ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 276, No. 1, January, pp. 172-179,199O

Application of an Efficient Strategy with a Phage X Vector for Constructing a Physical Map of the Amyloplast Genome of Sycamore (Acerpseudoplatanus)’ Jarunya Ngernprasirtsiri*32 *Research Institute Nagoya University,

and Hirokazu

Kobayashij-a3

for Biochemical Regulation, School of Agriculture, Chikusa, Nagoya 464-01, Japan

and tRadioisotope

Research Center,

Received May 2,1989, and in revised form August 21,1989

Amyloplasts were isolated from a heterotrophic culture cell line of a woody plant, sycamore (Acer pseudoplatanus), and their DNA was purified. Conventional procedures for making a physical map were not easily applicable to the amyloplast DNA, since the yield of DNA was too low and the presence of repeated sequences interfered with the analysis. Therefore, the pieces of amyloplast DNA starting with a few micrograms of DNA were cloned in the XFix vector, which is a derivative of XEMBL vectors improved for efficient cloning and gene walking. Cloned DNA fragments were randomly picked, mapped for restriction endonuclease sites by a refined procedure, and combined by overlapping their physical maps. The DNA library was also subjected to screening by gene walking using promoters recognized by T3 and T7 RNA polymerases in the vector to fill the gaps between sequences determined by overlapping the physical maps. In this way, we constructed the entire DNA library and the complete physical map of the amyloplast DNA. The sycamore amyloplast genome was composed of 141.7-kbp nucleotides with the same gene arrangement as that of tobacco 0 1990 Academic Press, Inc. chloroplasts.

The presence of DNA in chloroplasts was biochemically proved in the early 1960s (1,2); since then the nature of the DNA has been elucidated. The determination of the entire nucleotide sequences of chloroplast genomes from two plant species was completed in 1986 (3, ’ This research was supported by grants-in-aid from the Ministry of Education, Science and Culture of Japan (Monbusho), and by a grant from the Ishida Foundation (Nagoya). This is paper No. 83 in the series “Structure and Function of Chloroplast Proteins.” ’ Recipient of a predoctoral scholarship from the Hitachi Scholarship Foundation (Tokyo). 3 To whom correspondence should be addressed.

4), being a landmark in studies on chloroplast DNA. However, the biochemistry and molecular biology of plastids other than chloroplasts, namely nonphotosynthetic plastids, are still little known. The absence of photosynthetic activity in the nonphotosynthetic plastids made them less attractive. Furthermore, it was difficult to isolate nonphotosynthetic plastids, which were fragile enough to be destroyed during preparation due to the high content of starch. Plastids are organelles occurring specifically in plant cells, and a variety of them are known. Morphologically nondifferentiated plastids are called proplastids (5). As differentiated plastids, there are etioplasts in plants grown without light, chloroplasts possessing chlorophylls for photosynthesis, chromoplasts accumulating carotenoids, especially lycopene, and leucoplasts, which are colorless plastids (5). Amyloplasts, one of the plastids categorized as leucoplasts, lack pigments for photosynthesis and accumulate starch; they are present in storage tissues including endosperms, cotyledons, root caps, and tubers, as well as in cultured plant cells (5). Amyloplasts serve the human race in that they supply starch such as that in cereals and tubers. We chose cultured plant cells as experimental materials which we use relatively easily for several experimental purposes. The heterotrophically cultured cells of sycamore (plane maple, Acerpseudoplatanus), one of the most easily handled plant cultures (6), actually made it possible to isolate amyloplasts after sucrose starvation (7). This success let us develop molecular biology studies of amyloplasts. Genes and their expression in amyloplasts is an attractive research subject, because genes for photosynthesis do not seem to be expressed in amyloplasts (8). Cloning of the amyloplast DNA and construction of its physical map are necessary for further studies of the molecular mechanisms governing gene expression in amyloplasts. Conventional procedures for constructing

172 All

0003.9861/90 $3.00 Copyright 0 1990 by Academic Press, Inc. rights of reproduction in any form reserved.

PHYSICAL

MAP

OF AN AMYLOPLAST

physical maps were not easily applicable to amyloplast DNA due to the low yield of DNA and the presence of repeated sequences. Thus, in the present investigation we tried to improve the procedures in order to analyze the amyloplast genome. MATERIALS

AND

METHODS

Cell culture. A cell line derived from the cambial tissue of sycamore established by Lamport (6) was heterotrophically grown in a liquid medium supplemented with sucrose and 2,4-dichlorophenoxyacetic acid as described (8,9). Preparation of amyloplast DNA. Intact amyloplasts were isolated from the cultured sycamore cells at the exponential growth stage after sucrose starvation (7). Amyloplast DNA was purified as a closed circular DNA by CsCl density gradient centrifugation first with ethidium bromide and then with Hoechst 33258 as intercalating agenm as previously reported (7). Preparation of insert DNA for clon.ing. Amyloplast DNA (4.5 pg) was partially digested with 1 unit of Mb01 at 37°C. At 25, and 10 min after initiating digestion, 1.5 cg of DNA was taken from the reaction mixture and placed in a tube containing EDTA at a final concentration of 20 mM to stop the digestion. DNA (0.5 pg) of each digest was subjected to electrophoresis in 0.3% agarose gel to find the suitable digests composed of 9- to 22-kbp fragments. DNAs digested for 5 and 10 min were mixed and precipitated with ethanol. Those DNA fragments were subjected to filling-in of two nucleotides at the MboIcleaved site with dGTP and dATP. The resultant dipurine overhang, 5’.GA-3’, made it virtually impossible for the digested DNA fragments to religate themselves, thus lowering the frequency of insertion of rearranged multiple fragments. The filling-in reaction was carried out for 2 pg of the mixed digests with 10 mM dGTP, 10 mM dATP, 6 units of the Klenow fragment of Escherichia coli DNA polymerase I in a ligation buffer consisting of 50 mM Tris-Cl (pH 8), 7 mM MgCl,, and 1 mM dithiothreitol (DTT)4 at 25°C for 20 min, and stopped by heating at 68°C for 10 min. The sample was subjected to phenol-chloroform extraction and subsequent ethanol precipitation. Cloning by Wiz oector. The first two nucleotides at the ends of XhoI-digested XFix (Stratagene) were filled in with dTTP and dCTP to leave a 5’-TC-3’ overhang, which can combine with the partially filled-in Mb01 site of insert DNA fragments prepared as described above. With an equal molar ratio of the Mb01 inserts to the treated XFix arms, a ligation reaction was performed at 4°C for 12 h in the presence of 10 mM ATP with 3 units of T4 DNA ligase in the ligation buffer mentioned above. The ligation mixture was subjected directly to in vitro packaging by Gigapack Gold (Stratagene), which is essentially free from active EcoK or EcoB restriction systems. The packaged mixture was propagated on E. coli P2392 (P2 lysogen ofE. coli LE392). Microscale preparation of cloned h DNA. Recombinant plaques picked from the master stock plates were subjected to propagation of individual clone phage by the plate lysate method (10) in order to purify the DNA. The rapid purification of X DNA was performed using LambdaSorb phage absorbent (Promega Biotec). Partial digestion of cloned DNA with restriction enzymes. DNA preparations (1 pg each) from recombinant clones were digested with 1 unit of EcoRI, BarnHI, or Hind111 for 1, 2.5, 5, and 10 min at 37°C. The reaction was terminated by the addition of 5 hl of a stop solution consisting of 50 mM EDTA, 30% glycerol, 0.03% bromophenol blue, and 0.03% xylene cyanol. The above restriction Electrophoresis and Southern hybridization. enzyme digests were heated at 65°C for 5 min, and subjected to electro-

‘I Abbreviations used: DTT, dithiothreitol; RuBisCO, ribulose-l,5bisphosphate carboxylase/oxygenase; CF, , coupling factor l.

173

DNA

phoresis in 0.4% agarose gel together with X DNA nondigested (48.5 kbp) and DNA digested with Hind111 (23.1, 9.4,6.7, 4.1, 2.3, 2.0,0.56, and 0.13 kbp), with Sal1 (32.7, 15.3, and 0.50 kbp), and with SmaI (19.4, 12.2, 8.6, and 8.3 kbp), as DNA size markers. Electrophoresis was done until the 8.6-kbp fragment of &m&digested X DNA moved to the end of gel. The DNA fragments on the gel were transferred t,o Zeta-Probe membranes (Bio-Rad) by an alkaline procedure (11). In the Southern hybridization, the 3.5-kbp EcoRI fragment situated at the right end of X DNA was used as a probe. This fragment was purified from agarose gel using glass milk of Geneclean (BIO 101) and labeled with [rY-“‘P]dCTP by the Klenow fragment of E. coli DNA polymerase I and primers of a random primed DNA labeling kit (Boehringer). The membranes were exposed to Fuji X-ray film RXO-G with Fuji intensifying screen Grenex at -80°C. Among the signals of each cloned DNA digested for various periods as described above, the best partial digestion pattern was selected and processed to make the physical map of cloned DNA. Dataprocessing. The mobilities of individual bands on the autoradiograms were determined by a digitizer and its accompanying software using a NEC PC-9801 VM2 personal computer. Given the fact that the left and right arms of AFix are 20.0 and 9.0 kbp, respectively, the physical map of the insert DNA of each clone was elucidated. The individual physical maps were ordered and overlapped according to their relationship with neighbor clones in consideration of the consensus match of maps for the three restriction enzymes employed. Gene walking. To link the groups, the clones located at the terminals of each connected sequence were subjected to synthesis of endspecific probes using T3 or T7 promoter. The DNA was first digested independently with AluI, HueIII, and RsaI, and the reactions were terminated with 10 mM EDTA. The digests were mixed, treated with 0.2 mg/ml of proteinase K (DNase-free, Bethesda Research Laboratories) at 37°C for 30 min, extracted with phenol-chloroform, and precipitated with ethanol. The RNA polymerase reactions were carried out in 25 ~1 of a reaction mixture consisting of approximately 2 fig of the above restricted DNA templates, 40 mM Tris-Cl (pH 8.0), 8 mM MgCI,, 2 mM spermidine, 50 mM NaCI, 0.4 mM each of ATP, CTP, GTP, 2.5 PM [n-“LP]UTP (25 PCi, 925 kBq), 10 mM DTT, 25 units of RNase inhibitor from human placenta (Takara), and 10 units of T3 or T7 RNA polymerase at 37°C for 30 min. This procedure was repeated to confirm the physical map and ordering of each clone. Gene locations. To identify genes on the amyloplast DNA, highly purified DNA was completely digested with EcoRI, BnmHI, or Hind111 and subject.ed to Southern hybridization with specific gene probes. The following plasmids containing maize chloroplast DNA sequences cloned in Bogorad’s laboratory (12, 13) were employed: pZmc427 for the 32.kDa QH protein (psbA), the 0.6.kbp Hind111 fragment of pZmc527 for the o subunit of coupling factor 1 (CF,, atpA), pZmc556 for the apoprotein of P700 (psaA), pZmc747 for ribosomal protein S4 (rps4), pZR4876 for the p and t subunits of CF, (atpB,E), pZmc461 for the large subunit of ribulose-l$bisphosphate carboxylase/oxygenase (RuBisCO, rbcL), and pZmc532 for the 16 S rDNA. The clones of tobacco chloroplast DNA in M13mp18 phage (replicative form) from Sugiura’s laboratory (3, 14) were also employed for the e subunit [rpoA, 759.bp Sap1 fragment in Ps9], the fi subunit (rpoB, 525-bp HindII1 fragment in Bal2a), and the fl’subunit (rpoC, in Ba13, and rpo& in Ba4) of RNA polymerase. The DNA probes were labeled with [n-“‘P]dCTP by a random primed procedure as described above.

RESULTS Strategy

for constructing

a physical

map.

Conven-

tional procedures for making a physical map were not easily applicable to the amyloplast DNA due to the low yield of DNA and the intramolecular occurrence of repeated sequences which interfered with the analysis.

174

NGERNPRASIRTSIRI

AND

KOBAYASHI

A9 W = I

448.5 432.7 -23.1 * 19.4 - 15.3

w

E2

1

E5

i El5 $4

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,

1 E13al ElB 123t@&

4 12.2 4 9.4 :+ 8.6

Skbp

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kbp - 48.5 :g:: (IS.4 415.3 412.2 4 9.4 - 8.6

h,

T7 T7 a

I 8171 Bll I

,

Bl

Skbp

.

4

9kbp

t-J---T7 YC

--7-

- .-.----~

I

5kbp

I

FIG. 1. Examples of restriction map data showing overlaps. The DNA of recombinant phages X7, X8, and X9, which were randomly selected from the DNA library, were prepared by the microscale method described under Materials and Methods. One microgram of DNA was partially digested with EcoRI (A), BamHI (B), and Hind111 (C), and subjected to electrophoresis together with DNA size markers as indicated at the right in kilobase pairs. Southern hybridization was performed using the right-end probe of h DNA. The migration distance of each signal corresponding to size from the right end of recombinant h DNA was determined by a digitizer connected to a personal computer, and aligned to make a physical map of each clone. The restriction map is presented under the autoradiogram by horizontal bars: L and R indicate the left and right arms of recombinant phage, and T3 and T7 show the sides of T3 and T7 promoters located on the right and left arms of phage, respectively. The clone fragments were overlapped with specific restriction endonuclease sites and integrated into a longer sequence as shown at the bottom of each panel.

PHYSICAL 20

10

0

30

T71

50

40

I

MAP

OF AN AMYLOPLAST

60

80

70

90

175

DNA 100

110

120

130

140

kbp

No.

IT3

I 2

3 4 5 6 7 6 9 10 1; 12

13 14 15 16 I7

16

2 I

19 20

21

T7

III --. X

22 23 24 25

Cl

I II

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I

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28 29 30 31

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E3 [lOb]llb]321

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24 23b2Od26 31blEb35b 20b 35al80310 11362211 E2 1 E5 1151110dE9113~ I m I 111211 1 1131b lEBbh4h tli ~110[1914+E6~ 3341 I ) fl EcoRI 34 25 4cl 280200 16 300230 2Oc28b 4b IRb

IRo

FIG. 2. The integrated restriction map of amyloplast DNA with EcoRI. The restriction map data of a total of 35 recombinant clones are summarized. Scales are shown in kilobase pairs. The individual clones are represented by horizontal bars and placed according to coincidence of their borders with the neighboring clones under consensus maps for three restriction enzymes, EcoRI, BamHI, and KndIII. The data of hl to X25, which were randomly picked, were analyzed to sort first. The resultant integrated sequences were not connected to make an entire sequence with three gaps, namely X, Y, and Z. The final integrated data are shown at the bottom. See the text for details.

Therefore, we developed a procedure to overcome the difficulty. It consisted of six steps: (i) a library of the amyloplast DNA was prepared in the XFix vector, a derivative of XEMBL vectors which has been improved for efficient cloning and gene walking; (ii) the resultant recombinant clones were randomly picked and subjected to extraction of their DNA; (iii) the restriction enzyme sites of individual clones were mapped; (iv) the resulting physical maps were sorted by overlapping them; (v) gaps between linked large sequences were filled by finding clones covering them by gene walking with end-specific probes produced by T3 or T7 RNA polymerase; and (vi) the data were integrated into the whole map. Construction of a library and sorting of each cloned We cloned the pieces of amyloplast DNA startDNA. ing from 4.5 pugemploying hFix vector and a PB-lysogenie E. coli host, P2392. The efficiency of infection was 8.0 X 10’ plaques per microgram of DNA. By plaque hybridization with “2P-labeled amyloplast DNA, we found that all of the large plaques were recombinant. Twenty-

five of the recombinant plaques were randomly selected and their DNA was extracted. The region covered by X7, X8, and X9 is given in Fig. 1 as an example. The DNA was partially digested with EcoRI, BamHI, or HindIII, blotted on a membrane, and hybridized with the “‘P-labeled DNA fragment of the right end of X DNA. The restriction enzyme sites were mapped on each recombinant X DNA corresponding to the distance from the right end of X DNA to individual sites. The physical maps of the passenger DNA were combined by overlapping. The physical maps of 25 clones provided three linkage groups consisting of approximately 61, 36, and 30 kbp, respectively (Fig. 2). To link the groups, the Gene walking to fill the gaps. clones located at the terminals of each group were subjected to synthesis of end-specific probes using T3 or T7 RNA polymerase reaction (Fig. 2). The X26 to X28 were found to be hybridized with end-specific probes synthesized by T7 promoters of hl and X22. By the same principle, X29 and X30 were obtained by screening with the T7

176 e

NGERNPRASIRTSIRI EcoRI 12

0 3

4

5

6

7

6

FIG. 3. The location of genes pletely digested with EcoRI (A), bromide staining pattern, lanes rpoC,, rpoB, psaA, rps4, atpB,E, pairs.

9

10 1112

AND

KOBAYASHI

BamH I

1 2 3

@

4 5 6

7

6

9

10 11 12

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6 7 6

9 10 11 12

on restriction fragments of amyloplast DNA analyzed by Southern hybridization. Amyloplast DNA was comBamHI (B), and Hind111 (C), and subjected to electrophoresis in 0.7% agarose gel. Lane 1 shows an ethidium 2 to 12 present autoradiograms of the Southern hybridization with the following probes; psbA, atpA, rpo&, rbcL, rpoA, and 16 S rDNA, respectively. DNA size markers are indicated at the left of each panel in kilobase

promoter of X12 and the T3 promoter of X13. The h31 and X32 were chosen by screening with specific probes synthesized by T3 promoters of X18 and X19. The end probes were also able to be used to rescreen new clones to confirm the inverted repeat region between h19 and X22. The specific end probes of the T7 promoter of X19 and the T3 promoter of X22 were used to screen X33 to X35. We finally integrated the data as shown at the bottom of Fig. 2. The entire DNA library was thus constructed and, accordingly, the complete physical map of the amyloplast DNA was elucidated. Gene locations and the entire map. The location of genes on restriction fragments of amyloplast DNA were determined by Southern hybridization. The DNA was completely digested with EcoRI, BamHI, or HindIII, and subjected to Southern hybridization using gene probes of p&A, atpA, rpo&, rpoC,, rpoB, psaA, rps4, atpB,E, &CL, rpoA, and 16 S rDNA as shown in Fig. 3. The sizes of generated restriction fragments in relation to gene locations are summarized in Table I on the basis of the results shown in Figs. 2 and 3. The amyloplast DNA was composed of 53 EcoRI fragments longer than 0.5 kbp and 7 shorter ones migrating as 35 separate bands including duplication and triplication on the agarose gel, 32 (>0.5 kbp) and 2 (shorter) BamHI fragments visualized as 23 bands, and 18 (>0.5 kbp) and 2 (shorter) Hind111 fragments with 16 detectable bands. The entire physical map and gene location of sycamore amyloplast DNA are shown in Fig. 4. The amyloplast genome was concluded to consist of 141.7-kbp DNA with the same gene arrangement as tobacco chloroplast DNA (3,14).

DISCUSSION We constructed a complete physical map and an entire DNA library with a few micrograms of DNA using an improved procedure. Partial digestions of X recombinant DNA with restriction enzymes and subsequent Southern hybridization with a probe for the 3’ end of the right arm of the X vector allowed easy construction of the physical map of each cloned DNA, since we could optimize the intensity of radioactive bands by changing the time of exposure to X-ray film. In addition, when we had gaps which were not covered by the clones first selected, the present procedure with XFix was extremely convenient in comparison with conventional gene walking techniques (15-la), since subcloning is not required to make probes specific to the ends of inserts. Construction of other physical maps from cloned DNA has been attempted for E. coli genomes by the XEMBL4 vector (19) and by cosmid vectors (no), for the yeast genome by the XMG3 vector (al), and for the genome of the nematode Caenorhabditis elegans by the pJB8 cosmid vector (22). To our knowledge, no application of a vector possessing bacteriophage promoters, e.g., T3 and T7 promoters, for making end-specific probes such as hFix which we employed has been published for the purpose of elucidating a map of a genome. The DNA from nonphotosynthetic plastids have rarely been analyzed. Only one physical map is available from chromoplasts of the bell pepper Capsicum annuum (23). We found that the gene arrangement and size of sycamore amyloplast DNA were similar to those of tobacco chloroplasts (Fig. 4). The restriction profiles of

PHYSICAL

MAP

OF AN AMYLOPLAST TABLE

Sizes

177

DNA

I

and Coding Genes of Amyloplast DNA Fragments Generated by Complete Digestion with EcoRI, BarnHI, or HindIII”

EcoRI

kbp

El E2 E3 E4a,b E5 E6 E7 E8a E8b E9 ElOa ElOb Ella,c Ellb El2 E13a,b E14a,b El5 El6 El7 E18a-c El9 EBOa-c E20d E21 E22 E23a,b E24 E25 E26 E27 E28a,b E29 E30a,b ESla,b E32 E33a,b E34 E35a,b

8.5 7.1 5.9 5.2 5.0 4.9 4.5 4.3 4.3 3.8 3.7 3.7 3.6 3.6 3.4 3.2 3.0 2.9 2.7 2.5 2.2 2.1 2.0 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5

” Fragments

Gene

psaA

rps4 psbA 16SrDNA 16SrDNA rbcL atpB,E rpoC2 rpoG

rDoA

BamHI

kbp

Bl B2 B3a B3b B4a B4b B5 B6 B7 B8 B9

19.0 15.4 11.5 11.5 8.2 8.2 6.0 5.3 4.4 4.0 3.7

BlO Bll

3.5 3.2

B12 BlSa,b B14a,b B15a,b B16 B17 B18 BlSa,b B20 B2la,b B22a,b B23a,b

3.1 2.9 2.6 2.5 2.3 2.2 2.1 1.6 1.5 1.4 1.2 1.1

Gene rpoA psaA rps4 atpA 16SrDNA 16SrDNA

Hind111

kb

Hl

21.0

H2 H3a H3b

18.0 16.7 16.7

Gene atpB,E rbcL rpoA rpoB atpA ?-POG

rpoB

rpoc, rpoc, psbA atpB,E rbcL

rpoC:, atpB,E

H4 H5 H6 H7 H8 H9a H9b HlO Hll H12 H13 H14 H15 H16

14.6 12.8 6.2 5.6 5.4 4.8 4.8 3.4 3.0 2.9 2.5 1.3 1.1 0.9

rpoC$ psbA psaA rps4

16SrDNA 16SrDNA psbA

rpoB

were numbered in order of decreasing size. Fragments

less than 0.5 kbp were not included.

DNAs from amyloplasts and chloroplasts in heterotrophic and autotrophic culture cell lines of sycamore, respectively, were also compatible (24). Therefore, the nucleotide sequences among differentiated plastids are thought to be identical in each individual, excluding the reliability of DNA rearrangement during plastid development. None of the plastids from woody plants other than conifers (25) which belong to Gymnospermae has been analyzed on the basis of molecular biology. Sycamore is a wood, is perennial, and is classified to the infraclass Sapindaifloriidae, plastids from which have not been studied. Instead of being unique, the gene arrangement and size of sycamore plastid DNA are homologous to those of tobacco chloroplast DNA (3, 14). Therefore, genes

governing features characteristic of a wood are thought to be encoded in the nuclei. Plastid DNAs are thus relatively stable during evolution, and all genetic information encoded in plastid DNAs must be essential for growth. In Dicotyledoneae, plastid DNAs from the following infraclasses have identical gene arrangement and genome size: Sapindaifloriidae, e.g., sycamore (A. pseudoplatanus); Caryophylliidae, e.g., spinach (Spinacia oleratea) (26); and Corollifloriidae, e.g., tobacco (Nicotiana tabacum) (l), bell pepper (C. annuum) (23), tomato (Lycopersicon) (27), and Petunia (28). In contrast, the distinguishable properties of plastid DNAs were reported from the infraclasses Rosaefloriidae including Leguminosae, e.g., mung bean (Vigna radiata) (29,30), pea (Pi-

178

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82

$

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25 , Ztl’)

87 HI3

Ella 2a”

H12 I

El9 I96 HI1

El4a

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28a

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B4a

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I50

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2

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1 HlO\l~l~

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32

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>

FIG. 4. The entire physical map and gene locations of sycamore amyloplast DNA. The map is represented in linearized circular amyloplast DNA (141.7 kbp) is opened at the left border of an inverted repeat region (IRa). EcoRI, BamHI, and sites are shown. The sizes of restriction fragments are given in Table I. The bold lines indicate the approximate lengths of regions (IR). The genes were positioned by Southern hybridization. Approximate sizes of the genes are presented by bold gene symbols.

sum satiuum) (30), and soybean (Glycine) (29); and Dilleniidae including Brassicaceae, e.g., lettuce (Lactuca satiua) (30). Further analysis of plastid DNAs from other species thus may provide invaluable information on the taxonomical and evolutionary aspects of Dicotyledoneae. Elucidation of the physical map of sycamore amyloplast DNA and construction of its entire DNA library should facilitate precise analysis of regulatory mechanisms of gene expression in the amyloplasts. The hypothetical view of the suppression of transcription of photosynthesis genes through DNA methylation in the amyloplasts (24) could be further studied employing cloned DNA fragments obtained in the present investigation.

form, in which the Hind111 restriction the inverted repeat lines in&ding the

3. Shinozaki, K., Ohme, M., Tanaka, M., Wakasugi, T., Hayashida, N., Matsubayashi, T., Zaita, N., Chunwongse, J., Obokata, J., Yamaguchi-Shinozaki, K., Ohto, C., Torazawa, K., Meng, B. Y., Sugita, M., Deno, H., Kamogashira, T., Yamada, K., Kusuda, J., Takaiwa, F., Kato, A., Tohdoh, N., Shimada, H., and Sugiura, M. (1986) EMBO J. 5,2043-2049. H., Kohchi, T., Shirai,H., Sano,T., Sano, 4. Ohyama, K.,Fukuzawa, S., Umesono, K., Shiki, Y., Takeuchi, M., Chang, Z., Aota, S., Inokuchi, H., and Ozeki, H. (1986) Nature (London) 322,572-574. R. A. E. (1978) The Plastids: 5. Kirk, J. T. O., and Tilney-Bassett, Their Chemistry, Structure, Growth and Inheritance, p. 960, Elsevier/North-Holland Biomedical, Amsterdam. 6. Lamport, D. T. A. (1964) Exp. Cell Res. 33,195-206. 7. Macherel, D., Kobayashi, H., Akazawa, T., Kawano, S., and Kuroiwa, T. (1985) Biochem. Biophys. Res. Commun. 133,140-146. J., Macherel, D., Kobayashi, 8. Ngernprasirtsiri, T. (1988) Plant Physiol. 86,137-142.

H., and Akazawa,

9. Bligny, R. (1977) Plant Physiol. 59,502-505. ACKNOWLEDGMENTS We are much indebted to Dr. Takashi Akazawa for the use of facilities for the research and for his continuous encouragement throughout the work. We also express our hearty thanks to Drs. Lawrence Bogorad and Masahiro Sugiura for their permission to use their clones of maize and tobacco chloroplast DNAs, respectively, and to Drs. Julapark Chunwongse, Takashi Tsuge, and Alejandro M. Viale for their helpful technical suggestions and discussion. REFERENCES 1. Iwamura, T. (1960) Biochim. Biophys. Acta 42, 161-163. 2. Sager, R., and Ishida, M. (1963) Proc. N&l. Acad. Ski. USA 60, 725-730.

10. Maniatis T., Fritsch E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, p. 545, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 11. Reed, K. C., and Mann, D. A. (1985) Nucleic Acids Res. 13,72077221. K. M. T., Gobbins, E. J., and Bo12. Larrinua, I. M., Muskavitch, gorad, L. (1983) Plant Mol. Biol. 2,129-140. 13. Kobayashi, K., Bogorad, L., and Milles, D. M. (1987) Plant Physiol. 85, 757-767. 14. Sugiura, M., Shinozaki, K., Zaita, N., Kusuda, M., and Kumano, M. (1986) Plant Sci. 44,211-216. 15. Fritsch, 972.

E. F., Lawn, R. M., and Maniatis,

T. (1980) Cell 19,959-

PHYSICAL 16. Shimizu, A., Takahashi, Cell 28,499-506. 17. Eickbush,

OF AN AMYLOPLAST

N., and Yaoita, Y., and Honjo, T. (1982)

24.

P., and Hogness, D. S. (1983) J. Mol. Biol.

25.

K., and Isono, K. (1987) Cell 50,495-508.

26.

168,17-33. 19. Kohara, Y., Akiyama,

23. Gounaris,

F. C. (1982) Cell 29,633-643.

T. H., and Kafatos,

18. Bender, W., Spierer,

MAP

20. Knott, V., Rees, D. J. G., Cheng, Z., and Brownlee, Nucleic Acids Res. 16,2601-2612.

G. G. (1988)

21. Olson, M. V., Dutchik, J. E., Graham, M. Y., Brodeur, G. M., Helms, C., Frank, M., MacCollin, M., Scheinman, R., and Frank, T. (1986) Proc. Natl. Acad. Sci. USA 83,7826-7830. 22. Coulson, A., Sulston, J., Brenner, Natl. Acad. Sci. USA 83,782ll7825.

S., and Karn,

27. 28. 29.

J. (1986) Proc.

30.

DNA

179

I., Michalowski, C. B., Bohnert, H. J., and Price, C. A. (1986) Cur. Genet. 11,7-16. Ngernprasirtsiri, J., Kobayashi, H., and Akazawa, T. (1988) Proc. Natl. Acad. Sci. USA 85,4750-4754. Strauss, S. H., Palmer, J. I)., Howe, G. T., and Doerksen, A. H. (1988) Proc. Natl. Acad. Sci. USA 85,3898-3902. Westhoff, P., Alt, J., Nelson, N., and Herrmann, R. G. (1985) Mol. Gem Genet. 199,290-299. Piechulla, B., Imlay, K. R. C., and Gruissem, W. (1985) Plant Mol. Biol. 5, 373-384. Bovenberg, W. A., Howe, C. J., Kool, A. J., and Nijkamp, H. J. J. (1984) Curr. Genet. 8,283-290. Palmer, J. D., Singh, G. P., and Pillay, D. T. N. (1983) Mol. Gem Genet. 190,13-19. Palmer, J. D. (1985) Annu. Reu. Genet. 19,325-354.