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Physical and gene mapping of chloroplast DNA from Atriplex triangularis and Cucumis sativa

Physical and gene mapping of chloroplast DNA from Atriplex triangularis and Cucumis sativa Downloaded from https://academic.oup.com/nar/article/10/5/1593/1164751 by DeepDyve user on 17 September 2020 Volume 10 Number 51982 Nucleic Acid s Research Physical and gene mapping of cfaloroplast DNA from Atriplex triangularis and Cucumis sativa Jeffrey D.Palmer Carnegie Institution of Washington, Department of Plant Biology, 290 Panama St., Stanford, CA 94305, USA Received 2 December 1981; Revised and Accepted 28 January 1982 ABSTRACT A rapid and simple method for constructing restriction maps of large DNAs (100-200 kb) is presented. The utility of this method is illustrated by map- ping the Sal I, Sac I, and Hpa I sites of the 152 kb Atriplex triangularis chloroplast genome, and the Sal I and Pvu II sites of the 155 kb Cucumis sativa chloroplast genome. These two chloroplast DNAs are very similar in organization; both feature the near-universal chloroplast DNA inverted repeat sequence of 22-25 kb. The positions of four different genes have been localized on these chlo- roplast DNAs. In both genomes the 16S and 23S ribosomal RNAs are encoded by duplicate genes situated at one end of the inverted repeat, while genes for the large subunit of ribulose-l,5-bisphosphate carboxylaae and a 32 kilo- dalton photosystem II polypeptide are separated by 55 kb of DNA within the large single copy region. The physical and genetic organization of these DNAs is compared to that of spinach chloroplast DNA. INTRODUCTION The chloroplast genome of vascular plants consists of a single circular DNA molecule between 120 kb and 180 kb in size. The majority of chloroplast DNAs studied contain a large inverted repeat sequence of 22-25 kb, part of which codes for ribosomal RNA. Restriction maps which demonstrate the in- verted repeat organization have so far been constructed for chloroplast DNAs from corn (1), spinach (2), wheat (3), tobacco (4), petunia (5), mustard (6), mung bean (7), Oenothera (8), and Splrodela (9). Two exceptions to this pat- tern are chloroplast DNAs from pea (7), and broad bean (10), both of which lack one entire segment of the inverted repeat The relatively compact size, the absence of molecular heterogeneity, and the evolutionary conservatism of the chloroplast genome make it an ideal molecular tool for assessing evolutionary relationships among plants at practically all levels, from the intraspecific to the interdivisional. For many of these studies detailed physical maps of restriction endonuclease cleavage sites will be required. In this paper I present a strategy for © IRL Press Limited, 1 Falconberg Court. London W1 V 5FG, U.K. 1593 0305-1048/82/1005-1693S 2.00/0 Downloaded from https://academic.oup.com/nar/article/10/5/1593/1164751 by DeepDyve user on 17 September 2020 Nucleic Acids Research constructing restriction maps of large DNAs of the size of the chloroplast genome. This method is quite rapid, can be adapted to mapping a number of DNAs a t once, and requires only small amounts (<10yg) of chloroplast DNA. The utilit y of the method is illustrated by constructing restriction maps for the chloroplast genomes of Atriplex triangularis and Cucumis sativa. In ad- dition, the positions of four genes have been localized on the physical map of these DNAs. MATERIALS AND METHODS DNA Isolatio n Chloroplas t DNA wa s prepare d from one-week-old Cucumis sativ a cotyle- don s (cv. Bei t Alpha Mt.; seed obtained from FMC Corporation) , 3-week-old corn leaves (Zea mays, cv. Trojan; seed obtained from Pfizer Genetics) and spinach leaves (Spinacla oleracea, obtained from a local grocery market), by treatin g chloroplaats with DNase I according to the method of Kolodner and Tewari (11) . Chloroplas t DNA from Atriplex trianftularis was prepared by a modifica- tio n of the sucrose gradient technique described for the preparatio n of chlo- roplas t DNA from tobacco leaves (12,13). 10-100 gm of leaves are placed in 100-500 ml of ice-cold isolation buffer (0.35 M sorbitol , 50 mM Tris-HCl, ph 8.0, 5 mM EDTA, 0.12 BSA (w/v) , 15 mM (3-mercaptoethanol , 1 mM spermine, 1 mM spermidine ) and homogenized for 10-20 sec in a Waring blender or for 30-60 sec in a polytron homogenizer. The extrac t is filtered through cheese- clot h and miraclot h (Calbiochem) and centrifuged at 1000 g for 10-15 min a t 4°C. The pelle t is resuspended in 10 ml wash buffer (0.35 M sorbitol , 50 mM Tris-HCl, ph 8.0 , 25 mM EDTA, 1 mM spermine , 1 mM spermidine ) using a soft brush and loaded on a step gradient consisting of 18 ml of a 60Z sucrose so- lutio n and 7 ml of a 301 sucrose solution, each containing Tris, EDTA, sper- mine and spermidine at the same concentrations as in the wash buffer. The gradien t is placed in a SW-27 rotor and centrifuged at 25,000 RPM for 50 min a t 4°C. The chloroplas t band a t the 30Z-60Z interphase is removed, diluted with 3-10 volumes wash buffer, and centrifuged at 1,500 g for 10-15' at 4°C. Depending on it s size, the chloroplast pellet is resuspended in either 2 ml or 15 ml of wash buffer and one-tenth volume of a 10 mg/ml solution of Pro- nase (Calbiochem) is added. After 2 min a t room temperature one-fifth volume of lysis buffer (5X sodium sarcosinate (w/v), 50 mM Tris-HCl , ph 8.0, 25 mM EDTA) I s gently added. The tube is gently mixed by inverting several times and incubated at room temperature for 15' to several hours. 3.35 gm o r 23.0 1594 Downloaded from https://academic.oup.com/nar/article/10/5/1593/1164751 by DeepDyve user on 17 September 2020 Nucleic Acids Research gm solid CsCl (Kawecki Berylco Industries Inc.), EtBr to a final concen- tratio n of 200 yg/ml, and 50 mm Tris , ph 8.0, 25 mM EDTA to a final volume of 4.45 ml or 32.0 ml are added to the small (2 ml) or large (15 ml) chloro- plas t lysates, respectively. The small gradient is centrifuged in the TV-865 roto r (Sorvall) for 4-16 hr at 58,000 RPM (319,000 a ), while the large gradien t ia centrifuged in the TV-850 rotor (Sorvall) for 12-16 hr at 43,000 RPM (175,000 e ). The DNA from either a small or large initia l gradient is then banded a second time in the TV-865 rotor. Ethidium bromide is removed by three extractions with isopropanol saturated with NaCl and HO, and the DNA i s dialyzed against at least three changes of 2 I of 10 mM Tris , ph 8.0, 0. 1 mM EDTA over a period of 1-2 days. This method has proved extremely versatile in the purification of chloro- plas t DNA from well over 100 species of angiosperms, gynmosperms and ferns. The method gives higher yields (0.2 - 10 yg chloroplast DNA pe r gm F.W. of leaves ) than the DNase I procedure (11) and is applicable to a much wider range of plants, Including many for which i t is not possible to isolate any DNA usin g the DNase I treatment. The purity of the chloroplast DNA i s vari- abl e using the sucrose gradient method, but is generally high enough to en- abl e visualization of al l the chloroplast DNA restrictio n fragments on ethidium bromide-stained agarose gels. One final advantage is that nuclear DNA of high molecular weight can be obtained by resuspending the pelle t from th e sucrose gradient in wash buffer, and further treating this fraction in exactl y the same manner as the chloroplast fraction. Gels and Blots Chloroplas t DNA was digested with Sal I, Sac I, Hpa I or Pvu I I (New England Biolabs) according to the supplier's instructions. Between 0.2-0.5 yg DNA was loaded per lane on a 0.7% neutra l agarose (Sigma, Type I) hori- zonta l slab gel 0.4 x 20 x 40 cm in size. Electrophoresis was for 12-20 hrs a t 75 mA in 100 mM Tris-acetat e (ph 8.1), 1 mM EDTA. The gel was prepared for transfer to nitrocellulose filters according to Wahl et al . (14). Two filte r replicas of the same gel were prepared by blotting onto nitrocellu- los e filters placed on both sides of the gel exactly as described by Smith and Summers (15) except that the transfer buffer was 20 x SSC ( 3 mM Nad , 0.3 M trisodiu m citrate) . Preparatio n of P-Probes 2-5 yg intact chloroplast DNA was labeled with [a- P] dGTP (Araersham) by the nick-translation reaction according to Maniatis et al . (16) except tha t no DNase I was added. Chloroplast DNA prepared by the above methods 1595 Downloaded from https://academic.oup.com/nar/article/10/5/1593/1164751 by DeepDyve user on 17 September 2020 Nucleic Acids Research always has sufficient nicks to permit good Incorporation of P in such re- actions. In this case the amount of P in the reaction mixture was set at no more than 30 pCi/yg DNA In order to limit the specific activity, and hence prevent excessive degradation, of the labeled DNA. The nick-translation re- action was terminated by heating to 65° for 10' and restriction enzyme added after the buffer was adjusted to that prescribed for the enzyme. The P- labeled restriction fragments were separated by agarose (Sigma, Type I) gel electrophoresis and gel slices containing each fragment were cut out and placed in a 5 m l polypropylene tube. One ml of lx SSC was added and the tube boiled for 15' to melt the agarose and denature the DNA. The liquified aga- rose solution was then added to a bag containing the prehybridized filter (see below). Tobacco chloroplast 16S and 23S ribosomal ENAs were alkali hydrolyzed to a few hundred nucleotldes and labeled with [Y - P] ATP according to Maizels (17) . Filte r Hybridizations Nitrocellulose filters were placed In heat-sealable plastic bags which contained 4 x SSC, 50 mM phosphate buffer, 0.1Z SDS, 5 x Denhardt's solution (18), and 100 pg/ml sonicated calf thymus DNA, and the bags incubated for 14-16 hr In a shaking water bath at 65°C. P-labeled RNA or DNA was added and hybridization at 65°C allowed to proceed for two days. The filter s were washed In several changes of 2 x SSC, 0.1Z SDS over a period of 4-6 hr at 65°C and exposed to preflashed (19) Kodak XAR-5 film, using a Dupont Light- ning Plus intensifying screen, for 1-20 days at -70°C. RESULTS The restriction mapping strategy is to employ the complete set of frag- ments generated by a single restriction enzyme as hybridization probes against filters which contain single digests of chloroplast DNA produced by various other restriction enzymes and also double digests produced by each of the other enzymes plus the enzyme used to generate the probe fragments. Hybridization to single digests generates overlaps between probe and filter- bound fragments, while hybridization to double digests gives the precise lo- calization of cleavage sites within probe fragments. Atrlplex and Cucumis chloroplast DNAs were screened with 15 different six-base restriction enzymes in order to find enzymes which produce simple patterns In which all the fragments are well resolved. Three enzymes were chosen for the Atriplex mapping and two for the Cucimds mapping (Fig. 1). 1596 Downloaded from https://academic.oup.com/nar/article/10/5/1593/1164751 by DeepDyve user on 17 September 2020 Nucleic Acids Research - 2 Figure 1. 0.7Z agarose gel electrophoresis of Atriplex chloroplast DNA digested with (1) Sal I, (2) Sac I, and (3) Hpa I, and Cucumis chloroplast DN A digested with (4) Sal I and (5) Pvu II . Size scale at right is in kb. Arrow indicates a small amount of nuclear DNA present in the Atriplex chloroplast DNA preparation and resistant to digestion with Sal I as a con- sequence of its extensive methylation (24) . Sizes for the fragments shown in Fig. 1 are listed in Tables 1 and 2. When fragment stoichiometries are taken into account (Figs. 2 and 3) the size of the Atriplex chloroplast genome is estimated at 152 kb (Sal I: 152.7 kb; Sac I : 152.4 kb; Hpa I: 151.9 kb) and the Cucumis genome at 155 kb (Sal I: 155.7 kb; Pvu II : 154.5 kb). Fig. 4 shows the hybridization pattern of each of the Hpa I fragments of Atriplex chloroplast ENA to filters which contained both Sac land' Sac I-Hpa I digests. In a second experiment, the Atriplex Sal I fragments were used as probes against Sac I and Sac I-Sal I digests. Data from these two sets of hybridizations are summarized in Table 1. From the autoradiograms (Fig.4) it i s clear that some cross—contamination of smaller probe fragments occurred as a result of degradation of larger fragments. Cross-contamination with a giv- en fragment is greatest in the fragment next smaller in size and decreases in 1597 Downloaded from https://academic.oup.com/nar/article/10/5/1593/1164751 by DeepDyve user on 17 September 2020 Nucleic Acids Research Figure 2. Physical and genetic map of the Atriplex chloroplast genome. Restriction endonuclease cleavage sites were deduced from the data presented in Table 2, while locations of the four genes shown are from the hybridiza- tion data of Table 4. The two long, heavy black lines represent the extent of the inverted repeat segments, which, as defined by these mapping data are bounded by the sites between the 7.1 and 11.8 (8.6) kb Sac I fragments and the 10.7 and 0.9 (9.4) kb Sal I fragments. Including the 6.3 kb Sac I-Sal I fragment internal to the repeat, the minimal length of the inverted repeat is 24.1 kb (7.1 + 10.7 + 6.3 kb). Sal I fragments are shown on the outer circle, Sac I fragments on the middle circle, and Hpa I fragments on the in- ner circle. fragments further away. In some cases, when two probe fragments are very close in mobility, there may also be "upstream" contamination of the larger fragment by the smaller. When one assembles the filters in order of probe fragment size, true hybridization signals generally stand out and are easily recognized above the background of artefactual bands (Fig. 4) . The data of Table 1 yield the restriction map for the Atriplex chloro- plast genome (Fig. 2) . Note that the presence of the large inverted repeat introduces ambiguity in interpreting the single digest hybridizations but that this ambiguity is generally resolved by knowing the size of the double digest fragments for probe fragments which lie on the inverted repeat. While the Atriplex map was derived using probe fragments prepared with two dif- ferent restriction enzymes, almost all the mapping information could have been obtained using just a single set of probe fragments, e.g., the Hpa I fragments, by including Sal I and Sal I-Hpa I lanes on the filter. 1598 Downloaded from https://academic.oup.com/nar/article/10/5/1593/1164751 by DeepDyve user on 17 September 2020 Nucleic Acids Research Figure 3. Physical and genetic map of the Cucumia chloroplast genome. Cleavage sites and gene locations are from Tables 3 and 4. The two long, heavy black lines represent the extent of the inverted repeat segments, which, as defined by these mapping data, are bounded by the sites between the 6.8 and 24 kb Pvu II fragments and the 2.3 and 12.5 (18.5) Sal I fragments. Including the A. 9 kb Pvu II-Sal I fragment internal to the repeat, the mini- mal length of the inverted repeat is 14.0 kb (6.8 + 2.3 + 4.9 kb). Sal I fragments are shown on the outer circle and Pvu II fragments on the inner circle . Sal I and Pvu II sites for the Cucumis chloroplast genome were mapped by reciprocal sets of hybridizations of Sal I or Pvu II probe fragments to Sal I-Pvu II double digests plus either Pvu II or Sal I single digests, respec- tively . These experiments are summarized In Table 2 and the Cucumis restric- tion map is shown in Fig. 3. Note that the restriction mapping data do not allow unambiguous ordering of the three Pvu II fragments, two of 6.8 kb and one of 24 kb, which li e completely internal to the 48 kb Sal I fragment. The order of these three fragments was deduced on the basis of 16S ribosomal RNA hybridization (Table 3). In light of their similarities in physical organization (Figs.2 and 3),.it i s of interest to determine whether the arrangement of specific genes is also similar in the Atrlplex and Cucumis chloroplast genomes. To this end I have localized four different genes on the physical maps of Atriplex and CUC"HI1B chloroplast DNA. Fig. 5 showB the hybridization of probes for the genes for the 16S and 23S chloroplast ribosomal RNAs, the large subunit (LS) of ribu- lose-l,5-bisphophate carboxylase and a 32 kilodalton photosystem II polypep- tid e (PII) to nitrocellulose filters containing restriction digests of Atri- 1599 Downloaded from https://academic.oup.com/nar/article/10/5/1593/1164751 by DeepDyve user on 17 September 2020 Nucleic Acids Research 2J0 1.1 4 0 28 19.6 12.4 11.6 9.2 7.6 6.6 2.2 *| H S|H S|H S|*|H S|H S|H S| H SlH S H s| H S|H S|H S|*|H S|H S|H S|*|H S|H S|H Figure 4. Hybridization of P-labelled Atriplex Hpa I fragments to Atriplex (H) Hpa I-Sac I and (S) Sac I fragments separated on a 0.72 agarose gel and transferred to nitrocellulose filters. Numbers above the filters indicate the size in kb of the Hpa I fragments used as probes. Hae II I restriction fragments of phage 0X 174 DNA are in lanes marked "0". 1600 Downloaded from https://academic.oup.com/nar/article/10/5/1593/1164751 by DeepDyve user on 17 September 2020 Nucleic Acids Research Table 1 Summary of Atrlple x Restrictio n Mapping Hybridizations Probe DNA Filter-boun d WA Sal I Sac I Sac I-Sal I 18.4, 17.4, 11.8 , R 6 7.1 , 1 05 . 1 , 1 33 11.8 , 7.1 , 6.5, 6 .3 , 2 .0 5 6.1 , S 1 1.4, 1 05 1 . 4 , 1 22.6 8.0, 7.5 , 6.5 , 6.1 , 5.3, 2 .2 , .0 5 19.8 8.0, 5.7 , 5.5 , 3.2 , 1. 5 5.8 , 5.7,3.35 , 3 1 . 5 •2 , 16.2 9.7, 7.5 , 4.2 , 3. 4 9.7, 3.4 , 2.0, 0 . 8 15.2 18.4 , 17.4, 11.8 , 8.6 , 7. 1 11.8 , 7.1 , 6.3, 2 . 1 12.1 13.3 , 5. 5 10.4 , 1.85 10.7 18.4, 17.4 10.7 9. 4 18.4, 17.4, 4.2 , 3.8 , 1.35 3. 8 2.4 , 1.8, 1 IS 9 3. 1 13.3 3. 1 0. 9 18.4, 17.4 0. 9 Hpa I Sac I Sac I-Hpa I 4 0 18.4 , 17.4, 13.3 , 5.7 , 5.5 , 3 2 , 14.0, 13.3 , 12. 8 , 5 . 5 , 4 • 1, 3.2 , 1.5 1.5 28 18.4 , 17.4, 9.7 , 3.8 , 1 35 14.0, 12.8 , 4. 3 . 3 1.35 4.2 , 2 , •8 , 9 , 19.6 9.7, 7.5 , 5.3 , 1.4 7.5 , 4.4 , 3. 2 . 1 . 4 3.4 , 7 , 4 , 12.4 11.8 , 8.6, 1.05 11.8 , 2.2 , 0. 95 18.4, 17.4, 7. 1 7.0 , 4. 3 11.6 8.0, 5. 7 7.9 , 1.45 9. 2 7. 6 6.1 , 5.3 , 1.05 6.1 , 1.05 6. 6 8. 6 6. 5 2. 2 11.8 , 8. 6 11.8 , 2. 2 2. 0 5. 3 2. 0 1. 1 9. 7 1. 1 Table 2 Summary of Cucumis Restrictio n 1•lapping Hybridizations Probe DNA Filter-boun d DNA Pvu 11[ Sal I Pvu II - Sal I 4 7 48, 20.5, 18 .5 , 12.5 , 11.8, 2.3 18.5 , 15.6 , 12.5, 11.8, 4.9, 2.3 28 48, 18.5, 16 .6 , 12.5 , 2.3 18.5 , 12.5 , 4.9, 2.9, 2.3 24 4 8 24 13.8 16.6 13.8 10.2 11.6 10.2 9. 6 20.5 , 11.6 4. 9 8. 3 11.6 6.8. 1.5 6. 8 48 6. 8 Pvu II I Sal I Pvu II-Sal 48 47, 28, 24, 6. 8 24, 6 . 9 8, 4 20.5 47, 9.6 15.6, 4. 9 18.5 47, 28 18.5 , 12.5 16.6 28, 13.8 13.8 , 2. 9 12.5 47, 28 18.5 , 12.5 11.8 47 11.8 10.2, 9.6, 8 . 3 10.2 , 6.8 , 4.9 , 15 11.6 2. 3 47, 28 2. 3 1601 Downloaded from https://academic.oup.com/nar/article/10/5/1593/1164751 by DeepDyve user on 17 September 2020 Nucleic Acids Research Table 3. Summary of Gene Mapping Hybridizations Filter-boun d DNA Probe Atriple x triangularis Cucumi8 sativa Sac I-Hpa I Sac I-Sal ] Sa l Sac I [ Pvu I I Pvu II-Sal I I 47,28,6 .8 6.8, 4.9 48 16S rRNA 18.4, 17 .4 4.3 6.3 23S rRNA 7.1 7.0 7.1 6.8 6.8 LS 8.0 7.9 5.8 47 15.6 5 PI I 18.4 14.0 1.8 28 18.5 5 16S 23S LS PII Is H| s HJS H|S H|S H| Figure 5. Mapping Atriplex chloroplast genes. Probes used are P-labelled 16S and 23S tobacco chloroplast rlbosomal RNA, the 0.58 kb Pat I fragment of corn chloroplast DNA, which is located entirely within the translated region of the gene for the large subunlt (LS) of ribulose-l,5-blsphosphate carboxy- lase (25), and the 1.1 kb Sal I-Pst. I fragment of spinach chloroplast DNA (26) which contains most of the gene for a 32,000 dalton photosystem II polypep- tide (PII) (27) . Slices containing the corn and spinach fragments were cut out of polyacrylamide gels of total chloroplast DNA and the DNA eluted and nick-translated (16). Atrlplex chloroplast DNA was digested with Sac I (S) and Hpa I-Sac I (H), electrophoreaed on a 0.7Z agarose gel and transferred to nitrocellulose filters. 1602 Downloaded from https://academic.oup.com/nar/article/10/5/1593/1164751 by DeepDyve user on 17 September 2020 Nucleic Acids Research plex chloroplast DNA. Data from these, as well as other Atrlplex and Cucumls hybridizations, are suiranarized in Table 3 and the map locations of these genes are indicated on the restriction maps shown in Figs. 2 and 3. DISCUSSION The mapping strategy presented here is an amalgam of two approaches which have been commonly used to map large DNAB of the size of the chloroplast ge- nome. The first is the determination of fragment overlaps by hybridization of labelled fragments produced by one enzyme to filter-bound fragments pro- duced by a second enzyme (1,7,13). In the second method, fragment overlaps are recognized from common double digestion fragments produced by reciprocal digestions of individual fragments purified out of low-melting agarose gels (2,4,6,8,9). The approach taken in this study clearly incorporates the first of these approaches, by hybridizing purified fragments to single digests, and also the second, by hybridizing to double digests. This unified approach al- lows a complete map to be produced with reference to a single set of probe fragments. By adding more lanes to each filter replica, one can easily scale up the experiment in order to map more enzymes for one DNA, or even to map several DNAs at once. This feature should permit comparative restriction mapping, as exemplified by the evolutionary studies of Brown and coworkers on primate mi - tochondrial DNA (20,21), to be performed on the much larger chloroplast genome. An elegant approach which allows simultaneous determineatlon of all pos- sible combinations of fragment homologies for two sets of restriction frag- ments is the cross-hybridization procedure described by Sato et al. (22). In principle, i.e., by hybridizing "hot" gels to both single and double-digest gels, this procedure could be adapted to provide each of the two data sets generated by the method described in this paper. However, this would require a minimum of four gels, to map fragments produced by two enzymes, and pro- portionately more gels to map additional enzyme digests. The Atriplex and Cucumis chloroplast genomes are extremely similar in all aspects examined. They differ by only a few percent in sire, both feature the inverted repeat which contains the ribosomal genes, and the genes for IS and FII are in the same approximate locations in the two genomes. One ap- parent difference between the two genomes is the size of the inverted repeat - 24 kb in Atriplex, but only 14 kb in Cucumis. Restriction mapping neces- sarily yields a min-lmai estimate for the size of the repeat. Since the 12.5 1603 Downloaded from https://academic.oup.com/nar/article/10/5/1593/1164751 by DeepDyve user on 17 September 2020 Nucleic Acids Research Spinach 2.4 0.7 20.5 22.3 13.9 9.0 10.6 47.9 10.6 22. 6 2 9.4 16 10.7 15. 2 3 3 10.7 19.8 lil i2 1 0.9 3.1 Atriplex Figure 6. Comparison of Sal I cleavage sites in the Atriple x and spinach (2) chloroplast genomes. The two long lines represent the inverted repeat. and 18.5 k b Sal I fragments adjacent to the apparen t end o f the inverted re - peat cross-hybridize (Table 2) , it can b e concluded that the inverted repeat does extend into at least these two Cucumis fragments. Comparison of the Sa l I map o f Atriplex with that of spinach (2), a mem - ber of the same family (Chenopodiaceae), suggests that these two chloroplast genomes are colinea r in sequence (Fig. 6) . The slight size differences be - tween many of the fragment s which map to the same locations in the two ge- nomes probably reflect small deletion/insertions, known to occur quite fre - quently during chloroplast genome evolution (23), rather than restriction site changes near the end s of fragments. Accordingly, these small size dif - ferences have been neglected in aligning the two maps . He have recently performed heterologous hybridizations using cloned mung bean restriction fragments and hav e found that the spinac h and Cucumis ge - nomes share a common sequence arrangement around their entire circumference (Palmer and Thompson , sub. for publ. ) . Thus it appears that all three of these chloroplast DNAs, from spinach, Atriplex and Cucumis, share the same basic pattern of sequence organization. ACKNOWLEDGEMENTS I am grateful to William F. Thompson for providin g laboratory facilities and support throughout this research. I thank D. Bourque for hi s generous gift of tobacco ribosomal RNA an d R. Jorgensen and D. Stein for their assis- tance in some of the DNA labeling reactions. I also appreciate the critical readings of this manuscript by W . F. Thompson, R. Jorgensen, and M. Zolan. This is CIW-DPB Publication #767. 1604 Downloaded from https://academic.oup.com/nar/article/10/5/1593/1164751 by DeepDyve user on 17 September 2020 Nucleic Acids Research REFERENCES 1. Bedbrook, J. R. and Bogorad, L. (1976) Proc. Natl. Acad. Sci. USA 73, 4309-4313 . 2 . Herrmann, R. G., Whitfeld, P. R. and Bottomley, W. (1980) Gene 8, 179- 191 . 3 . Bowman, C. M., Roller, B., Delius, H. and Dyer, T. A. (1981) Molec. Gen. Genet. 183, 93-101. 4 . 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Gruenbaum, Y., Naveh-Many, T. , Cedar, H. and Rozin, A. (1981) Nature 292, 860-862. 25 . Mclntosh, L., Poulson, C. and Bogorad, L. (1980) Nature 288, 556-560. 26. Driesel, A. J. , Crouse, E. J., Gordon, R., Bohnert, H. J., Herrmann, R. G., Steinmetz, A., Mubumbila, M. , Reller, M. , Burkard, G. and Weil, J . H. (1979) Gene 6, 285-306. 27. Driesel, A. J., Spelrs, J. and Bohnert, H. J. (1980) Biochim. Biophys. Acta 610, 297-310. 1605 Downloaded from https://academic.oup.com/nar/article/10/5/1593/1164751 by DeepDyve user on 17 September 2020 Nucleic Acids Research http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

Physical and gene mapping of chloroplast DNA from Atriplex triangularis and Cucumis sativa

Palmer, Jeffrey, D.
Nucleic Acids Research , Volume 10 (5) – Mar 11, 1982
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Oxford University Press
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© IRL Press Limited
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0305-1048
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10.1093/nar/10.5.1593
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Abstract

Downloaded from https://academic.oup.com/nar/article/10/5/1593/1164751 by DeepDyve user on 17 September 2020 Volume 10 Number 51982 Nucleic Acid s Research Physical and gene mapping of cfaloroplast DNA from Atriplex triangularis and Cucumis sativa Jeffrey D.Palmer Carnegie Institution of Washington, Department of Plant Biology, 290 Panama St., Stanford, CA 94305, USA Received 2 December 1981; Revised and Accepted 28 January 1982 ABSTRACT A rapid and simple method for constructing restriction maps of large DNAs (100-200 kb) is presented. The utility of this method is illustrated by map- ping the Sal I, Sac I, and Hpa I sites of the 152 kb Atriplex triangularis chloroplast genome, and the Sal I and Pvu II sites of the 155 kb Cucumis sativa chloroplast genome. These two chloroplast DNAs are very similar in organization; both feature the near-universal chloroplast DNA inverted repeat sequence of 22-25 kb. The positions of four different genes have been localized on these chlo- roplast DNAs. In both genomes the 16S and 23S ribosomal RNAs are encoded by duplicate genes situated at one end of the inverted repeat, while genes for the large subunit of ribulose-l,5-bisphosphate carboxylaae and a 32 kilo- dalton photosystem II polypeptide are separated by 55 kb of DNA within the large single copy region. The physical and genetic organization of these DNAs is compared to that of spinach chloroplast DNA. INTRODUCTION The chloroplast genome of vascular plants consists of a single circular DNA molecule between 120 kb and 180 kb in size. The majority of chloroplast DNAs studied contain a large inverted repeat sequence of 22-25 kb, part of which codes for ribosomal RNA. Restriction maps which demonstrate the in- verted repeat organization have so far been constructed for chloroplast DNAs from corn (1), spinach (2), wheat (3), tobacco (4), petunia (5), mustard (6), mung bean (7), Oenothera (8), and Splrodela (9). Two exceptions to this pat- tern are chloroplast DNAs from pea (7), and broad bean (10), both of which lack one entire segment of the inverted repeat The relatively compact size, the absence of molecular heterogeneity, and the evolutionary conservatism of the chloroplast genome make it an ideal molecular tool for assessing evolutionary relationships among plants at practically all levels, from the intraspecific to the interdivisional. For many of these studies detailed physical maps of restriction endonuclease cleavage sites will be required. In this paper I present a strategy for © IRL Press Limited, 1 Falconberg Court. London W1 V 5FG, U.K. 1593 0305-1048/82/1005-1693S 2.00/0 Downloaded from https://academic.oup.com/nar/article/10/5/1593/1164751 by DeepDyve user on 17 September 2020 Nucleic Acids Research constructing restriction maps of large DNAs of the size of the chloroplast genome. This method is quite rapid, can be adapted to mapping a number of DNAs a t once, and requires only small amounts (<10yg) of chloroplast DNA. The utilit y of the method is illustrated by constructing restriction maps for the chloroplast genomes of Atriplex triangularis and Cucumis sativa. In ad- dition, the positions of four genes have been localized on the physical map of these DNAs. MATERIALS AND METHODS DNA Isolatio n Chloroplas t DNA wa s prepare d from one-week-old Cucumis sativ a cotyle- don s (cv. Bei t Alpha Mt.; seed obtained from FMC Corporation) , 3-week-old corn leaves (Zea mays, cv. Trojan; seed obtained from Pfizer Genetics) and spinach leaves (Spinacla oleracea, obtained from a local grocery market), by treatin g chloroplaats with DNase I according to the method of Kolodner and Tewari (11) . Chloroplas t DNA from Atriplex trianftularis was prepared by a modifica- tio n of the sucrose gradient technique described for the preparatio n of chlo- roplas t DNA from tobacco leaves (12,13). 10-100 gm of leaves are placed in 100-500 ml of ice-cold isolation buffer (0.35 M sorbitol , 50 mM Tris-HCl, ph 8.0, 5 mM EDTA, 0.12 BSA (w/v) , 15 mM (3-mercaptoethanol , 1 mM spermine, 1 mM spermidine ) and homogenized for 10-20 sec in a Waring blender or for 30-60 sec in a polytron homogenizer. The extrac t is filtered through cheese- clot h and miraclot h (Calbiochem) and centrifuged at 1000 g for 10-15 min a t 4°C. The pelle t is resuspended in 10 ml wash buffer (0.35 M sorbitol , 50 mM Tris-HCl, ph 8.0 , 25 mM EDTA, 1 mM spermine , 1 mM spermidine ) using a soft brush and loaded on a step gradient consisting of 18 ml of a 60Z sucrose so- lutio n and 7 ml of a 301 sucrose solution, each containing Tris, EDTA, sper- mine and spermidine at the same concentrations as in the wash buffer. The gradien t is placed in a SW-27 rotor and centrifuged at 25,000 RPM for 50 min a t 4°C. The chloroplas t band a t the 30Z-60Z interphase is removed, diluted with 3-10 volumes wash buffer, and centrifuged at 1,500 g for 10-15' at 4°C. Depending on it s size, the chloroplast pellet is resuspended in either 2 ml or 15 ml of wash buffer and one-tenth volume of a 10 mg/ml solution of Pro- nase (Calbiochem) is added. After 2 min a t room temperature one-fifth volume of lysis buffer (5X sodium sarcosinate (w/v), 50 mM Tris-HCl , ph 8.0, 25 mM EDTA) I s gently added. The tube is gently mixed by inverting several times and incubated at room temperature for 15' to several hours. 3.35 gm o r 23.0 1594 Downloaded from https://academic.oup.com/nar/article/10/5/1593/1164751 by DeepDyve user on 17 September 2020 Nucleic Acids Research gm solid CsCl (Kawecki Berylco Industries Inc.), EtBr to a final concen- tratio n of 200 yg/ml, and 50 mm Tris , ph 8.0, 25 mM EDTA to a final volume of 4.45 ml or 32.0 ml are added to the small (2 ml) or large (15 ml) chloro- plas t lysates, respectively. The small gradient is centrifuged in the TV-865 roto r (Sorvall) for 4-16 hr at 58,000 RPM (319,000 a ), while the large gradien t ia centrifuged in the TV-850 rotor (Sorvall) for 12-16 hr at 43,000 RPM (175,000 e ). The DNA from either a small or large initia l gradient is then banded a second time in the TV-865 rotor. Ethidium bromide is removed by three extractions with isopropanol saturated with NaCl and HO, and the DNA i s dialyzed against at least three changes of 2 I of 10 mM Tris , ph 8.0, 0. 1 mM EDTA over a period of 1-2 days. This method has proved extremely versatile in the purification of chloro- plas t DNA from well over 100 species of angiosperms, gynmosperms and ferns. The method gives higher yields (0.2 - 10 yg chloroplast DNA pe r gm F.W. of leaves ) than the DNase I procedure (11) and is applicable to a much wider range of plants, Including many for which i t is not possible to isolate any DNA usin g the DNase I treatment. The purity of the chloroplast DNA i s vari- abl e using the sucrose gradient method, but is generally high enough to en- abl e visualization of al l the chloroplast DNA restrictio n fragments on ethidium bromide-stained agarose gels. One final advantage is that nuclear DNA of high molecular weight can be obtained by resuspending the pelle t from th e sucrose gradient in wash buffer, and further treating this fraction in exactl y the same manner as the chloroplast fraction. Gels and Blots Chloroplas t DNA was digested with Sal I, Sac I, Hpa I or Pvu I I (New England Biolabs) according to the supplier's instructions. Between 0.2-0.5 yg DNA was loaded per lane on a 0.7% neutra l agarose (Sigma, Type I) hori- zonta l slab gel 0.4 x 20 x 40 cm in size. Electrophoresis was for 12-20 hrs a t 75 mA in 100 mM Tris-acetat e (ph 8.1), 1 mM EDTA. The gel was prepared for transfer to nitrocellulose filters according to Wahl et al . (14). Two filte r replicas of the same gel were prepared by blotting onto nitrocellu- los e filters placed on both sides of the gel exactly as described by Smith and Summers (15) except that the transfer buffer was 20 x SSC ( 3 mM Nad , 0.3 M trisodiu m citrate) . Preparatio n of P-Probes 2-5 yg intact chloroplast DNA was labeled with [a- P] dGTP (Araersham) by the nick-translation reaction according to Maniatis et al . (16) except tha t no DNase I was added. Chloroplast DNA prepared by the above methods 1595 Downloaded from https://academic.oup.com/nar/article/10/5/1593/1164751 by DeepDyve user on 17 September 2020 Nucleic Acids Research always has sufficient nicks to permit good Incorporation of P in such re- actions. In this case the amount of P in the reaction mixture was set at no more than 30 pCi/yg DNA In order to limit the specific activity, and hence prevent excessive degradation, of the labeled DNA. The nick-translation re- action was terminated by heating to 65° for 10' and restriction enzyme added after the buffer was adjusted to that prescribed for the enzyme. The P- labeled restriction fragments were separated by agarose (Sigma, Type I) gel electrophoresis and gel slices containing each fragment were cut out and placed in a 5 m l polypropylene tube. One ml of lx SSC was added and the tube boiled for 15' to melt the agarose and denature the DNA. The liquified aga- rose solution was then added to a bag containing the prehybridized filter (see below). Tobacco chloroplast 16S and 23S ribosomal ENAs were alkali hydrolyzed to a few hundred nucleotldes and labeled with [Y - P] ATP according to Maizels (17) . Filte r Hybridizations Nitrocellulose filters were placed In heat-sealable plastic bags which contained 4 x SSC, 50 mM phosphate buffer, 0.1Z SDS, 5 x Denhardt's solution (18), and 100 pg/ml sonicated calf thymus DNA, and the bags incubated for 14-16 hr In a shaking water bath at 65°C. P-labeled RNA or DNA was added and hybridization at 65°C allowed to proceed for two days. The filter s were washed In several changes of 2 x SSC, 0.1Z SDS over a period of 4-6 hr at 65°C and exposed to preflashed (19) Kodak XAR-5 film, using a Dupont Light- ning Plus intensifying screen, for 1-20 days at -70°C. RESULTS The restriction mapping strategy is to employ the complete set of frag- ments generated by a single restriction enzyme as hybridization probes against filters which contain single digests of chloroplast DNA produced by various other restriction enzymes and also double digests produced by each of the other enzymes plus the enzyme used to generate the probe fragments. Hybridization to single digests generates overlaps between probe and filter- bound fragments, while hybridization to double digests gives the precise lo- calization of cleavage sites within probe fragments. Atrlplex and Cucumis chloroplast DNAs were screened with 15 different six-base restriction enzymes in order to find enzymes which produce simple patterns In which all the fragments are well resolved. Three enzymes were chosen for the Atriplex mapping and two for the Cucimds mapping (Fig. 1). 1596 Downloaded from https://academic.oup.com/nar/article/10/5/1593/1164751 by DeepDyve user on 17 September 2020 Nucleic Acids Research - 2 Figure 1. 0.7Z agarose gel electrophoresis of Atriplex chloroplast DNA digested with (1) Sal I, (2) Sac I, and (3) Hpa I, and Cucumis chloroplast DN A digested with (4) Sal I and (5) Pvu II . Size scale at right is in kb. Arrow indicates a small amount of nuclear DNA present in the Atriplex chloroplast DNA preparation and resistant to digestion with Sal I as a con- sequence of its extensive methylation (24) . Sizes for the fragments shown in Fig. 1 are listed in Tables 1 and 2. When fragment stoichiometries are taken into account (Figs. 2 and 3) the size of the Atriplex chloroplast genome is estimated at 152 kb (Sal I: 152.7 kb; Sac I : 152.4 kb; Hpa I: 151.9 kb) and the Cucumis genome at 155 kb (Sal I: 155.7 kb; Pvu II : 154.5 kb). Fig. 4 shows the hybridization pattern of each of the Hpa I fragments of Atriplex chloroplast ENA to filters which contained both Sac land' Sac I-Hpa I digests. In a second experiment, the Atriplex Sal I fragments were used as probes against Sac I and Sac I-Sal I digests. Data from these two sets of hybridizations are summarized in Table 1. From the autoradiograms (Fig.4) it i s clear that some cross—contamination of smaller probe fragments occurred as a result of degradation of larger fragments. Cross-contamination with a giv- en fragment is greatest in the fragment next smaller in size and decreases in 1597 Downloaded from https://academic.oup.com/nar/article/10/5/1593/1164751 by DeepDyve user on 17 September 2020 Nucleic Acids Research Figure 2. Physical and genetic map of the Atriplex chloroplast genome. Restriction endonuclease cleavage sites were deduced from the data presented in Table 2, while locations of the four genes shown are from the hybridiza- tion data of Table 4. The two long, heavy black lines represent the extent of the inverted repeat segments, which, as defined by these mapping data are bounded by the sites between the 7.1 and 11.8 (8.6) kb Sac I fragments and the 10.7 and 0.9 (9.4) kb Sal I fragments. Including the 6.3 kb Sac I-Sal I fragment internal to the repeat, the minimal length of the inverted repeat is 24.1 kb (7.1 + 10.7 + 6.3 kb). Sal I fragments are shown on the outer circle, Sac I fragments on the middle circle, and Hpa I fragments on the in- ner circle. fragments further away. In some cases, when two probe fragments are very close in mobility, there may also be "upstream" contamination of the larger fragment by the smaller. When one assembles the filters in order of probe fragment size, true hybridization signals generally stand out and are easily recognized above the background of artefactual bands (Fig. 4) . The data of Table 1 yield the restriction map for the Atriplex chloro- plast genome (Fig. 2) . Note that the presence of the large inverted repeat introduces ambiguity in interpreting the single digest hybridizations but that this ambiguity is generally resolved by knowing the size of the double digest fragments for probe fragments which lie on the inverted repeat. While the Atriplex map was derived using probe fragments prepared with two dif- ferent restriction enzymes, almost all the mapping information could have been obtained using just a single set of probe fragments, e.g., the Hpa I fragments, by including Sal I and Sal I-Hpa I lanes on the filter. 1598 Downloaded from https://academic.oup.com/nar/article/10/5/1593/1164751 by DeepDyve user on 17 September 2020 Nucleic Acids Research Figure 3. Physical and genetic map of the Cucumia chloroplast genome. Cleavage sites and gene locations are from Tables 3 and 4. The two long, heavy black lines represent the extent of the inverted repeat segments, which, as defined by these mapping data, are bounded by the sites between the 6.8 and 24 kb Pvu II fragments and the 2.3 and 12.5 (18.5) Sal I fragments. Including the A. 9 kb Pvu II-Sal I fragment internal to the repeat, the mini- mal length of the inverted repeat is 14.0 kb (6.8 + 2.3 + 4.9 kb). Sal I fragments are shown on the outer circle and Pvu II fragments on the inner circle . Sal I and Pvu II sites for the Cucumis chloroplast genome were mapped by reciprocal sets of hybridizations of Sal I or Pvu II probe fragments to Sal I-Pvu II double digests plus either Pvu II or Sal I single digests, respec- tively . These experiments are summarized In Table 2 and the Cucumis restric- tion map is shown in Fig. 3. Note that the restriction mapping data do not allow unambiguous ordering of the three Pvu II fragments, two of 6.8 kb and one of 24 kb, which li e completely internal to the 48 kb Sal I fragment. The order of these three fragments was deduced on the basis of 16S ribosomal RNA hybridization (Table 3). In light of their similarities in physical organization (Figs.2 and 3),.it i s of interest to determine whether the arrangement of specific genes is also similar in the Atrlplex and Cucumis chloroplast genomes. To this end I have localized four different genes on the physical maps of Atriplex and CUC"HI1B chloroplast DNA. Fig. 5 showB the hybridization of probes for the genes for the 16S and 23S chloroplast ribosomal RNAs, the large subunit (LS) of ribu- lose-l,5-bisphophate carboxylase and a 32 kilodalton photosystem II polypep- tid e (PII) to nitrocellulose filters containing restriction digests of Atri- 1599 Downloaded from https://academic.oup.com/nar/article/10/5/1593/1164751 by DeepDyve user on 17 September 2020 Nucleic Acids Research 2J0 1.1 4 0 28 19.6 12.4 11.6 9.2 7.6 6.6 2.2 *| H S|H S|H S|*|H S|H S|H S| H SlH S H s| H S|H S|H S|*|H S|H S|H S|*|H S|H S|H Figure 4. Hybridization of P-labelled Atriplex Hpa I fragments to Atriplex (H) Hpa I-Sac I and (S) Sac I fragments separated on a 0.72 agarose gel and transferred to nitrocellulose filters. Numbers above the filters indicate the size in kb of the Hpa I fragments used as probes. Hae II I restriction fragments of phage 0X 174 DNA are in lanes marked "0". 1600 Downloaded from https://academic.oup.com/nar/article/10/5/1593/1164751 by DeepDyve user on 17 September 2020 Nucleic Acids Research Table 1 Summary of Atrlple x Restrictio n Mapping Hybridizations Probe DNA Filter-boun d WA Sal I Sac I Sac I-Sal I 18.4, 17.4, 11.8 , R 6 7.1 , 1 05 . 1 , 1 33 11.8 , 7.1 , 6.5, 6 .3 , 2 .0 5 6.1 , S 1 1.4, 1 05 1 . 4 , 1 22.6 8.0, 7.5 , 6.5 , 6.1 , 5.3, 2 .2 , .0 5 19.8 8.0, 5.7 , 5.5 , 3.2 , 1. 5 5.8 , 5.7,3.35 , 3 1 . 5 •2 , 16.2 9.7, 7.5 , 4.2 , 3. 4 9.7, 3.4 , 2.0, 0 . 8 15.2 18.4 , 17.4, 11.8 , 8.6 , 7. 1 11.8 , 7.1 , 6.3, 2 . 1 12.1 13.3 , 5. 5 10.4 , 1.85 10.7 18.4, 17.4 10.7 9. 4 18.4, 17.4, 4.2 , 3.8 , 1.35 3. 8 2.4 , 1.8, 1 IS 9 3. 1 13.3 3. 1 0. 9 18.4, 17.4 0. 9 Hpa I Sac I Sac I-Hpa I 4 0 18.4 , 17.4, 13.3 , 5.7 , 5.5 , 3 2 , 14.0, 13.3 , 12. 8 , 5 . 5 , 4 • 1, 3.2 , 1.5 1.5 28 18.4 , 17.4, 9.7 , 3.8 , 1 35 14.0, 12.8 , 4. 3 . 3 1.35 4.2 , 2 , •8 , 9 , 19.6 9.7, 7.5 , 5.3 , 1.4 7.5 , 4.4 , 3. 2 . 1 . 4 3.4 , 7 , 4 , 12.4 11.8 , 8.6, 1.05 11.8 , 2.2 , 0. 95 18.4, 17.4, 7. 1 7.0 , 4. 3 11.6 8.0, 5. 7 7.9 , 1.45 9. 2 7. 6 6.1 , 5.3 , 1.05 6.1 , 1.05 6. 6 8. 6 6. 5 2. 2 11.8 , 8. 6 11.8 , 2. 2 2. 0 5. 3 2. 0 1. 1 9. 7 1. 1 Table 2 Summary of Cucumis Restrictio n 1•lapping Hybridizations Probe DNA Filter-boun d DNA Pvu 11[ Sal I Pvu II - Sal I 4 7 48, 20.5, 18 .5 , 12.5 , 11.8, 2.3 18.5 , 15.6 , 12.5, 11.8, 4.9, 2.3 28 48, 18.5, 16 .6 , 12.5 , 2.3 18.5 , 12.5 , 4.9, 2.9, 2.3 24 4 8 24 13.8 16.6 13.8 10.2 11.6 10.2 9. 6 20.5 , 11.6 4. 9 8. 3 11.6 6.8. 1.5 6. 8 48 6. 8 Pvu II I Sal I Pvu II-Sal 48 47, 28, 24, 6. 8 24, 6 . 9 8, 4 20.5 47, 9.6 15.6, 4. 9 18.5 47, 28 18.5 , 12.5 16.6 28, 13.8 13.8 , 2. 9 12.5 47, 28 18.5 , 12.5 11.8 47 11.8 10.2, 9.6, 8 . 3 10.2 , 6.8 , 4.9 , 15 11.6 2. 3 47, 28 2. 3 1601 Downloaded from https://academic.oup.com/nar/article/10/5/1593/1164751 by DeepDyve user on 17 September 2020 Nucleic Acids Research Table 3. Summary of Gene Mapping Hybridizations Filter-boun d DNA Probe Atriple x triangularis Cucumi8 sativa Sac I-Hpa I Sac I-Sal ] Sa l Sac I [ Pvu I I Pvu II-Sal I I 47,28,6 .8 6.8, 4.9 48 16S rRNA 18.4, 17 .4 4.3 6.3 23S rRNA 7.1 7.0 7.1 6.8 6.8 LS 8.0 7.9 5.8 47 15.6 5 PI I 18.4 14.0 1.8 28 18.5 5 16S 23S LS PII Is H| s HJS H|S H|S H| Figure 5. Mapping Atriplex chloroplast genes. Probes used are P-labelled 16S and 23S tobacco chloroplast rlbosomal RNA, the 0.58 kb Pat I fragment of corn chloroplast DNA, which is located entirely within the translated region of the gene for the large subunlt (LS) of ribulose-l,5-blsphosphate carboxy- lase (25), and the 1.1 kb Sal I-Pst. I fragment of spinach chloroplast DNA (26) which contains most of the gene for a 32,000 dalton photosystem II polypep- tide (PII) (27) . Slices containing the corn and spinach fragments were cut out of polyacrylamide gels of total chloroplast DNA and the DNA eluted and nick-translated (16). Atrlplex chloroplast DNA was digested with Sac I (S) and Hpa I-Sac I (H), electrophoreaed on a 0.7Z agarose gel and transferred to nitrocellulose filters. 1602 Downloaded from https://academic.oup.com/nar/article/10/5/1593/1164751 by DeepDyve user on 17 September 2020 Nucleic Acids Research plex chloroplast DNA. Data from these, as well as other Atrlplex and Cucumls hybridizations, are suiranarized in Table 3 and the map locations of these genes are indicated on the restriction maps shown in Figs. 2 and 3. DISCUSSION The mapping strategy presented here is an amalgam of two approaches which have been commonly used to map large DNAB of the size of the chloroplast ge- nome. The first is the determination of fragment overlaps by hybridization of labelled fragments produced by one enzyme to filter-bound fragments pro- duced by a second enzyme (1,7,13). In the second method, fragment overlaps are recognized from common double digestion fragments produced by reciprocal digestions of individual fragments purified out of low-melting agarose gels (2,4,6,8,9). The approach taken in this study clearly incorporates the first of these approaches, by hybridizing purified fragments to single digests, and also the second, by hybridizing to double digests. This unified approach al- lows a complete map to be produced with reference to a single set of probe fragments. By adding more lanes to each filter replica, one can easily scale up the experiment in order to map more enzymes for one DNA, or even to map several DNAs at once. This feature should permit comparative restriction mapping, as exemplified by the evolutionary studies of Brown and coworkers on primate mi - tochondrial DNA (20,21), to be performed on the much larger chloroplast genome. An elegant approach which allows simultaneous determineatlon of all pos- sible combinations of fragment homologies for two sets of restriction frag- ments is the cross-hybridization procedure described by Sato et al. (22). In principle, i.e., by hybridizing "hot" gels to both single and double-digest gels, this procedure could be adapted to provide each of the two data sets generated by the method described in this paper. However, this would require a minimum of four gels, to map fragments produced by two enzymes, and pro- portionately more gels to map additional enzyme digests. The Atriplex and Cucumis chloroplast genomes are extremely similar in all aspects examined. They differ by only a few percent in sire, both feature the inverted repeat which contains the ribosomal genes, and the genes for IS and FII are in the same approximate locations in the two genomes. One ap- parent difference between the two genomes is the size of the inverted repeat - 24 kb in Atriplex, but only 14 kb in Cucumis. Restriction mapping neces- sarily yields a min-lmai estimate for the size of the repeat. Since the 12.5 1603 Downloaded from https://academic.oup.com/nar/article/10/5/1593/1164751 by DeepDyve user on 17 September 2020 Nucleic Acids Research Spinach 2.4 0.7 20.5 22.3 13.9 9.0 10.6 47.9 10.6 22. 6 2 9.4 16 10.7 15. 2 3 3 10.7 19.8 lil i2 1 0.9 3.1 Atriplex Figure 6. Comparison of Sal I cleavage sites in the Atriple x and spinach (2) chloroplast genomes. The two long lines represent the inverted repeat. and 18.5 k b Sal I fragments adjacent to the apparen t end o f the inverted re - peat cross-hybridize (Table 2) , it can b e concluded that the inverted repeat does extend into at least these two Cucumis fragments. Comparison of the Sa l I map o f Atriplex with that of spinach (2), a mem - ber of the same family (Chenopodiaceae), suggests that these two chloroplast genomes are colinea r in sequence (Fig. 6) . The slight size differences be - tween many of the fragment s which map to the same locations in the two ge- nomes probably reflect small deletion/insertions, known to occur quite fre - quently during chloroplast genome evolution (23), rather than restriction site changes near the end s of fragments. Accordingly, these small size dif - ferences have been neglected in aligning the two maps . He have recently performed heterologous hybridizations using cloned mung bean restriction fragments and hav e found that the spinac h and Cucumis ge - nomes share a common sequence arrangement around their entire circumference (Palmer and Thompson , sub. for publ. ) . Thus it appears that all three of these chloroplast DNAs, from spinach, Atriplex and Cucumis, share the same basic pattern of sequence organization. ACKNOWLEDGEMENTS I am grateful to William F. Thompson for providin g laboratory facilities and support throughout this research. I thank D. Bourque for hi s generous gift of tobacco ribosomal RNA an d R. Jorgensen and D. Stein for their assis- tance in some of the DNA labeling reactions. I also appreciate the critical readings of this manuscript by W . F. Thompson, R. Jorgensen, and M. Zolan. This is CIW-DPB Publication #767. 1604 Downloaded from https://academic.oup.com/nar/article/10/5/1593/1164751 by DeepDyve user on 17 September 2020 Nucleic Acids Research REFERENCES 1. Bedbrook, J. R. and Bogorad, L. (1976) Proc. Natl. Acad. Sci. USA 73, 4309-4313 . 2 . Herrmann, R. G., Whitfeld, P. R. and Bottomley, W. (1980) Gene 8, 179- 191 . 3 . Bowman, C. M., Roller, B., Delius, H. and Dyer, T. A. (1981) Molec. Gen. Genet. 183, 93-101. 4 . 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Nucleic Acids Research – Oxford University Press

Published: Mar 11, 1982

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