCtGAA

1050ACTGTAGCTA GAGCAGCTAT CTCTATAGCT GCCAGTAAAA TGCCCATACG AAGTCAATTT CTTCGATTAGACaaTtGCTA GAcaAGCaAT gcgcATtGCa GCttaTAAAA TGCCagTAaa AAcaaAATTT tTaacAaaAa

1120AGATATAGAA CCCCAAAAAG GAATATTGAA GATAAAAAAC CCCTAGTTTT TCTTTCTGGA AAGACAATAT

13 ORF2 1190TTCTTTCATC CTTTTGCATT TGAAATAACA AATGGAAACC AAATAATATG ATTCAACCTC AGACCCTTTT

1260AAATGTAGCG CGATATGCTC GTATTGGTGA TGTTATTATT GCTGTAATCA AAGACGCAGT GCCCCAAATG

1330CGATATGCTC GTATTGGTGA TGTTATTATT GCTGTAATCA AAGACGCAGT GCCCCAAATG CCTCTAGAAA

1368GATCCGAAGT AATTCGAGCT GTAATTGTAC GTACACGT

B -20 30

GGAACTAAG AAGGAACCAT CGACTATAAC CCC AAA AGA ACT AGA TTT CGT AAA CAA CAT AGAZea mays P K R T R F R K Q H RNicotiana tabacwm MLS P K R T R F R K H RChlZamydomonas reinhardii MLS P K R T K F R K P H REscherichia coZi MLQ P K R T|K IF R K | H K

60 90GGA AGA ATG AAG GGA AAA TCC TGC CGA GGC AAT CAT ATT TGT TTT GGT AGA TAT GCTG R M K G K S C N H I C F G RG R M K GI H R G N H I S F GKOY AGL H L R GJ A T R G V F G DF tG ~R|N R 0 L A Q - J T D V S F G S F G

120CTT CAA GTA CTT GAA CCC GCT TGG ATC ACC GCG AGA CAG ATA GAA GCA GGA CGA AGAL Q V L E P A W I T A R Q I E A G R RL Q A L E P A W I T S R Q I E A G R RL Q A I T S JR Q I E A C, R R

K 5A V G 0 R LTA R Q I K A A R P

150 180GTA ATG ACA CGA TAT GCA CGT CGT GGT GGA AAA ATA TGG GTG CGT ATA TTT CCC GACrA M T R Y A R R G G K I W V R I F P DHAM T R N A R R G G K I W V R I F P DV L IT R N V R G0 0 LrnW I P I F P D[A M T R A VKI DI V

210 240AAA CCG GTT ACA ATA AGA CCC ACA GAA ACA CGT ATG GGC TCG GGA AAG GGA TCC CCCK P V T I T E T R M G S G K G S PK P V T L RP A E T R M G S G K G S PK A T M R A C T R M G S G K 0 ALK P I IE K j L A V C V

270 300C:AA. ACA TOG GTA GCC GTT GTT AAA CCA GOT CGA ATA CTT TAT GAA ATG AGC GGA GTAE Y W V A V V K P G R I L Y E M S G VE Y W V A V V K P G R I L Y E M G G V0 Y W V A I L Y E M Q G V|E Y W V AI L I Q P 0 K V L Y E M D G V

330 360TCC GAA ACT GTA OCT AGA GCA GCT ATC TCT ATA GCT GCC AGT AAA ATG CCC ATA CGA|S El T V A R A A I S I A A S K M P I RP

E N I A R R A I S L A A S K M P I RE N I A R Q A H P I A A Y K M P V KE1SE L | A R K A F K L AA A KK

390 432AGT CAA TTT CTT CGA TTA GAG ATA TAG AACCCCAAAA AGGAATATTG AAGATAAAAA ACCCCTS Q F R L E I -a-T Q F I I ST F LI T K T VT T S V T K T V M

FIG. 4. (A) Nucleotide sequence of the 1368-bp Eco x of maize chloroplast DNA. Horizontal lines are drawn above regions related to theyeast ARS consensus sequence, to the conserved elements I and II found in C. reinhardtii ARS fragments, and to the 19-bp consensus sequence(ARC) found in C. reinhardtii cpDNA fragments that promote autonomous replication in the alga. Underlined regions represent sequences thatcan form stable stem-loop structures. Starts and reading directions of the two ORFs (ORF 1 and ORF 2) are marked. The sequence of C.reinhardtii cpDNA D-loop region (nucleotides -17 to +403 in ref. 22) is aligned below its homologous region in maize cpDNA. Conservednucleotides are indicated by capital letters and differences by small letters in the C. reinhardtii sequence. No gaps were introduced for thealignment. (B) Nucleotide sequence of ORF 1 and flanking regions (nucleotides 628-1094 in A). The sequence of the nontranscribed strand ofORF 1 is arranged in codons, and the corresponding amino acid sequence is shown below. Translation was started upstream of the first ATGcodon (indicated by a double line above the triplet) to maximize homology with the primary structures of the ribosomal protein L16 from tobacco(23), C. reinhardtii (22), and E. coli, which are aligned below the maize gene. Conserved amino acids are boxed. Numbering starts at the firsttranslated triplet (CCC). The arrow indicates the position of the 1020-bp intron site in tobacco L16 gene.

197

8 ..-.-w a x

II I~ I

Proc. Natl. Acad. Sci. USA 84 (1987)

homologous to E. coli ribosomal protein L16 (Fig. 4B).Moreover, the similarity between maize open reading frame(ORF) 1 and E. coli L16 can be maximized at the amino acidlevel (53% homology) by taking into account the 13 codonsimmediately upstream of ORF 1 (see Fig. 4B) although noinitiation codon is present. In Spirodela oligorhiza (24),Marchantia (25), and tobacco (23), there is a large intronbetween the first three coding triplets and the remainder ofthe coding sequence of the L16 gene. We, therefore, proposethat the reading frame starting at position 657 in Fig. 4Acorresponds to the C-terminal portion of the L16 protein genein maize cpDNA with its first codons located beyond the 5'end of Eco x. Making this assumption, alignments of thededuced amino acid sequence with those derived from C.reinhardtii and tobacco result in 68% and 85% homology,respectively (Fig. 4B). Unlike the L16 genes of higher plants,the C. reinhardtii gene lacks introns (22).Eco x also contains the initial portion of a second ORF

located 100 bp downstream from ORF 1 (Fig. 4A). Itsdeduced translation product is 54% homologous with E. coliribosomal protein L14 and 82% homologous to tobacco L14(23). This second ORF is absent from the correspondingposition in C. reinhardtii cpDNA (22).

DISCUSSION

Two regions of the maize chloroplast chromosome about 180°apart (and each near an end of one of the large invertedrepeated segments) serve as active templates for DNAsynthesis in a heterologous assay containing a partiallypurified DNA polymerase of pea chloroplasts. Two otherDNA sequences (pZr 36 and pZmc 573 in Fig. 1) are alsoactive. The remainder of the 94% of the maize chloroplastchromosome analyzed supports little if any DNA synthesis.The region of major activity-a stretch including BamHIfragments 17', 22', and 9'-extends over 9 kb, but thetemplate activity of fragments just outside this region falls offprecipitously. The most active region after dissection intosmaller fragments is Eco x, which contains sequences presentin Bam 17' as well as some in Bam 6 (Fig. 1). A furtherargument suggesting that Eco x does contain an origin for thein vivo replication of maize cpDNA comes from its hybrid-ization and sequence homology to a cloned portion of C.reinhardtii chloroplast DNA that has been shown to form aD-loop (3, 4). Surprisingly, the maize cpDNA sequence thatis most active in the enzymatic assay and its homologue in C.reinhardtii are within the coding sequence for ribosomalprotein L16.Although the preceding data strongly suggest that the

regions promoting high DNA polymerase activity in vitro areassociated with the sites for the in vivo initiation of DNAsynthesis, the final proof that these sequences are true originsof replication must come from the identification of D-loopregions in the maize chloroplast chromosome.The exact sequence elements that are required to initiate

chloroplast DNA replication are not known. Generally, A+Trich, stable stem-loop structures with a promoter that canfunction as a template primer are thought to be properties ofgood candidate sequences for DNA replication origins. Onthe other hand, maize cpDNA sequences that have arsactivity, e.g., BamHI fragment 10, show only base levelactivity as templates in our assays (5 x 103 cpm, Fig. 1) anddid not hybridize to Chlamydomonas D-loop DNA. Con-versely, Eco x contains sequence elements that resembleyeast and C. reinhardtii ARS and autonomously replicatingconsensus sequences, respectively (Fig. 4A), but this frag-ment did not show any ARS activity in our experiments.We have used supercoiled DNA as templates; when

nicked, circular DNA was used, the specificity of replication

was drastically reduced. The activity is not dependent uponthe addition of ribonucleotide triphosphates. A more purifiedDNA polymerase derived from these extracts can use DNaseI-digested calf thymus DNA as a template (6) but shows nopolymerase activity with the supercoiled templates providedin our experiments. From this we conclude that the specificreplication observed in these experiments depends uponother proteins in the extract in addition to those requiredsimply for DNA synthesis.

The authors thank Drs. Richard Kolodner, Madeline Wu, BarbaraKoller, and Jean Rochaix for their cooperation and helpful discus-sions while this work was in progress and Ms. Mshikka Chakrabortyfor her expert technical assistance. B.G. was supported by NationalInstitutes of Health Fellowship F32-GM08475 and N.C. was sup-ported by International Congress of Scientific Unions/United Na-tions Educational, Scientific, and Cultural Organizations Distin-guished Fellowship in Science (contract SC/RP/201125.4/A). Thisresearch was supported in part by research grants to L.B. fromNational Science Foundation and the Maria Moors Cabot Founda-tion for Botanical Research of Harvard University and by a grant toK.K.T. from the National Institutes of Health (GM3372503).

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198 Genetics: Gold et al.