Current Genetics

Current Genetics 3, 1 8 9 - 2 0 4 (1981)

© Springer-Verlag 1981

A Physical Map of Nicotiana tabacum Plastid DNA Including the Location of Structural Genes for Ribosomal RNAs and the Large Subunit of Ribulose Bisphosphate Carboxylase/Oxygenase Patrick Seyer 1, Klaus V. Kowallik 2 , and Reinhold G. Herrmann 2 1

Laboratoire de Biochimie Fonctionnelle des Nantes, 13288 Marseille Cedex 2, France 2 Botanisches Institut der Universit~it, Universit~itsstr. 1,4 Diisseldorf 1, FRG

Summary

1) Tobacco plastids contain a homogeneous population of double-stranded circular DNA molecules, 101 Megadalton (160 kbp) in circumference. In neutral CsC1 equilibrium gradients, this DNA displays a density of 1.697 g • cm -3 which is equivalent to an average base composition of 37.7 mole-% G+C. 2) A restriction endonuclease fragment map of the tobacco plastid chromosome is presented for the enzymes Bgl I, Sal I, Xho I and Pvu II which together dissect the DNA molecule into about 60 fragments. The map was derived by sequential digestion employing the previously described Seaplaque technique. The tobacco plastid chromosome has an anatomy similar to that of many other higher plants; the circular DNA is segmentally organized into two unique sequence segments of approximately 24 and 95 kbp separated on each side by a large inverted duplication of at least 20.4 kbp. 3) Saturation and blot hybridization showed that the genes for the 16S and 23S pt-rRNAs are duplicated. Each copy of the inverted repeat contains one set of rRNA genes; about 26 kbp (short distance) separate the sets from each other. 4) Cloned fragments of spinach ptDNA nick translated to high specific activity in vitro were used to probe the location of the large subunit gene of ribulose

Offprint requests to: P. Seyer, Laboratoire Physiologic Cel-

lulaire V6g6tale, Universit6de Grenoble BP 53X, 38041 Grenoble Cedex, France Abbreviations: kbp - kilobase pairs; ptDNA - plastid DNA,

chloroplast DNA; nucDNA - nuclear DNA; mtDNA - mitochondrial DNA; cDNA - Copy DNA; RuBPcase/oase - ribulose 1,5-biophosphate carboxylase/oxygenase; LSU - large subunit; EDTA - ethylene diamine tetraacetic acid (disodium salt); SDS sodium dodecylsuffate

bisphosphate carboxylase/oxygenase on tobacco ptDNA. A 3.5 kbp-long fragment of the large singlecopy region of the tobacco chromosome is complementary to structural sequences of the spinach gene. 5) Mapping and hybridization data suggest that the tobacco and spinach ptDNAs share striking similarities in anatomy and sequence. Key words: Tobacco plastid DNA - physical map rRNA gene mapping - RuBPcase/oase large subunit gene

Introduction

Cooperative interaction of nuclear and organellar genetic systems in autotrophic eukaryotic cells can be influenced by external as well as by internal factors. The expression of nuclear and plastid genes which contribute to thylakoid biogenesis following etiolation appears to be under positive light control (Siddel and Ellis 1975; Apel and Klopstech 1978; Bedbrook et al. 1978; Grebanier et al. 1979; Weinbaum et al. 1979). We have recently demonstrated that the tobacco AG 14 cell line which is able to grow in suspension culture in the absence of cytokinin requires both continuous illumination plus the presence of this phytohormone for differentiation of chloroplasts (Seyer et al. 1975; for review of this subject see Parthier 1979). This development may be accompanied by differential expression of the plastome (plastid genome) since distinct polypeptides are synthesized within plastids during this phase (Lescure 1978). A physical map of tobacco ptDNA would be of great use in the study of phytohormone effects and desirable also for a variety of other reasons. The genus Nicotiana has been extensively characterized taxonomi0172-8083/81/0003/0189/$ 03.20

190 cally, genetically and biochemically, and its tissue and protoplast culture are well developed. R e c e n t examination o f structure and genetics o f RuBPcase/oase, a k e y e n z y m e in CO 2 fixation and photorespiration, by Wildman and coworkers which disclosed the dual origin o f multimeric plastid c o m p o n e n t s as one means o f p l a s t o m e / genome c o o p e r a t i o n (reviewed in B o t t o m l e y 1980) suggests that this material should provide insights into general problems o f plant molecular biology such as the evolution and genetic organization o f plastomes. Plastomes specify a n u m b e r o f significant c o m p o n e n t s , including the structural R N A s for the organelle-located translational machinery and polypeptides associated w i t h p h o t o s y n t h e t i c processes (for review see H e r r m a n n and Possingham 1980). In this paper we describe the isolation o f circular D N A molecules f r o m t o b a c c o chloroplasts and the construction of a detailed restriction endonuclease fragment map for this D N A species. The m a p provides insight i n t o the structural organization o f the t o b a c c o plastid chrom o s o m e and has served as a basis for the localization o f genes of the 16S and 23S R N A species f r o m plastid ribosomes, o f the large subunit o f RuBPcase/oase, and for comparison with physical maps f r o m o t h e r dicotyledon ptDNAs. Preliminary reports o f these results were presented at t h e 31st Mosbach C o l l o q u i u m "Biological Chemistry o f Organelle F o r m a t i o n " ( H e r r m a n n et al. 1980a), the first G o r d o n Conference on " P l a n t Molecular Biology" and the 6th A n n u a l EMBO S y m p o s i u m at Heidelberg.

Material and Methods Tobacco (Nicotiana tabacurn var. Wisconsin 38) and spinach (Spinacia oIeracea var. Monopa) plants were grown in soil in green houses supplemented with 14 h artificial illumination during the winter season. Before collecting leaf tissue, the material was placed in the dark for 48 h to deplete it of starch.

Preparation of Chloroplasts and Chloroplast DNA. Chloroplasts were prepared by the rapid procedure of Walker (1971) with the modifications detailed in Schmitt and Herrmann (1977), and purified by centrifugation in continuous sucrose gradients (Herrmann 1981). The organelles were lysed with detergent and Proteinase K in the presence of isopycnic sucrose (Herrmann 1981) and the DNA was collected from the lysate into a CsC1 cushion by centrifugation (Herrmann et al. 1975). The entire DNA-containing region of each tube was detected by ethidium bromide fluorescence after spotting 1 #1 aliquots of gradient fractions onto Whatman GF/A filter discs as described (Herrmann et al. 1980b). After dialysis the DNA was extracted with phenol, dialyzed in 1 mMTris/HCl, 0.2 mM EDTA, pH 8.0, concentrated I0- to 20-fold in vacuo and stored refrigerated (Herrmann 1981). Restriction Endonuclease Digestion; Agarose GelEleetrophoresis. Restriction endonucleases Bgl I, Xho I, Pvu II and Kpn I were purchased from New England Biolabs (Beverly, Mass. USA) and

P. Seyer et al.: Physical Map of Tobacco Plastid DNA used for digestion of 0.5-4 ~zg DNA in 4 0 - 6 0 ~1 assays under the conditions recommended by the supplier. Sal I was prepared according to Arrand et al. (1978) and incubated in the Xho I buffer. Sufficient enzyme was added in each instance to give a complete reaction. Two procedures for double digestions were utilized. The first method involved sequential digestion, with the second enzyme being added together with additional salt to optimize its activity (Kpn I/Sal I, Bgl I/Sal I, Pvu II/Sal I); in the second method both nucleases were used simultaneously in the buffer for the enzyme tolerating the lowest salt concentration. Horizontal agarose slab gels (20 x 20 x 0.4 cm; 0.3-2.5% agarose depending on the expected fragment sizes) were prepared in 40 mM Tris/acetate, 20 mM Na acetate, 1 mM EDTA, 0.5 /~g ethidium bromide/m1, pH 7.4, as described previously except that the fluorochrome was present during agarose dissolution (Herrmann et al. 1980b). After the application of an initial potential of 3 V/cm for 15 rain at 15 °C to achieve sharpening of bands upon entry of fragments into the gel, the voltage was reduced to 1 (large) or 1.5 V/era (small fragments) and was maintained for 14-18 h until the tracking dye (bromophenol blue) migrated to the bottom of the gel. The gels were transilluminated with short-wave UV and the fluorescence fragment patterns photographed with Polaroid 665 film. Four sets of molecular weight standards were included on each gel for size calibration, lambda DNA, lambda DNA digested with Eco RI or Hind III (all Boehringer-Mannheim, Germany) or Hae III digests of (bX 174 RF DNA (New England Biolabs). A combination of these standards and five concentrations of agarose (single digests: 0.45, 1.2 and 2.1%; double digests: 0.65, 1.2 and 2.5%) allows reasonably accurate size determinations for fragments from 0.1 to 50 kbp even in the non-linear gel ranges. The standards were also included into the samples to account for migration differences due to salt content of the assays. To achieve accurate sizes the gels were relaxed on a thin liquid film on the transillumination plate before photographic documentation. The fragment molecular weights were determined from enlarged photographs of the gels; densitometric scans were taken from negative using a Joyce-Loeble microdensitometer. Because of limitations in the accuracy of their molecular weight determinations, largest fragments were sized by summing their secondary fragments from sequential digestions. Each fragment band is designated by the capital letter of the respective enzyme and consecutively numbered by decreasing size. Secondary fragments are characterized by small Latin letters, alphabetically ordered by decreasing size. Non-identical fragments of multiple bands are distinguished by small letter subscripts. Our previously described technique for restriction cleavage site mapping (Herrmann et al. 1980c; Herrmann and Whitfeld 1981) was used to construct the physical map of tobacco ptDNA. It is based on agarose types like Seaplaque (Marine Colloids Inc., Rockland, Maine USA) which liquefy at or below 70 °C, maintain sol state at or below 37 °C, and lack impurities which inhibit the activity of restriction endonucleases. The method is fast, requires little DNA, and obviates problems of random homology that may be associated with the cRNA or cDNA mapping techniques.

Plasmid DNA Preparation. Spinach ptDNA and plasmid pBR 322 DNA digested with either Sal I, Pst I or Barn HI were covalently joined with DNA ligase, and the recombinant molecules were used to transform E. coil The strain C 600 r , m (hsr-, hsm-, t h r - , leu-) served as recipient. Two positive colonies carrying sequences of the structural gene for the large subunit of RuBPcase/oase, pWHsp 105 (insert: ptDNA fragment

191

P. Seyer et al.: Physical Map of Tobacco Plastid DNA spinach Pst 1-5) and pWHsp 403 (insert: ptDNA fragment Barn HI-3), were employed in our experiments. The transformed strains were grown in L broth in the presence of 30 ~tg ampicillin per ml (strains harboring Bam HI fragments) or 10 gg tetracycline/ml (strains carrying Pst I fragments). Supercoiled plasmid DNA was isolated from scaled-up cleared lysates (Bazaral and Helinski 1968) by ethidium bromide/CsC1 buoyant density centrifugation (Radloff et al. 1967) and used in all experiments. A detailed description of the procedures will be presented elsewhere (Schedel et al., in preparation). The outlined experiments were performed under P2/EK1 containment conditions as specified by the German-Guidelines for Research Involving Recombinant DNA.

Nick Translation. For the preparation of cDNA, multiple restriction endonuclease assays were run on slab gels of SeaPlaque agarose over the full width of the gel. The fragment-carrying gel pieces were excised, melted at 70 °C for 120 s and extracted with buffer-saturated phenol at 32 °C. After concentration of the aqueous phase with iso-butanol (Stafford and Bieber 1975) the fragments were ethanol precipitated for 1 h at - 7 0 °C in the presence of 100 mM potassium acetate and collected by centrifugation (10,000 x g, 40 rain). They were washed twice with 75% ethanol, once with 96% ethanol, briefly dried in a vacuum chamber and dissolved in a small volume of double-distilled water. Nick translation was performed essentially as described by Rigby et al (1977). DNA polymerase I from E. coli and deoxyribonuclease I were obtained from Boehringer Mannheim, FRG, and (c~-32p) deoxyribonucleotide triphosphates from NEN. The reaction mixture was extracted twice with equilibrated phenol, applied to a 7 ml Sephadex G50 (medium, Pharmacia) column and developed with distilled water. The excluded radioactive peak fractions (specific activities 1 - 4 x 108 cpm/~zg) were combined and used for hybridization.

Preparation of rRNA, Radioiodination of RNA. The rRNAs were prepared from unbroken spinach chloroplasts by first pelleting a 70S ribosomal fraction through a discontinuous sucrose gradient (Driesel et al. 1979). The ribosomal fractions were lysed with SDS, extracted twice with two volumes of redistilled phenol, and the RNA precipitated in 70% ethanol overnight at - 2 0 °C. The RNA species were separated by centrifugation in a 5-30% sucrose gradient for 16 h at 30,000 rpm and 2 °C (SW 40 Ti rotor; see Figs. 3 - 6 in Herrmann et al. 1976). After fractionation, appropriate RNA fractions were ethanol-precipitated, successively washed with 70% aqueous ethanol, 96% ethanol, dried and radioiodinated in vitro (Commerford 1971). The specific activity of 16S and 23S rRNA was 1-3 x 106 cpm/gg.

Fragment Transfer, Strip Hybridizations. DNA was transferred from agarose slab gels onto nitrocellulose filters (Sartorius 11308, pore size 0.15) according to Southern (1975). The filters were briefly soaked in annealing buffer modified to reduce background by adding ATP or yeast tRNA and incubated in sealed plastic bags with continual shaking for 12 h at 60 °C in 3 x SSC or in 0.4 x SSC, 0.02% polyvinylpyrrolidone, 0,02% Ficoll, 0.02% bovine serum albumine and 10 mM ATP (Denhardt 1966) with 0.5 ~g radioactive RNA or cDNA per filter respectively. Following hybridization they were washed extensively in 4 x SSC at 50 °C and room temperature and, after drying, exposed to X-ray film for 1 - 4 days at - 7 0 °C using tungsten or titanium intensifying screens.

Analytical

OTtracentrifugation. Analytical density gradient equilibrium centrifugation in CsC1 of native, denatured and

reassociated DNA was performed as described by Herrmann et al. (1975). Micrococcus lysodeikticus [= luteus) DNA p = 1.731 g-em - 3 , served as an internal density marker. Occasionally the centrifuge ceils were overloaded with 5-14 /sg DNA to detect contaminating mtDNA.

Electron Microscopy. Gradient purified ptDNA was dialysed against 10 mM Tris/HC1, 2 mM EDTA, pH 8.0, and mounted for electron microscopy by spreading from a solution containing 0.5 gg DNA/ml, 50 #g cytochrome c/ml (Calbiochem,grade A), 10 m g EDTA, 100 mM Tris/HCl, pH 8.5, and formamide onto an aqueous hypophase. As previously, circular DNA from Acanthamoeba easteIlanii mitochondria (contour length 12.7 urn), donated by Dr. H.-J. Bohnert, was used as internal marker for length calibration (Herrmann et al. 1975). The monolayer was picked up with collodium-coated copper grids, stained, shadowed and examined in the Philips electron microscope EM 300 as described by Herrmann et al. (1975). Length distributions were obtained from projected negatives using a Numonics XY-stage with a digitizing board calculator.

Results A. Preparation o f Plastid DNA The most c o m m o n l y employed procedure for isolating intact DNA molecules from chloroplasts involves preinc u b a t i o n of organdies in DNAase to remove contaminating n u c D N A before the organelles are lysed (Wells and Birnstiel 1969). Although p t D N A of good quality has been prepared from a variety of organisms in this way, in our hands the m e t h o d gave a low yield and DNA o f o n l y moderate molecular weight from tobacco chloroplasts. Staining the chloroplast preparation with the trypanocide fluorochrome DAPI (James and Jope 1979) showed that, although most organelles were morphologically intact, they had lost their DNA during the nuclease i n c u b a t i o n . It is likely that the secondary metabolites o f Nicotiana tissues alter envelope permeability, thus rendering the p t D N A susceptible to nuclease digestion. To circumvent this problem, we adapted a procedure developed for Oenothera ( H e r r m a n n 1977, 1 9 8 l ; Gordon et al. 1981a). After purification o f plastids in continuous sucrose gradients, the p t D N A was extracted in the presence o f isopycnic sucrose to reduce the violence of lysis and subsequent shear forces. Phase contrast and fluorescence microscopy showed that most o f the gradient purified organelles had lost their morphological integrity b u t had n o t released all stromal protein. The fractions did n o t contain m i t o c h o n d r i a b u t were n o t completely devoid o f nuclear fragments. As would be expected, the DNA was moderately c o n t a m i n a t e d with n u c D N A which varied from one preparation to another, b u t in general was 10% or less (see Sections B, 2 and 3). The DNA was purified from the lysate b y overnight centrifugation into a CsC1 cushion and/or b y phenol extraction ( H e r r m a n n 1981). The final, concentrated DNA samples gave stable, highly viscous and occasionally some-

192

P. Seyeret al. : PhysicalMap of Tobacco Plastid DNA

17. Characterization of Tobacco Plastid DNA

iJiNill ~! iliiiilli~!:.i!!'!!!!~i~!

;ii[fiii[?~ 1.697

1.731

1.713 1,731

1.700

1.731

Fig. 1. a - c . Photoelectric scans at 262 n m of (a) native (b) heat denatured and (c) reanealed D N A from Nicotiana tabacum chloroplasts in b u o y a n t neutral CsC1 at 44,000 revs./min and 25 °C. The field is directed to the right. Densities are given in g • cm - 3 . In each case t h e marker band on t h e right at 1.731 g c m - 3 represents Micrococcus lysodeikticus (luteus) DNA

Fig. 2. Slab gel (0.5% SeaKem agarose) electrophoresis of Nico. tiana tabaeum ptDNA after digestion with restriction endonucleases {a) Bgl I, (b) Pvu II, {e) Kpn I, {d) Sal I and (eJ Xho I. Sizes of fragments are given in kbp. The smallest fragments in track b, d and e have migrated out of the gel (ef. Fig. 5). The arrows indicate the diffuse band of contaminating nucDNA

(see text) what turbid solutions indicating the presence of nonnucleic acid material. However, neither non-nucleic acid inpurities nor the small degree of nucDNA contamination affect the physical mapping of ptDNA restriction sites and gene localization (Gordon et al. 1981 a).

1. Buoyant Density Analysis. Since the buoyant density of tobacco ptDNA has been subject to dispute (Whitfeld and Spencer 1968; Tewari and Wildman 1970), we analysed our DNA preparations by CsC1 density gradient equilibrium centrifugation (Fig. 1). Total cellular DNA and DNA from chloroplast preparations gave sharp and unimodal bands at a peak density of 1.697 +0.001 g'cm - 3 (Fig. la). This density corresponds to 37.7 mole-% G+C, on the basis of the relationship between nucleotide composition and buoyant density in CsC1 (Schildkraut et al. 1962) and disregarding the presence of modified bases (Kirk 1967). No distinct species was discernible in any DNA preparation at a density expected for mtDNA (1.706 g. cm - 3). Heat denaturation of both DNAs caused a density shift of +16 mg .cm- a characteristic of duplex DNA (Fig. lb). The relatively narrow banding of the single-stranded form indicated that the DNA could not be extensively nicked. Reannealed ptDNA displayed a single component expectedly at a density position close to that of native DNA (Fig. lc; a slight skewing of the peak to the heavier side that was occasionally noted is indicative of a second density mode). In contrast, the density of total cellular DNA remained almost unchanged under the chosen reannealing conditions (experiment not shown). Thus by reassociation criteria ptDNA could be distinguished from whole-cell DNA. Restriction Endonuclease Analysis, Sequence Complexity. To estimate sequence complexity and for physical mapping analysis, tobacco ptDNA was digested with various site-specific restriction endonucleases. Five hexanucleotide-recognizing enzymes were selected: Bgl I, Pvu II, Kpn I, Sal I and Xho I. Each of these enzymes generates relatively few fragments resulting in an easyly resolvable pattern in primary and double digests. Furthermore, sum of the number of primary fragments from two enzymes usually equals the number of secondary fragments produced in double digests indicating that their cuts are well-distributed along the molecule. Agarose slab gel electrophoresis of primary and secondary fragments produced by digestion of tobacco ptDNA with these enzymes is illustrated by Figs. 2 and 5. Bgl I, Sal I, Pvu II, Xho I and Kpn I generate 8 (10), 11 (11), 12 (14), 18 (24) and 13 (15) size classes (fragments) respectively. Sizes, numbers and multiplicity of all fragments that have been detected in 0.4-2.5% agarose gels are listed in Table 1. Summing the sizes of the individual fragments in each single or double digest series provides estimates for the sequence complexity of tobacco ptDNA. The values varied from 99.7-102 Md or 158-163 kbp with an average of 101 Md or 160 kbp which is in accord with the

P. Seyer et al.: Physical Map of Tobacco Plastid DNA

193

Table 1. Sizes (in kbp) and stoichiometries (in brackets) of primary and secondary fragments of tobacco ptDNA obtained by digestion with the restriction endonucleases Sal I, Bgl I, Pvu II, Xho I, Sal I + Bgl I, Sal I + Pvu II and Sal I + Xho I Primary band Nr.

Sal I kbp

Bgl I kbp

Pvu II kbp

Xho I kbp

Secondary band Nr.

Sal I/Bgl I kbp

Sal I/Pvu II kbp

Sal I/Xho I kbp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

27 23.8 22.2 19.7 16.7 15.2 13.2 11.4 5.6 2.9 0.65

39.7 29.4 (2x) 19.7 11.6 9.2 7.1 (2x) 3.9 2.1

42.9 19.8 17.5 14.3 12.1 10.3 10.0 8.6 6.35 4.1 (2x) 3.5 2.5 (2x)

21.9 17.9 15.55 12.7 (2x) 11.75 10.5 9.2 8.9 5.8 5.4 3.8 3.1 (2x) 3.0 (3x) 2.6 2.1

a b c d e f g h i j k 1 m n o p q r s t u v w x y z

25.1 19.0 15.9 (2x) 14.3 (2x) 10.0 9.2 7.9 5.6 4.0 (2x) 3.0 (2x) 2.3 2.1 1.9 1.4 1.3 1.0 0.65

14.9 13.5 (2x) 12.1 10.6 (2x) 9.4 8.7 (2x) 6.3 5.7 5.5 4.9 (3x) 4.2 (3x) 3.5 3;2 2.9 2.4 (2x) 1.55 0.65

14.0 10.2 9.1 (2x) 8.7 7.6 7.5 (2x) 7.0 (2x) 6.5 5.9 5.6 (3x) 5.2 4.2 3.8 3.3 3.0 (2x) 2.95 (2x) 2.4 2.1 (2x) 1.9 1.5 (2x) 0.82 0.76 0.63 0.56 0.44 0.35

16

1.55

17 18

0.8 (3x) 0.7

Sum:

158.35

159.2

158.55

160.65

contour lengths o f circular DNA molecules from tobacco chloroplasts.

3. Structure. The narrow equilibrium banding of DNA (Fig. 1a) and the approximate stoichiometric appearance even of the largest restriction fragments amounting to more than one fourth of the circle contour length (Fig. 2) suggested that our tobacco ptDNA was not appreciably fragmented. To evaluate the presence o f the large circular molecules, tobacco ptDNA was extensively dialysed and prepared for electron microscopy b y cytochrome spreading. Examination of this material showed that a substantial percentage o f DNA, 4 0 - 7 0 % in several separate preparations, consisted of relaxed circular molecules with a homogeneous size distribution and a corrected mean contour length of 48.5 -+ 1.6 /~m. This size is equivalent to a molecular weight o f 101 x 106 dalton or 160 kbp. The equivalence o f the circle circumference and the sequence complexity determined b y restriction analysis proves that this DNA molecule is a monomer. Fig. 3 shows an electron micrograph o f such a molecule. A histogram o f 44 randomly selected, well-spread circular molecules is presented in Fig. 4. One open circle having a contour length o f 109 # m was found which

161.85

163.4

160.36

probably represents the infrequent class of unicircular dimers. The remaining DNA was linear and heterogeneous in size. A b o u t 10% exceeded in size the circular monomer, and may be attributed primarily to nuclear contamination as mentioned above (cf. Fig. 2).

C The PhysicalMap The strategy employed to order restriction fragments has been detailed elsewhere (Herrmann et al. 1980 c; Herrmann and Whitfeld 1981). Derivation o f the tobacco ptDNA map was based primarily on the identification o f the overlapping piece produced in reciprocal experiments by sequential digestion of individual fragments with the endonuclease pairs Sal I/Xho I, Sal I/Bgl I or Sal I/Pvu II. Only in ambiguous instances were other combinations o f these enzymes evaluated. Primary restriction digests of tobacco ptDNA were first electrophoreticaUy fractionated on Seaplaque tube gels. Each individual DNA band was excised, then directly digested with the reciprocal enzyme after agarose liquefication and the resulting products were immediate-

194

P. Seyer et aL: Physical Map of Tobacco Plastid DNA Table 2. Relationship of primary and secondary fragments produced by digestion of tobacco ptDNA with Sal I and Xho I (cf. Fig. 5, A-D) Secondary fragment Sal I/Xho I

Primary fragment from which secondary fragments are derived Sal I

Fig. 3. Electron micrograph of a circular DNA molecule obtained from Nicotiana tabacum chloroplasts. The bar indicates 1 t~m

a b Ca, b(2X) d e fa, b(2x) g (2x) h i Ja, b,b(3x) k 1 m n o (2x) p (2x) q ra, b (2x) s t u (2x) v w x y z

2 1 4, 6 3 8 5, 5 1, 4 1 2 3, 7, 9* 6 3 7 2 1,4 3, 7 8 5, 10 6 8 1,4 10 3 11 7 11

Xho I 1 6* 3, 7* 8* 2 1, 5 4, 4 3 9* 2, 4, 4 2 5 11" 10 12", 12" 13g, 13g 13 a 14, 15" 10 16" 17g, 17g 17a 18" 13 a 14 17 a

Fragment stoichiometries in brackets * Primary fragments lacking a cleavage site for the reciprocal enzyme

I0, u 0

E

5.

45

50

55

pm

Fig. 4. Length distribution (ungorrected) of open circular DNA molecules prepared from tobacco chloroplasts. After correction for the internal size standard, the average size is 48.5 ~m

ly analysed b y slab gel electrophoresis. Single and double digests of total ptDNA were included in parallel tracks for band identification. Various agarose concentrations were used to resolve small and large fragments respectively. This approach is illustrated b y Fig. 5; the relationships between primary and secondary fragments are tabu-

lated for each enzyme pair (Tables 2 - 4 ) . The map locations o f fragments can be deduced from the Table columns, first proceding up and down to find the subfragments of a chosen primary fragment (vertical reading) and subsequently back and forth to relate corresponding fragments from a given enzyme pair (horizontal reading). Reading should be continuous and consistent with one fragment arrangement. Due to a fortunate constellation of bi- and trimolar bands, the chosen enzyme combinations presented no mapping difficulties: In double digests many multiple bands contain either an undigested primary fragment, arise from one primary fragment, or the secondary fragments o f multiple primary fragments allow only one possibility of combination.

The Sal I/Xho I map: The Sal I/Xho I series allows the unambiguous ordering o f two large groups o f fragments. The first o f these segments can beconstructed starting with X-5. The only two secondary fragments, 1 and fa,

P. Seyer et al.: Physical Map of Tobacco Plastid DNA obtained after cleaving this primary fragment with Sal I (vertical reading), overlap with S-3 and S-5 respectively (horizontal reading). These Sal primary fragments must therefore be placed adjacent to each other. S-5 gives rise to two bands, one of which is bimolar. This stoichiomerry is evident from densitometry of double and sequential digests (Fig. 5, B), calculating the S-5 molecular weight from its secondary fragments (Table 1) and from comparison with the reciprocal series (Fig. 5, A). Thus S-5 is composed of two terminal fragments, fa and fb, of different sequence but equal in size plus an internal piece, the primary fragment X-15 (re). Subfragment fb extends into X-1 which upon cleavage with Sal I also gives rise to fragment a and overlaps S.2. Digestion of S-2 with Xho I produces subfragments n and i in addition to a. The reciprocal series illustrates that i is identical to the uncleaved primary fragment X-9 and therefore located centrally within S-2. Fragment n is terminal and overlaps with X-10. This fragment in turn extends into S-6 via the common secondary fragment s. S-6 also gives rise to subfragments Ca and k, of which the former represents the uncleared primary fragment X-7 (Fig. 5, A); k forms the junction to X-2. S-9 links S-6 and S-8; X-2 forms the homologous overlap: it encompasses S-9 (Ja), shares k with S-6 and e with S.8. S-8 includes X-16 (t) and with its segment q overlaps into one fragment of the triple band X-13 (X-13a). The two other fragments are an identical pair (X-13b) and are shown below to be part of an inverted duplication. X-13 a overlaps S-8 and S-11, the latter being the smallest primary fragment in the Sal I series. The overlap sequence to S-11 is x. S-11 contains an asymetric Xho I cut which also results in the secondary fragment z. This smallest subfragment (0.08 kbp) is the only one missing from the digest profiles. It can be indirectly inferred from the size reduction of S-11 and, by similar reasoning, must also derive from X-17 a (Fig. 5, C), one fragment of the triple band X-17, in the reciprocal series. (As will be outlined subsequently, the bimolar identical fragments X-17 b are again part of an inverted repeat.) The juxtaposition of S-8-11-10 is confirmed directly by the Bgl I series (Fig. 5, G and H). The larger secondary fragment of X-17a, v, is part of S-10. The second Xho fragment of S-10, r u, must overlap into X-14 which also possesses one Sal I site. X-14 must therefore be positioned adjacent to X-17 a. Its other secondary fragment, y, is derived from S-7 in the reciprocal experiment. Taken together, these results give a colinear arrangement of 9 of the 11 Sal fragments: S-3-

5-2-6-9-8-

11 - 1 0 - 7 - ,

and of 11 of the 23 Xho fragments: X-5-1514 - .

1-9-

10-7-2-16-13

a-178-

195 The secondary fragments can also be linearly arranged as follows: - 1-fa-ra-

fb--a--i--n--S--Ca--k--ja--e-t--q--x--z--v--r b --y-The ordering of the second set of fragments, including both unique and repetitive sequences, can be determined by the following reasoning: S-1 gives rise to five secondary fragments, b, g, h, o and u (Fig. 5B, D). Of these, g and h are terminal fragments overlapping into X-4 and X-3, respectively; b, o and u remain uncleared in the reciprocal series and thus represent the primary fragment X-6, one of the bimolar primary fragments X-12 and one of the bimolar primary fragments X.17 b. Their arrangement within S-1 has been deduced using Pvu II (see Table 4). Continuing on one side, X-3 also generates fragment c b which in the reciprocal experiment is derived from S-4 (see above). Xho I cuts this fragment in turn into the secondary pieces g (terminal), o and u (again identical with X-12 and X-17 b respectively). This leads to a symmetrical arrangement represented by a central region of unique sequence flanked by a duplicated segment in inversed orientation - g - o - u - b - h - Cb -u-o-gThe further arrangement of the Sal fragments confirms the existence of a large (repetitive) inversion in the tobacco plastid chromosome. Both S-1 and S-4, overlap into ?(-4 which is a bimolar band of two identical fragments. When X-4 is cleaved with Sal I, it produces two bimolar, identical products (g and Jb), which in the reciprocal experiment are obtained from $ 4 or S-1 (g) and from S-3 or S-7 (Jb) respectively. S-3 contains one of the two molar fragments Jb, the one molar primary fragments X-18 (w), X-8 (d), one of the bimolar primary fragments X-13 b (p) and the terminal fragment 1 which overlaps into S-5 as outlined above. S-7 in turn contains the terminal fragments Jb and y (linking to S-10 as outlined above) and harbors one of the bimolar primary fragments X-13 b (p) as well as the one molar primary fragment X-11. This extends the inverted symmetrical structure to p - Jb -- g -- o -- u -- b - h - cb - u - o g - - J b - - P and leads to a circular arrangement of all the fragments encountered. Thus, it appeared that the tobacco plastid chromosome is organized into four well defined segments as has been previously shown for other plastid chromosomes (see Discussion). The Bgl I and Pvu II maps: The enzymes Bgl I and tMa II were employed to confirm and refine the Sal I/Xho I map. Representative gels of these series are illustrated by Fig. 5 G, H and I respectively. In the Bgl series (Table 3), S-2 contains B-8 (secondary fragment 1) and overlaps into B-5 (common secondary fragment g) which itself is also related to S-6 (u). The latter primary fragment extends into B-2. The band B-2

196

P. Seyer et al.: Physical Map of Tobacco Plastid DNA

Fig. 5. A-I. Agarose slab gel electrophoresis of secondary digests of Xho I (A and C) and Sal I (B and D) primary fragments of tobacco ptDNA. The (horizontal) tube gel patterns at the top of subfigures A and B illustrate the respective primary digests. The slab gel tracks are marked with the respective fragment number (cf Table 1). The centrally positioned reference digests are (from left) Xho I, Xho I + Sal I and Sal I. Subfigures E and G, F and H represent a similar analysis of Bgl I and Sal I primary fragments respectively; subfigure I illustrates the pattern obtained by digesting Sal I primary fragments with Pvu II. Gels A, B, E, F and I: 0.7% agarose, gels C, D, G and H: 1.7% agarose. A few cross-contaminations resulting from the primary gels, or partial digestion products are marked by points at the left of the respective band. Incompletely digested residual, primary fragments that can serve as internal standards in the alignment of fragments are occasionally indicated by a r r o w

consists of two non-identical primary fragments of almost equal size. Its cleavage with Sal I generates five secondary fragments, c, da, b, f, h in stoichiometric ratio 1 : 2 : 1 : 1 and their molecular weights comprise a length twice that of the fragment. The only possible arrangements of these five subfragments are c + d a and d b +

h + f, h being the primary fragment S-9 which in fact has above been localized as the linking fragment between S-6 (db_) and S-8 (f). This implies that the two B-2a secondary fragments overlap S-2 (da) and S-5 (e) again in accordance with the previously established Sal/Xho map. B-3 extends into S-5 as well as S-3 which contains part of

P. Seyer et al,: Physical Map of Tobacco Plastid DNA

one copy of the duplication confirming that S-5 and S-3 are neighbours (B-5 secondary fragments o and b, respectively). The other secondary fragment of S-8, k, extends into B-7 which in turn contains the small primary fragment S-11 and overlaps S-10 with its subfragment p. The fragment B-4 brackets S-10 and 8-7 which extends into the other copy of the duplication. The Bgl series also confirms that S-1 and S-4 are adjacent and reveals the existence of an inverted repeat via B-6. A similar set of experiments was carried out with the Pvu II/Sal I double digest which further increased the resolution of the map, particularly within the inverted

197

repeat. The relationships of primary and secondary fragments in this series are listed in Table 4. Figure 6 presents the composite map drawn to scale for the cut positions of these four enzymes.

D. Physical Mapping of Genes L Localization of the Genes for 16S and 23S rRNA. To identify the fragments encoding sequences for pt-rRNA species, we used Southern's filter hybridization technique essentially as described in our previous work (Whitfeld et

198

P. Seyer et al.: Physical Map of Tobacco Plastid DNA

Table 3. Relationship of primary and secondary fragments produeed by digestion of tobacco ptDNA with Sal I and Bgl I (ef. Fig. 5, E - H ) Secondary fragment Sal I/Bgl I

Primary fragment from which secondary fragments are derived Sal I

a b Ca, b (2x) da, b (2x) e f g h i (2x) j (2x) k

1 3 4, 5 2, 6 7 8 2 9* .1, 4 3, 7 8

Table 4. Relationship of primary and secondary fragments produced by digestion of tobacco ptDNA with Sal I and Pvu II (el. Fig. 5, I) Secondary fragment Sal I/Pvu II

Bgl I

Primary fragment from which secondary fragments are derived Sal I

1 3 1, 2 a 2a, 2b 4 2b 5 2b 6, 6 6, 6 7

a ba, b (2x) c da, b (2x) e fa, b (2x) g h i Ja, b, b (3x) ka, b, b (3x)

1 3, 7* 8* 2, 5 3 2, 4 6 9* 6 1, 2, 4 1,4,6 5

1

2

8*

1

m n o p q

10 6 5 10 11'

4 5 3 7 7

m n o p q

Fragment stoichiometries in brackets * Primary fragment lacking a cleavage site for the reciprocal enzyme

5 10' 1, 4 1 11"

Pvu II 4* 1, 3 1 2, 2 5 7, 8* 9* 1 6 1, 3, 6 1, 10", 10" 11" 5 1 12", 12" 7 1

Fragment stoiehiometries in brackets * Primary fragment lacking a cleavage site for the reciprocal enzyme

Fig. 6. The physical map of the Nicotiana tabacum plastid chromosome. The fragment nomenclature used is outlined in Material and Methods. The upper part of the Figure represents the larger singlecopy, the lower the smaller single-copy region. The bold region and the expanded parts show the inverted duplication. The symbols indicate cleavage sites of v = Sail, v = BglI, I = Xho land (> = Pvu II. The positions of the genes for 16S and 23S rRNA (shaded area) and the large subunit (LSU) of RuBPcase/oase are indicated. The smallest DNA segments to which hybridization was observed is marked. See text and Tables 1 - 4 for details of map construction and gene localization

P. Seyer et al.: Physical Map of Tobacco Plastid DNA

199

Fig. 7. Strip filter hybridization of radioactively labelled 16S and 23S pt-rRNA (spinach) to various restriction fragment patterns of tobacco ptDNA. The DNA fragments were separated by electrophoresis on 0.6% agarose gels, denatured, transferred to nitrocellulose paper by Southern blotting and the filters incubated with the radioactive RNA probes. On the left are the respective gel patterns, and on the right, the corresponding autoradiographs obtained with {a) 16S and (b) 23S rRNA

al. 1978; Gordon et al. 1981a). High molecular weight tobacco ptDNA was hydrolysed to completion by various restriction enzymes. The resulting fragments were separated by electrophoresis through agarose slab gets, directly transferred to nitrocellulose sheets and annealed with radioio~linated 16S or 23S rRNA prepared from spinach chloroplast ribosomes. Fig. 7 illustrates the results obtained with the enzymes Bgl I, Sal I, Xho I, Pvu II and Kpn I. After cleavage with the endonuclease Bgl I, only one fragment, B-l, was found to hybridize with both rRNA species. This large fragment encompasses the entire small single-copy segment of the chromosome as well as approximately one third of each of the two inversely oriented copies of the adjacent repeats (Fig. 6, see foregoing Section). The endonuclease Sal I dissects this Bgl fragment within the unique sequences to produce two secondary fragments, a and Ca, differing in size and overlapping with S-1 and S-4, respectively (Fig. 5 E and F, Fig. 6, Table 3). Incubation of a2SI-rRNA with the Southern blots of the Sal I fragments generated two prominent

bands of radioactivity corresponding in mobility to these two primary fragments. The finding that both 16S and 23S RNA bind to these fragments indicated the presence of two distinct rDNA regions, as known for other higher plant ptDNAs. Additional information bearing on this point could be obtained by analysis of the Pvu II, Xho I and Kpn I fragmentation patterns. Three distinct Pvu II bands were found to hybridize to 16S rRNA. The most prominent band showing sequence homology is the bimolar P-12 fragment. The unique fragments P-1 and P-3 hybridized less intensely. These unique fragments symmetrically span the junction from P-12 on both sides into the large single-copy segment and each one thus includes two thirds of one copy of the duplicated segment (Fig. 6). This establishes the existence of two, widely distant 16 S rRNA genes in the tobacco plastid chromosome which start in P-1 and P-3, respectively, and procede into P-12 in each copy of the duplication. Differences in the transfer efficiency between large and small fragments from agarose gels pre-

200 elude a more accurate assignment of the gene sequences on the basis of hybridization intensities. The 23S RNA exclusively hybridized to P-IO. This fragment is repetitive and its two copies are inversely arranged neighbouring P-12, respectively. This places the genes for the large rRNA chains symmetrically on the molecule between the 16S rRNA genes and the small single-copy region. Hybridization performed with Xho I fragmentation patterns are consistent with these findings: the 16S rRNA sequences'are complementary to the bimolar fragment X-4, and the 23S RNA hybridized to X-12 and X-17 b (the latter has migrated off the gel bottom in Fig. 7). Two fragments of the trimolar X-17 band derive from P-IO and constitute the junction between X-12 and ?(-3 on one copy of the repeat and between X-12 and X-6 on the other towards the small single-copy region. Both, X-3 and X-6 give an autoradiographic signal. Collectively these data allow the conclusion that the rRNA genes of tobacco ptDNA are organized into two sets each of which contains one gene for 23S and 16S RNA. The intramolecular position of these sets is widely distant; they are inversely oriented and are part of the large inverted duplication of the molecule (Fig. 6). One copy of the 16S rRNA is represented by the fragment order 1)-3 - P-12 (X-4 - X-12) and the other copy by P-1 - P-12 (X-4 - X-12). The 23S rRNA genes lie on the P-10 fragment on both sides of the circle (X-I2 X-17 - X-3 on one copy and X-12 - X-17 - X-6 on the other). The secondary fragment bounded by the cuts X-4/X-12 and P-12/P-10 does not hybridize and must therefore be part of a spacer region.

2. Localization of the Gene for the Large Subunit of RuBPcase/oase. The eukaryotic RuBPcases/oases are multimeric soluble plant proteins which control key reactions of the photosynthetic carbon reduction cycle and photorespiration. Due to their abundance and the dual genetic origin of their polypeptides (Chen et al. 1977), these proteins have been the focus of considerable scientific research. The enzymes are composed of two subunits of different sizes, each present eight-fold per molecule (Baker et al. 1977). The smaller subunit is of nuclear origin and imported into the organelle from the cytosol, while the large, catalytic subunit (LSU) is plastome-coded (reviewed by Bottomley 1980). Based on a polypeptide molecular weight of 52 kd, the minimum coding length for LSU is about 1.6 kbp or about 1% of the chromosome contour length. We have recently mapped the structural gene for LSU to the larger of the two unique-sequence regions of the spinach plastid chromosome (Herrmann et al. 1980a; Schedel et al., in preparation). The fragments Bam-3 (B-3) and Pst-5 (P-5) carrying all or part of the LSU gene respectively have been cloned using pBR 322 as a vec-

P. Seyer et al.: Physical Map of Tobacco Plastid DNA tot have taken advantage of the availability of these clones and of the fact that the LSU gene is phylogenetically conserved to probe the position of this gene as well as surrounding sequences in tobacco ptDNA. The experiments follow the general design used to locate the gene in Oenothera ptDNAs (Schedel et al., in preparation). Both B-3 and P-5 from the spinach plastome yield 4 secondary fragments upon digestion with Kpn I. The B-3 secondary fragments have sizes of 0.8, 1.6 (the primary Kpn I fragment which encodes most of the LSU structural gene), 4.2 and 5.1 kbp, while the P-5 secondary fragments have sizes of 0.7, 4.2, 5.1 and 2.1 kbp. The fragments occur in stoichiometric amounts and map in the order given. These secondary fragments as well as the entire B-3 or P-5 were labelled in vitro to high specific activity by nick translation (Rigby et al. 1977) and hybridized to Southern blots of tobacco ptDNA fragments. Hybridization was visualized by autoradiography and the gene position deduced from the map position and molecular weights of the hybridizing fragments. Figure 8 shows the tobacco ptDNA geI patterns after digestion with one of five endonucleases, as well as hybridization patterns of the 32P-labelled spinach B-3 primary fragment and its individual Kpn I secondary fragments. The fragments hybridized to common or related fragments in all digests. Vector DNA labelled by nick translation hybridized to itself but did not react either with the ptDNA digest nor to the B-3 or P-5 fragments derived from the chimeric plasmids (experiment not shown). The cDNA of the entire spinach B-3 fragment hybridized to tobacco fragments B-2/B-5, S-6/S-2, P-6/P-9, X-7/X-10 as well as K-5/K-6 and K-9. These fragments share a common segment which allows immediate recognition of the signal position within the large singlecopy region of the chromosome. To locate the gene and its surrounding sequences more accurately, the Kpn I subfragments of spinach B-3 or P-5 were hybridized to Pvu II, Xho I and Kpn I fragmentation patterns of tobacco ptDNA. The 1.6 kbp spinach secondary fragment hybridized to tobacco fragments corresponding to P-9, X-7 (and K-6) which therefore should carry most, if not all of the LSU structural gene. Again, all these fragments share a common segment. When the adjacent spinach 4.2 kbp secondary fragment is hybridized to Xho I fragments, a single autoradiographic signal was observed (?(-7). Against the Pvu II digest two signals were apparent (P-9/P-6). These resuits are again consistent with the mapping data. The terminal spinach 5.1 kbp secondary fragment hybridized to DNA fragments in the same region (P-6; X-7/X- 10). Hybridization with B-5 would be predicted and can be seen under somewhat less stringent reannealing conditions (experiment not shown). Thus all data can be accomodated in the tobacco map which should contain

P. Seyer et al.: Physical Map of Tobacco Plastid DNA

201

Fig. 8. Localization of the RuBPcase/oase LSU gene in tobacco ptDNA. DNA of the chimeric plasmid pWHsp 403 obtained from spinach ptDNA (insert Barn HI-3 fragment) was used as sequence probe. The excised insert or its four products obtained by further digestion with Kpn I were fractionated by Seaplaque electrophoresis. After nick-translation these fragments were hybridized against various restriction patterns of total tobacco ptDNA. In each set the left pattern is the ethidium bromide stained gel of tobacco ptDNA (0.6% SeaKem agarose). The autoradiographs on the right show the hybridization with the entire insert fragment and subsequently its Kpn subfragments in order of their position in the spinach ptDNA map. The fragment sizes in kbp are given on top. The 1.6 kbp fragment carries most of the structural sequence for LSU. The hybridizing tobacco DNA fragments are numbered in decreasing size. The hybridization patterns indicate that tobacco and spinach ptDNA contain extensive homology

substantial homology in this region to the spinach plastome (see Discussion).

Discussion

This paper adds tobacco 'Nicotiana tabacurnj to the dozen other plant species from which plastid chromosomes have been isolated in intact form (for review see Herrmann and Possingham 1980). Although only 70% or less o f the DNA has been isolated as circles, the coincidence of the size determined by electron miroscopy and by restriction endonuclease analysis indicates that tobacco ptDNA exists as one molecular species with an average monomer size of 160 kbp (48.5 ~tm). This conclusion is substantiated by the construction of a physical map for this DNA which always resulted in a circular structure. The DNA species also shares size, base composition, easiness of reassociation and molecular anatomy with many other higher plant ptDNAs. More than 20% of its sequences are repetitive. We attribute the linear molecules in our preparations to random breaks in the large fragile circular molecules. When these molecules exceed the monomer length, they probably represent nuclear contamination. Bulk nucDNA was removed by isopycnic centrifugation instead of

DNAase treatment of chloroplasts prior to lysis. The effectiveness of this procedure was evident by highcomplexity DNA found in the chlorophyll-free gradient pellet. However, fluorochrome staining disclosed that its removal was incomplete. Only with plant species that possess pt- and nucDNA differing in buoyant density can this contamination be demonstrated directly by equilibrium centrifugation (Gordon et al. 1981a). No small circles of 0 . 5 - 4 ~m in circumference reported by Wong and Wildman (1972) to occur in tobacco chloroplast were observed although those should have been overrepresented since our isolation procedures select against large molecules. This sizes o f plastid chromosomes reported to date range from 120 to 200 kbp. Those of vascular plants fall into a relatively constant size range and tobacco ptDNA with its 160 kbp is no exception to this. The accuracy of size determinations by gel techniques is limited primarily by the choice of the enzyme and the difficulty in calculating the molecular weights of large fragments. Sizes of ptDNA determined with the aid of commonly encountered enzymes like EcoRI or Barn HI are generally underestimations. These endonucleases produce a large number of small fragments and under the electrophoretic conditions usually employed these fragments escape detection. In contrast the four site specific restriction

202 endonucleases Bgl I, Sal I, Pvu II and Xho I chosen here for sizing tobacco ptDNA generate only a relatively small number of fragments (Table 1)and within each single and double digest series gave coinciding sizes within the errors of the method. It is thus likely that these represent the entire tobacco plastid chromosome. If there were small undetected fragments, together they can account for only a negligible fraction of the chromosome. When the necessary precautions were taken (appropriate gel concentrations, voltage gradients and secondary magnification of prints, internal size standardization and relaxation of gels), sizes of individual fragments varied usually less than 3% from one experiment to another. Since small changes in opposite directions cancel each other out, slight variability should not significantly affect contour length determinations. Differences up to 0.5 kbp were occasionally noted when sizes of large primary fragments were compared with the sum of their secondary fragments. In two such cases sizes of large fragments were adjusted to coincide with data obtained from secondary fragments. Hence the absolute length given may be slightly in error but this does not significantly affect the relative order of restriction sites. The fragments produced by digestion of tobacco ptDNA with Bgl I, Sal I, Pvu II or Xho I have been analysed electrophoretically and their location on the chromosome has been determined using sequential digestion as the major tool for map construction. The data obtained with the three pairs of enzymes are mutually consistent, and are further confirmed by specific hybridization experiments. The tobacco ptDNA shares a characteristic anatomy with plastid chromosomes of many other higher plants. It is organized into 4 well-defined segments: a long duplicated inversion interspersed with two single-copy regions differing in size. The Xho map allows definition of the limits between the repeat and the large single-copy region within 0.7 kbp because the small fragment X-18 adjacent to the repetitive fragment X-13 is unique. (X11 is the corresponding unique fragment on the opposite site.) In the other direction towards the small singlecopy region, the delimitation is less accurate. The bimolar primary fragment P-10 is definitely a part of the repeat but a portion of the adjacent fragment P-7 and an equivalent stretch of P-4 may also contain some repetitive sequences. Therefore the minimum length of one repeat copy is 20.4 kbp and defined by the fragment sum X-4, ?(-12, X-17 b plus the rest of P-10. The maximum size of the small single-copy segment is 24.3 kbp (P-4 plus P-7), while the size of the large singlecopy region is about 95 kbp. The segment sizes of tobacco ptDNA are similar to those of other dicotyledon plastid chromosomes. The corresponding figures in spinach are 23.8 (2x), 20.6 and 85.6 kbp, those in Oenothera plastome IV DNA 23.8 (2x), 22.2 and 90.4 kbp (Herr-

P. Seyer et al.: PhysicalMap of Tobacco Plastid DNA mann et al. 1980a; Gordon et al. 1981 a). In the five basic Euoenothera ptDNAs such changes result from multiple insertions/deletions indicating that the slight variations in contour lengths between the DNAs are real (Herrmann et al. 1980a; Gordon et al. 1981b). As with other higher plant ptDNA the only remaining ambiguity is the alignment of the two single-copy regions relative to each other (Herrmann and Possingham 1980). It should be noted that Jurgenson and Bourque (1980)have recently presented a restriction site map of tobacco ptDNA which coincides with ours in the overall anatomy of the molecule but differs in circumference (146 kbp), number and arrangement of Sal I and Xho I fragments. It is not possible to reconcile their arrangement with ours since detailed mapping data are not yet available. The comparison of the tobacco map with those of spinach (Herrmann and Possingham 1980) and the five basic Euoenothera plastomes (Gordon et al. 1981 a, b) indicates that the overall organization of these DNAs is remarkably similar notwithstanding the fact that these species belong to three different dicotyledon genera and that the restriction patterns of their ptDNA have rarely any fragment in common as far as size is concerned. Nevertheless the fragment patterns obtained with particular endonucleases exhibit general similarities and the size distribution of fragments at analogous map positions often corresponds as does the gene order probed (Herrmann et al. 1980a). A few examples may illustrate this. Concentrating on tobacco and spinach only, it is possible to align their maps using for example the smallest Sal I primary fragment (band S-11) which is generated from both DNAs. In both instances this fragment is positioned in the large single-copy segment proximal (about 5 kbp away) from one copy of the repeat. In both instances, this fragment lacks sites for many hexanucleotide-recognizing enzymes but it is asymmetrically cut by Xho I, with the smaller of the subfragments being located nearer to the repeat (for spinach see Herrmann and Possingham 1980). The evolutionary conservation of primary sequences for rRNAs, their arrangement in sets and possibly polycistronic expression in plastid chromosomes are well known. Plastid rRNAs from a wide variety of higher plants have been shown to have a high degree of sequence homology (Ingle et al. 1970; Thomas and Tewari 1974) and all ptDNAs studied have the rRNA cistrons in close proximity separated by a spacer (reviewed in Herrmann and Possingham 1980; for tobacco data see Kusuda et al. 1980 and Jurgenson and Bourque 1980; these data are in good agreement with ours except for the location of two Pvu II cuts - cf. Fig. 4 in Kusuda et al.). This intercistronic spacer ranges in size from 0.28 (Euglena, Orozco et al. 1980) to 2.4 kbp. In tobacco this spacer is 2.1 kbp long (Kusuda et al. 1980) sim-

P. Seyer et al.: Physical Map of Tobacco Plastid DNA ilar in size to that of maize (Bedbrook et al. 1977), larger than that of spinach (1.8 kbp, Bohnert et al. 1979) and smaller than that of Oenothera (up to 2.4 kbp, Gordon et al. 1981a, b). In spinach (Driesel et al. 1979; Bohnert et al. 1979), maize (Koch et al. 1980) and Euglena (Orozco et al. 1980) the spacer has been shown to carry tRNA genes that differ in the presence or absence of intervening sequences. Transcription in spinach chloroplasts, as in E. coli (Lund et al. 1976), proceeds from the 16S towards the 23S rRNA gene and yields a common precursor molecule of 2.7 Md molecular weight (Bohnert et al. 1976). The similarity in molecular anatomy suggests that this expression ofrDNA units is common to all plastomes. As seen in the spacer, dosage and intrachromosomal arrangement of rDNA equivalents may be more variable. The pt-rRNA genes in many higher plants including tobacco exist in duplicated sets as inverted repeats. The two units are symmetrically arranged and the ends of their 5S rRNA genes (for tobacco see Kusuda et al. 1980; Takaiwa and Sugiura 1980a, b) are separated from each other by approximately 20-24 kbp. However in Fabaceae the precence of only one set (Vicia faba, Koller and Delius 1980) or two rDNA equivalents in tandem (Pisum sativum, Kolodner and Tewari 1979) has recently been reported. Evolutionary conservation of sequences and moIecular anatomy extends to other regions of plastid chromosomes as well. Using the above mentioned Sal fragment as reference the position of the LSU gene is the same. In tobacco, most or all of this gene could be located on a 3.5 kbp fragment bordered by the cleavage sites X-2/X-7 and P-6/P-9. To determine its location cloned spinach ptDNA fragments about 12kbp in size and known to carry the 1.6 kbp structural gene for LSU (Herrmann et al. 1980a; Schedel et al., in preparation) were utilized as sequence probes. Hybridization of se. condary fragments from this plastome sequence to restricted tobacco ptDNA disclosed not only conservation of the LSU structural gene but also of 10 kbp neighbouring sequences with regard to physical map order, relative sizes and clustering of Kpn I sites (Fig. 8, the predicted Kpn fragment order is K-9 - K-6 - K-5). This suggests that this part of the large single-copy region, comprising more than 10% of the total plastid chromosome, is conserved (cf. also Coen et al. 1977 for maize). A third example is provided by the Kpn fragment patterns. These patterns of the ptDNA from tobacco, spinach and Oenothera are all characterized by a large bimolar band composed of two fragments of almost identical size but differing in nucleotide sequence, designated K-2a and I(-2 b. One of these fragments, K-2a, is composed of the entire small single-copy region plus left- and righthand repetitive sequences including part of the rRNA operon (the terminal part of the 23S rRNA genes and the 4.5S and 5S rRNA genes). In all

203 instances a small Kpn primary fragment of about 0.7 kbp, located within the 23S rRNA structural gene, separates fragment K-2b in one of the repeats and K-1 in the other from K-2a. Both K-2b and K-1 carry the initial part of the 23S rRNA gene, the intercistronic spacer plus the 16S rRNA gene (Fig. 7), and extend symmetrically into the large single-copy region. In spinach and tobacco K-2b encompasses the previously mentioned Sal marker (S-11), while the K-1 fragment on the opposite site contains the 32 kd photosystem II polypeptide (Bedbrook et al. 1978) at the repeat border. Again the position of this gene is conserved (to be published). Other examples can be seen in the Pvu II, Xho I and Bgl I series. The tobacco configuration P-10 ( 2 x ) P-11-P-12 (2x) (Fig. 2) is reflected in ~the spinach fragment patterns P-8 (2x) - P-9 - P-10 (2x) (see Fig. 2 in Herrmann and Possingham 1980). The order X-4 (2x) - X-12 (2x) - X-17 (2x) - X-3 or X-6 in tobacco has an equivalent in the spinach chromosome X-2 (2x) - X-14 (2x) - X-3 or X-5 (Crouse et al. 1978). Once again, equivalent cleavage sites are located at equivalent map positions. From the comparison of these maps, even at this early stage of exploration, it appears that the molecular anatomy even of distant plastomes is largely conserved while primary sequences undergo fairly rapid evolution. These changes are caused by relatively small insertions and deletions (Herrmann et al. 1980a, Gordon et al. 1981b) and possibly by nucleotide substitution if number of cleavage sites for a particular enzyme is altered between different ptDNAs. Such changes occur in all four segments of ptDNA. However, most of them were found within the small single-copy segment in particular between distant organisms (Herrmann and Possingham 1980) and in two regions of the large single-copy segment which lie near the repeat in the related Euoenothera plastomes. The molecular organization of the rDNA operon indicates a hierarchy and mosaic of evolutionary events (see above). The intergeneric homologies in plastome organization suggest also that plastome evolution is directed, possibly associated with nuclear coevolution since interspecific genome/plastome hybrids of Oenothera (Herrmann et al. 1980a) and of tobacco (Frankel et al. 1979) can show a variety of developmental disturbances. Mapping of the species-specific differences in restriction patterns (for tobacco see Atchison et al. 1976; Frankel et al. 1979; Scowcroft 1979) and searching for strategic DNA regions may aid in resolving the paradox plastome similarity, yet specificity in genome/plastome cooperation.

Acknowledgements. The authors thank Dr. Barabara Sears for stimulating discussions and advice in the preparation of this manuscript. We are grateful to the highly competent technical assistance of Ms. Barbara Schiller and Ms. Monika Streubel and to Mr. Schiltz, lnstitut Francais du Tabac, Bergerac,France, for

204 providing us with tobacco seeds. P. S. gratefully acknowledges the financial aid received from DFG and CNRS. This study was supported by DFG grants (He 693) and by Forschungsmittel des Landes Nordrhein/Westfalen.

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Communicated by F. Kaudewitz Received March 31, 1981